CN115197307B - Protein IbGER5 for regulating stress resistance of plants, coding gene and application thereof - Google Patents

Abstract

The invention discloses the protein IbGER5 for regulating the stress resistance of plants, its encoding gene and application. Specifically disclosed are proteins whose amino acid sequence is SEQ ID No. 1, related biological materials, and applications thereof in regulating plant salt tolerance and/or drought resistance. In the present invention, by introducing the IbGER5 gene derived from sweet potato into the recipient control Lizixiang, transgenic sweet potato plants with overexpression and interference expression of the IbGER5 gene have been obtained. Experiments have shown that under adversity stress (such as salt stress and/or drought stress) conditions The salt and drought resistance of the transgenic sweet potato plants overexpressing the IbGER5 gene is significantly reduced, and the salt and drought resistance of the transgenic sweet potato plants that interfere with the expression of the IbGER5 gene (RNAi) is significantly improved, indicating that the IbGER5 protein and its coding gene provided by the present invention have Regulating the function of plant salt tolerance and/or drought resistance can be widely used in sweet potato anti-reverse gene breeding.

Description

Protein IbGER5 regulating plant stress resistance and its coding gene and application

technical field

The invention belongs to the field of biotechnology, and in particular relates to a protein IbGER5 regulating plant stress resistance, its coding gene and its use.

Background technique

Sweet potato (Ipomoea batatas (L.) Lam.) is an important food, feed and industrial fuel crop, which has both health care and medicinal value, and is widely planted throughout my country. With the continuous reduction of arable land, sweet potatoes are mainly planted on marginal lands such as drought or salinization, while the salinity of saline-alkali land in my country is 0.6-10%. The range of arid areas is wide, and the salinization of arable land and water resources The lack seriously affects the normal growth of crops and brings great difficulties to the further development of sweet potato production. Therefore, cultivating new high-quality sweet potato varieties with strong salt tolerance and drought resistance has become one of the important measures to promote the development of sweet potato industry.

Although conventional sweet potato breeding technology has played an important role in variety improvement, due to the problems of self-incompatibility, unstable hybrid offspring, lack of germplasm resources, and long breeding cycle, traditional hybrid breeding methods are difficult to breed. New sweet potato varieties with strong salt and drought resistance, and the use of genetic engineering methods can overcome the barriers of species isolation and gene linkage in conventional breeding, and can improve the characteristics of sweet potato from the molecular level. It is a feasible way to cultivate high-quality sweet potato varieties at present. It has potential significance in promoting the breeding and production of sweet potato varieties. Mining important salt-tolerant and drought-resistant genetic resources plays a key role in cultivating new varieties of salt-tolerant and drought-resistant plants. The development and utilization of salt-tolerant and drought-resistant plants has immeasurable ecological, economic and social benefits.

Contents of the invention

The technical problem to be solved by the invention is how to regulate the stress resistance of plants (such as sweet potato). The technical problems to be solved are not limited to the described technical subjects, and those skilled in the art can clearly understand other technical subjects not mentioned herein through the following description.

In order to solve the above-mentioned technical problems, the present invention firstly provides the application of proteins or substances that regulate the activity and/or content of the proteins, and the applications can be any of the following:

D1) the application of proteins or substances that regulate the activity and/or content of the proteins in regulating the stress resistance of plants;

D2) Application of proteins or substances that regulate the activity and/or content of the proteins in the preparation of products that regulate plant stress resistance;

D3) Application of proteins or substances that regulate the activity and/or content of said proteins in cultivating stress-resistant plants;

D4) Application of proteins or substances that regulate the activity and/or content of said proteins in the preparation of products for cultivating stress-resistant plants;

D5) Application of protein or substances regulating the activity and/or content of said protein in plant breeding;

The name of the protein is IbGER5, which can be any of the following:

A1) amino acid sequence is the protein of SEQ ID No.1;

A2) A protein having more than 80% identity and the same function as the protein shown in A1) obtained by substituting and/or deleting and/or adding amino acid residues to the amino acid sequence shown in SEQ ID No.1;

A3) A fusion protein with the same function obtained by linking tags at the N-terminal and/or C-terminal of A1) or A2).

In order to make the protein in A1) easy to purify or detect, the amino-terminus or carboxy-terminus of the protein composed of the amino acid sequence shown in the sequence listing as SEQ ID No.1 can be connected with a tag protein.

The tagged protein includes but not limited to: GST (glutathione thiol transferase) tagged protein, His6 tagged protein (His-tag), MBP (maltose binding protein) tagged protein, Flag tagged protein, SUMO tagged protein, HA tag protein, Myc-tagged protein, eGFP (enhanced green fluorescent protein), eCFP (enhanced cyan fluorescent protein), eYFP (enhanced yellow-green fluorescent protein), mCherry (monomeric red fluorescent protein) or AviTag-tagged protein.

Those skilled in the art can easily use known methods, such as directed evolution or point mutation methods, to mutate the nucleotide sequence encoding protein IbGER5 of the present invention. Those nucleotides that have been artificially modified and have 75% or more identity with the nucleotide sequence of the protein IbGER5 isolated in the present invention, as long as they encode the protein IbGER5 and have the function of the protein IbGER5, are all derived from the present invention Nucleotide sequences and are equivalent to the sequences of the present invention.

The identity of 75% or more may be 80%, 85%, 90% or more.

Herein, identity refers to the identity of amino acid sequence or nucleotide sequence. Amino acid sequence identities can be determined using homology search sites on the Internet, such as the BLAST webpage of the NCBI homepage. For example, in advanced BLAST2.1, by using blastp as the program, set the Expect value to 10, set all Filters to OFF, use BLOSUM62 as Matrix, and set Gap existence cost, Per residue gap cost and Lambda ratio to 11, 1 and 0.85 (default value) and search to calculate the identity of the amino acid sequence, then the value (%) of the identity can be obtained.

Herein, the above 80% identity may be at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98% or 99% identity.

Herein, the substance that regulates the activity and/or content of the protein may be a substance that regulates the expression of a gene encoding the protein IbGER5.

In the above, the substance that regulates gene expression can be a substance that performs at least one regulation in the following 6 kinds of regulation: 1) regulation performed at the gene transcription level; 2) regulation performed after the gene transcription ( That is, the regulation of the splicing or processing of the primary transcript of the gene); 3) the regulation of the RNA transport of the gene (that is, the regulation of the mRNA of the gene from the nucleus to the cytoplasm); 4) regulation of the translation of the gene; 5) regulation of the degradation of the mRNA of the gene; 6) regulation of the post-translation of the gene (that is, regulation of the activity of the protein translated by the gene ).

The substance that regulates gene expression can specifically be the biological material described in any one of the following B1)-B3).

Further, the substance for regulating gene expression may be a substance (including a nucleic acid molecule or a vector) that improves or up-regulates the expression of the gene encoding the protein IbGER5.

Further, the substance regulating gene expression can also be a substance (including nucleic acid molecule or vector) that inhibits or reduces or down-regulates the expression of the gene encoding the protein IbGER5.

In the above application, the protein IbGER5 can be derived from sweet potato (Ipomoea batatas (L.) Lam.).

Further, the protein IbGER5 may be a protein IbGER5 regulating plant stress resistance.

Further, the protein IbGER5 may be a protein IbGER5 that regulates the salt tolerance and drought resistance of plants.

The present invention also provides the application of biological materials related to the protein IbGER5, and the application can be any of the following:

E1) application of biological materials related to said protein IbGER5 in regulating plant stress resistance;

E2) Application of biological materials related to the protein IbGER5 in the preparation of products regulating plant stress resistance;

E3) application of biological materials related to the protein IbGER5 in cultivating stress-resistant plants;

E4) Application of biological materials related to the protein IbGER5 in the preparation of products for cultivating stress-resistant plants;

E5) Use of biological material related to said protein IbGER5 in plant breeding;

The biological material can be any of the following B1) to B8):

B1) nucleic acid molecule encoding the protein described in claim 1 or 2;

B2) suppress or reduce the nucleic acid molecule of the coding gene expression of protein described in claim 1 or 2;

B3) an expression cassette containing the nucleic acid molecule of B1) and/or B2);

B4) a recombinant vector containing the nucleic acid molecule described in B1) and/or B2), or a recombinant vector containing the expression cassette described in B3);

B5) A recombinant microorganism containing the nucleic acid molecule described in B1) and/or B2), or a recombinant microorganism containing the expression cassette described in B3), or a recombinant microorganism containing the recombinant vector described in B4);

B6) the transgenic plant cell line containing the nucleic acid molecules described in B1) and/or B2), or the transgenic plant cell line containing the expression cassette described in B3), or the transgenic plant cell line containing the recombinant vector described in B4);

B7) Transgenic plant tissue containing the nucleic acid molecule described in B1) and/or B2), or a transgenic plant tissue containing the expression cassette described in B3);

B8) A transgenic plant organ containing the nucleic acid molecule described in B1) and/or B2), or a transgenic plant organ containing the expression cassette described in B3).

In the above application, the nucleic acid molecule can be any of the following:

C1) the coding sequence is the DNA molecule shown in the 1-204th positions of SEQ ID No.2 or SEQ ID No.2;

C2) nucleotide sequence is the DNA molecule shown in the 1-204th positions of SEQ ID No.2 or SEQ ID No.2.

The amino acid sequence encoded by the DNA molecule (IbGER5 gene) shown in SEQ ID No.2 is the protein IbGER5 of SEQ ID No.1.

The nucleotide sequence shown in SEQ ID NO.2 is the nucleotide sequence of the protein IbGER5 coding gene (CDS).

B1) The nucleic acid molecule can also include a nucleic acid molecule obtained through codon preference modification on the basis of the nucleotide sequence shown in SEQ ID No.2.

B1) The nucleic acid molecule also includes a nucleic acid molecule that is more than 95% identical to the nucleotide sequence shown in SEQ ID No.2 and derived from the same species.

The protein IbGER5 gene (IbGER5 gene) of the present invention can be any nucleotide sequence that can encode the protein IbGER5. Considering the degeneracy of codons and the preference of codons in different species, those skilled in the art can use codons suitable for the expression of specific species as needed.

The expression cassette includes a promoter, a nucleic acid molecule encoding the protein IbGER5 and a terminator, the promoter can be a CaMV35S promoter, a NOS promoter or an OCS promoter, and the terminator can be a NOS terminator or an OCS polyA terminator.

The nucleic acid molecule described herein can be DNA, such as cDNA, genomic DNA or recombinant DNA; the nucleic acid molecule can also be RNA, such as gRNA, mRNA, siRNA, shRNA, sgRNA, miRNA or antisense RNA.

The vectors described herein are well known to those skilled in the art, including but not limited to: plasmids, bacteriophages (such as λ phage or M13 filamentous phages, etc.), cosmids (ie Cosmids), Ti plasmids or viral vectors. Specifically, it can be pMD19-T vector and/or pCAMBIA1300 vector and/or pFGC5941 vector.

An existing plant expression vector can be used to construct a recombinant expression vector containing the IbGER5 gene. The plant expression vectors include, but are not limited to, binary Agrobacterium vectors and vectors that can be used for plant microprojectile bombardment. The plant expression vector may also include the 3' untranslated region of the foreign gene, that is, the polyadenylation signal and any other DNA fragments involved in mRNA processing or gene expression. The polyadenylic acid signal can guide polyadenylic acid to be added to the 3' end of the mRNA precursor, such as including but not limited to Agrobacterium crown gall tumor induction (Ti) plasmid gene (such as nopain synthase Nos gene), plant gene (such as soybean storage protein gene) 3' transcribed untranslated regions have similar functions.

When using the IbGER5 gene to construct a recombinant plant expression vector, any enhanced promoter or constitutive promoter can be added before its transcription initiation nucleotide, including but not limited to the cauliflower mosaic virus (CaMV) 35S promoter , maize ubiquitin promoters (ubiquitin), which can be used alone or in combination with other plant promoters; in addition, when using the gene of the present invention to construct plant expression vectors, enhancers, including translation enhancers or transcription enhancers, can also be used These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence to ensure correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes (GUS genes, luciferase gene, etc.), antibiotic markers with resistance (gentamycin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

Using any vector that can guide the expression of exogenous genes in plants, the IbGER5 gene or gene fragments provided by the present invention are introduced into plant cells or recipient plants, and transgenic plants with altered stress resistance can be obtained. The expression vector carrying the IbGER5 gene can transform plant cells or tissues by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrical conductivity, and Agrobacterium-mediated, and transform the transformed plant tissues grow into plants.

The microorganisms described herein may be yeast, bacteria, algae or fungi. Among them, the bacteria can be from Escherichia, Erwinia, Agrobacterium, Flavobacterium, Alcaligenes, Pseudomonas (Pseudomonas), Bacillus (Bacillus), etc. Specifically, it can be Escherichia coli DH5α and/or Agrobacterium tumefaciens EHA105.

The recombinant vector can specifically be the recombinant vector pCAMBIA1300-GFP-IbGER5 and/or pFGC5941-IbGER5.

Described recombinant vector pCAMBIA1300-GFP-IbGER5 is that the fragment (small fragment) between the KpnI of pCAMBIA1300-GFP carrier and the SalI recognition site is replaced with the DNA fragment of SEQ ID No.2 in the sequence listing, keeps pCAMBIA1300 The other sequences of the -GFP vector remain unchanged, and the resulting recombinant expression vector is obtained. The recombinant vector pCAMBIA1300-GFP-IbGER5 expresses the protein IbGER5 shown in SEQ ID No.1 in the sequence listing. The recombinant vector pCAMBIA1300-GFP-IbGER5 has an expression cassette, the coding gene of CaMV35S promotor, IbGER5 protein (SEQ ID No.2) and the coding gene of green fluorescent protein (GFP) are arranged in the nucleotide sequence of expression cassette.

The pCAMBIA1300-GFP vector is based on pCAMBIA1300, and the CaMV 35S fragment is inserted between the EcoRI and SacI recognition sites of the pCAMBIA1300 vector, and the GFP fragment is inserted between the SalI and PstI recognition sites of the pCAMBIA1300 vector.

The recombinant vector pFGC5941-IbGER5 replaces the small fragment between the XhoI and SwaI recognition sequences of the pFGC5941 carrier with the DNA molecule fragment shown in the 1st to 204th positions of SEQ ID No.2 from the 5' end in the sequence listing, which will restrict The fragment (small fragment) between the endonuclease BamHI and XbaI recognition sites is replaced by the nucleotide sequence, which is the reverse of the DNA molecule shown in the 1st to 204th positions from the 5' end of SEQ ID No.2 in the sequence listing The complementary sequence keeps other sequences of the pFGC5941 vector unchanged to obtain a recombinant expression vector. The recombinant vector pFGC5941-IbGER5 interferes with the protein IbGER5 shown in SEQ ID No.1 in the expression sequence list. The recombinant vector pFGC5941-IbGER5 has an expression cassette, and the nucleotide sequence of the expression cassette has the CaMV35S promoter, the coding gene fragment of the IbGER5 protein and the OCS polyA terminator. The recombinant microorganism can be obtained by introducing the recombinant vector into the starting microorganism.

The recombinant microorganism can specifically be recombinant Agrobacterium EHA105/pCAMBIA1300-GFP-IbGER5 and/or EHA105/pFGC5941-IbGER5.

The recombinant Agrobacterium EHA105/pCAMBIA1300-GFP-IbGER5 is a recombinant bacterium obtained by introducing the recombinant vector pCAMBIA1300-GFP-IbGER5 into Agrobacterium tumefaciens EHA105.

The recombinant Agrobacterium EHA105/pFGC5941-IbGER5 is a recombinant bacterium obtained by introducing the recombinant vector pFGC5941-IbGER5 into Agrobacterium tumefaciens EHA105.

The present invention also provides a method for cultivating a transgenic plant, the method comprising increasing and/or reducing the content and/or activity of the protein IbGER5 in the target plant to obtain the transgenic plant.

In the above method, the increase of the content and/or activity of the protein IbGER5 in the target plant can be achieved by increasing the expression of the gene encoding the protein IbGER5 in the target plant.

In the above method, the increase of the expression level of the gene encoding the protein IbGER5 in the target plant can be achieved by introducing the gene encoding the protein IbGER5 into the target plant.

In the above method, the reduction of the content and/or activity of the protein IbGER5 in the target plant is achieved by reducing the expression of the gene encoding the protein IbGER5 in the target plant.

In the above method, the reduction of the expression of the gene encoding the protein IbGER5 in the target plant can be the use of gene mutation, gene knockout, gene editing or gene knockout technology to make the gene encoding the protein IbGER5 in the genome of the target plant active decline or inactivation.

Further, the use of gene knockout technology to reduce or inactivate the activity of the gene encoding the protein IbGER5 in the genome of the target plant can be carried out by using an RNA interference vector, and the RNA interference vector contains a nucleotide sequence of SEQID No.2 DNA molecules shown at positions 1-204.

In the above method, the transgenic plant can be a plant with altered stress resistance, further, the plant with altered stress resistance can be reduced (down-regulated) in stress resistance (such as salt tolerance and/or drought resistance) and/or or improved (up-regulated) transgenic plants.

The transgenic plant with reduced stress resistance is a transgenic plant with lower stress resistance than the target plant.

The transgenic plant with improved stress resistance is a transgenic plant (stress-resistant plant) whose stress resistance is higher than that of the target plant.

In the above method, the gene encoding the protein IbGER5 can be any of the following:

F1) coding sequence is the DNA molecule of SEQ ID No.2;

F2) nucleotide sequence is the DNA molecule of SEQ ID No.2;

F3) coding sequence is the DNA molecule shown in the 1st to 204th positions from the 5' end of SEQ ID No.2;

F4) nucleotide sequence is the DNA molecule shown in the 1st to 204th positions from the 5' end of SEQ ID No.2.

Specifically, in one embodiment of the present invention, said improving the expression of the gene encoding the protein IbGER5 in the target plant is achieved by introducing the DNA molecule shown in SEQ ID No.2 into the target plant.

Specifically, in one embodiment of the present invention, the reduction of the expression level of the gene encoding the protein IbGER5 in the target plant is performed by using the DNA at positions 1 to 204 from the 5' end shown in SEQ ID No.2 The molecule and the DNA molecule of the reverse complementary sequence are introduced into the target plant to achieve.

In one embodiment of the invention, the method for cultivating transgenic plants comprises the steps of:

(1) constructing a recombinant vector comprising a DNA molecule shown in SEQ ID NO.2;

(2) introducing the recombinant vector constructed in step (1) into the target plant (such as crops or sweet potatoes);

(3) Obtaining the transgenic plant through screening and identification.

The introduction refers to introduction by recombinant means, including but not limited to transformation mediated by Agrobacterium, biolistic methods, electroporation, in planta technology, and the like.

In one embodiment of the present invention, the method for cultivating transgenic plants (stress-resistant plants, such as drought-resistant plants and/or salt-tolerant plants) comprises the following steps:

(1) Construct the RNA interference vector pFGC5941-IbGER5 that suppresses the expression of the target gene IbGER5 gene;

(2) introducing the RNA interference vector pFGC5941-IbGER5 constructed in step (1) into the target plant (such as crops or sweet potatoes);

(3) Obtaining stress-resistant plants with higher stress resistance than the target plant through screening and identification.

In the above method, the plant can be any of the following:

G1) monocot or dicot;

G2) Convolvulaceae plants;

G3) Plants of the genus Ipomoea batatas;

G4) plants of the sweet potato group;

G5) Sweet potato.

The sweet potato can specifically be a sweet potato variety Lizixiang.

The protein IbGER5, and/or, the biological material is also within the protection scope of the present invention.

Herein, the stress resistance may be salt tolerance and/or drought resistance.

Herein, the plant may be a crop (such as a crop).

The present invention also provides the application of the method for cultivating transgenic plants in creating plants with altered stress resistance, and/or the application in plant breeding or improvement of plant germplasm resources.

The plants with altered stress resistance can be plants with increased or decreased stress resistance, such as drought-resistant plants, salt-tolerant plants, etc., but not limited thereto.

Modulating plant stress resistance as described herein may be up-regulating (increasing) or down-regulating (decreasing) plant stress resistance.

Further, the regulating plant stress resistance may be up-regulating (increasing) or down-regulating (decreasing) the salt tolerance and/or drought resistance of sweet potato.

Herein, the transgenic plant is understood to include not only the first-generation transgenic plant obtained by transforming the target plant with the IbGER5 gene or knocking out the IbGER5 gene, but also its progeny. The gene may be propagated in the species, or transferred into other varieties of the same species, including in particular commercial varieties, using conventional breeding techniques. The transgenic plants include seeds, callus, whole plants and cells.

In the present invention, the IbGER5 gene that regulates plant stress resistance derived from sweet potato (Ipomoea batatas (L.) Lam.) is introduced into the recipient plant sweet potato variety Lizixiang to obtain a transgenic sweet potato plant that overexpresses the IbGER5 gene. The plants were identified for salt tolerance and drought resistance, and the results of various physiological and biochemical indicators showed that compared with the non-transgenic control sweet potato variety Lizixiang (WT), the transgenic sweet potato plants overexpressing the IbGER5 gene were more resistant to salt stress and/or drought stress. Under these conditions, no matter in vitro identification, hydroponic identification or soil cultivation identification results, the stress resistance of sweet potato was significantly reduced, that is, the salt and drought resistance of transgenic sweet potato plants overexpressing the IbGER5 gene was significantly reduced.

The present invention introduces the IbGER5 gene segment derived from sweet potato (Ipomoea batatas (L.) Lam.) to regulate plant stress resistance into the recipient plant sweet potato variety Lizixiang, and obtains a transgenic sweet potato plant that interferes with the expression of the IbGER5 gene. The transgenic plants were identified for salt tolerance and drought resistance, and the results of various physiological and biochemical indicators showed that compared with the non-transgenic control sweet potato variety Lizixiang (WT), the transgenic sweet potato plants that interfered with the expression of the IbGER5 gene were more resistant to salt stress and/or drought stress. Under the conditions, the in vitro identification improves the stress resistance of the sweet potato, that is, the salt tolerance and drought resistance of the transgenic sweet potato plants that interfere with the expression of the IbGER5 gene are improved.

In summary, the IbGER5 protein of the present invention and its coding gene IbGER5 can regulate the stress resistance (such as salt tolerance and/or drought resistance) of plants, by reducing the content and/or activity of the IbGER5 protein in the target plant (such as inhibiting the IbGER5 gene expression) to breed salt- and/or drought-tolerant plants. The IbGER5 protein and its coding gene provided by the invention have important theoretical significance and application value in regulating the salt and drought resistance of sweet potato.

Description of drawings

Figure 1 is the PCR detection electrophoresis of the transgenic plants. Wherein, swimming lane M is the Maker band, and swimming lane W is the band of negative control (water); Swimming lane P is positive control (recombinant plasmid pCAMBIA1300-GFP-IbGER5 (A in Fig. 1) or pFGC5941-IbGER5 (B in Fig. 1) ) band; Swimming lane WT is the band of contrasting sweet potato Chestnut fragrant plant; Swimming lane OE-G1, OE-G2, OE-G4, OE-G34, OE-G53 is the sweet potato quasi-transgenic plant transformed pCAMBIA1300-GFP-IbGER5 Bands; lanes Ri-G6 and Ri-G7 are the bands of pseudo-transgenic sweet potato plants transformed with pFGC5941-IbGER5.

Fig. 2 is a graph showing the identification results of salt tolerance and drought resistance of transgenic plants under in vitro culture.

Fig. 3 is a graph showing the results of identification of salt tolerance and drought resistance of transgenic plants under hydroponic conditions.

Fig. 4 is a graph showing the results of drought resistance identification of transgenic plants under soil cultivation conditions.

Fig. 5 is a diagram showing the measurement results of physiological and biochemical indexes of transgenic plants after salt stress and drought stress treatment.

Detailed ways

The present invention will be further described in detail below in conjunction with specific embodiments, and the given examples are only for clarifying the present invention, not for limiting the scope of the present invention. The examples provided below can be used as a guideline for those skilled in the art to make further improvements, and are not intended to limit the present invention in any way.

The experimental methods in the following examples, unless otherwise specified, are conventional methods, carried out according to the techniques or conditions described in the literature in this field or according to the product instructions. The materials and reagents used in the following examples can be obtained from commercial sources unless otherwise specified.

The sweet potato lines Lizixiang and ND98 in the following examples are described in the following literature: Zhang Huan. Salt tolerance transcriptome analysis of sweet potato and cloning and functional verification of stress resistance related genes IbBBX24 and IbCPK28. Doctoral dissertation of China Agricultural University, 2017. The public can obtain it from the Laboratory of Sweet Potato Genetics and Breeding of China Agricultural University to repeat this experiment.

The pMD19-T vector in the following examples is a product of Treasure Bioengineering (Dalian) Co., Ltd., and the product catalog number is 6013. The pCAMBIA1300 vector is a product of Shanghai Lianmai Bioengineering Co., Ltd., and the product catalog number is LM1375. The pFGC5941 vector is a product of Beijing Huayueyang Biotechnology Co., Ltd., and the product catalog number is VECT0360.

The EcoRI enzyme in the following examples is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD0274. SacI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD1133. KpnI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD0524. SalI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD0644. PstI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., the product catalog number is FD0614. XhoI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD0694. SwaI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD1244. BamHI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD0054. XbaI enzyme is a product of Thermo Fisher Scientific (China) Co., Ltd., and the product catalog number is FD0684. Escherichia coli DH5α is a product of Shenzhen Kangti Life Science and Technology Co., Ltd., the product catalog number is KTSM101L, and Agrobacterium tumefaciens EHA105 is a product of Beijing Qingke Biotechnology Co., Ltd., and the product catalog number is TSC-A03.

The following examples adopt SPSS statistical software to process the data, and the experimental results are represented by mean ± standard deviation, using T test, P < 0.05 (*) represents a significant difference, P < 0.01 (**) represents a very significant difference Differences in gender were analyzed by one-way analysis of variance, and different letters indicate statistically significant differences.

Quantitative experiments in the following examples, unless otherwise specified, were set up to repeat the experiments three times, and the results were averaged.

Embodiment 1, the application of protein IbGER5 in regulating the stress resistance of sweet potato

The inventors of the present application isolated and cloned a sweet potato gene from the sweet potato line ND98, and named it IbGER5. The nucleotide sequence of the coding sequence (CDS) of the gene IbGER5 is shown in SEQ ID NO.2; the amino acid sequence encoded by the gene IbGER5 is the protein IbGER5 of SEQ ID NO.1, and the SEQ ID NO.1 consists of 283 amino acid residues.

1. Construction of plant expression vectors

A, construction of recombinant vector pCAMBIA1300-GFP-IbGER5

According to the coding sequence of the sweet potato IbGER5 protein nucleotide (SEQ ID NO.2), the primer sequences for amplifying the complete coding sequence were designed, and the forward and reverse primers were respectively introduced into KpnI and SalI restriction sites. The primer sequences were as follows:

IbGER5-OE-KpnI:

5'-TACGAATTCGAGCTC GGTACC ATGGAGCCAAAAGGAGAAGAAT-3' (the underlined part is the KpnI restriction site),

IbGER5-OE-SalI:

5'-CTTGCATGCCTGCAG GTCGAC GTTAGCAGTAGCAGGTTGTGACA-3' (the underlined part is the SalI restriction site).

Use the double-stranded DNA molecule shown in SEQ ID NO.2 in the artificially synthesized sequence list as a template, and use IbGER5-OE-KpnI and IbGER5-OE-SalI as primers for PCR amplification, and then connect the product to the pMD19-T vector On the above, the recombinant vector was obtained, named pMD-IbGER5, and the sequencing of M13-F/R (5'-GTAAAACGACGGCCAGT-3'/5'-CAGGAAACAGCTATGAC-3') was carried out to ensure the reading frame and enzyme of sweet potato IbGER5 protein nucleotides The cut site is correct.

The plant expression vector pCAMBIA1300 was double digested with restriction enzymes EcoRI and SacI, and a fragment of about 8948 bp was recovered. Primers (5'-CGAGCTCTCTCTATCTAAACATCTCTCTCTGACC-3' and 5'-CGGAATTCGCTGTGAGAGCAGATGAGGTTC-3') were cloned from the vector pCAMBIA1300 carrying EcoRI and the 770bp CaMV 35S fragment of the SacI restriction site, and after the fragment was recovered, the two fragments were connected to obtain the recombinant plasmid pCAMBIA1300-1.

Recombinant plasmid pCAMBIA1300-1 was digested with restriction endonucleases SalI and PstI, and a fragment of about 9708 bp was recovered. Primers (5'-GCGTCGACATGGTGAGCAAGGGCGAG-3' and 5'-AACTGCAGTTACTTGTACAGCTCGTCCATGC-3') were cloned to carry SalI and PstI enzymes The 720bp GFP fragment at the cutting site was recovered and the two fragments were connected to obtain the recombinant plasmid pCAMBIA1300-GFP.

The pCAMBIA1300-GFP recombinant vector was digested with KpnI and SalI, and the large fragment of the vector was recovered. The recombinant vector pMD-IbGER5 was digested with restriction endonucleases KpnI and SalI, and a small DNA fragment of about 852bp was recovered. The recovered vector was large The fragment was connected with a small DNA fragment to obtain the recombinant vector pCAMBIA1300-GFP-IbGER5, which is the target plasmid. The target plasmid was transformed into Escherichia coli DH5α, cultured at 37°C for 20 hours, PCR analysis and enzyme digestion identification of the recombinant vector pCAMBIA1300-GFP-IbGER5, and sequencing verification. The sequencing results showed that the sequence shown in SEQ ID No.2 in the sequence listing was inserted between the KpnI and SalI restriction sites of the vector pCAMBIA1300-GFP, indicating that the recombinant vector was constructed correctly.

The recombinant vector pCAMBIA1300-GFP-IbGER5 replaces the fragment (small fragment) between the KpnI and SalI recognition sites of the pCAMBIA1300-GFP carrier with a nucleotide sequence, which is the DNA fragment of SEQ ID No.2 in the sequence table, keeping pCAMBIA1300-GFP The other sequences of the vector remain unchanged, and the recombinant expression vector is obtained. The recombinant vector pCAMBIA1300-GFP-IbGER5 expresses the protein IbGER5 shown in SEQ ID No.1 in the sequence listing.

The recombinant vector pCAMBIA1300-GFP-IbGER5 has an expression cassette, the coding gene of CaMV35S promotor, IbGER5 protein (SEQ ID No.2) and the coding gene of green fluorescent protein (GFP) are arranged in the nucleotide sequence of expression cassette.

B. Construction of recombinant vector pFGC5941-IbGER5

With the double-stranded DNA molecule shown in SEQ ID NO.2 in the artificially synthesized sequence listing as a template, with IbGER5-Ri-UF (XhoI): 5'-TTTGGAGAGGACACG CTCGAG ATGGAGCCAAAAGGAGAAG AATCA-3' (the underline is a restriction enzyme Recognition sequence of XhoI) and IbGER5-Ri-UR (SwaI): 5'-AAGAAATTCTTACAC ATTTAAAT AGGTTGAGGGTGGAAATCTTGG-3' (the underline is the recognition sequence of restriction endonuclease SwaI) as primers for PCR amplification, and the recovery of about 200bp small For the fragment, the plant expression vector pFGC5941 was double-digested with restriction endonucleases XhoI and SwaI, and a large vector fragment of about 10 kb was recovered, and the recovered large vector fragment was connected with a small DNA fragment to obtain the recombinant vector pFGC5941-IbGER5-U1.

With the double-stranded DNA molecule shown in SEQ ID NO.2 in the artificially synthesized sequence listing as a template, with IbGER5-Ri-DF (BamHI): 5'-AATTTGCAGGTATTT GGATCC AGGTTGAGGGTGGAAATCTTGG-3' (the underline is the restriction enzyme BamHI recognition sequence) and IbGER5-Ri-DR (XbaI): 5'-GGTCTTAATTAACTC TCTAGA ATGGAGCCAAAAGGAGAAGAATCA-3' (the underline is the recognition sequence of restriction endonuclease XbaI) as primers for PCR amplification, and a small fragment of about 200bp was recovered , use restriction endonucleases BamHI and XbaI to double-digest the plant expression vector pFGC5941-IbGER5-U1, recover a large vector fragment of about 10kb, connect the recovered large vector fragment with a small DNA fragment, and obtain the recombinant vector pFGC5941-IbGER5, namely target plasmid. Sequencing results showed that the DNA molecule shown in the 1st to 204th positions from the 5' end of the sequence shown in SEQ ID No.2 in the sequence table, BamHI and XbaI enzymes were inserted between the XhoI and SwaI restriction sites of the vector pFGC5941 The reverse complementary sequence of the DNA molecule shown in the 1st to 204th positions from the 5' end of the sequence shown in SEQ ID No. 2 in the sequence listing is inserted between the cutting sites.

The recombinant vector pFGC5941-IbGER5 replaces the small fragment between the XhoI and SwaI recognition sequences of the pFGC5941 vector with the DNA molecule fragment shown in the 1st to 204th position of SEQ ID No.2 from the 5' end in the sequence listing, and the restriction internal The fragment (small fragment) between the recognition sites of Dicer BamHI and XbaI is replaced by the nucleotide sequence, which is the reverse complementary sequence of the DNA molecule shown in the 1st to 204th positions from the 5' end of SEQ ID No.2 in the sequence listing , keeping the other sequences of the pFGC5941 vector unchanged to obtain a recombinant expression vector. The recombinant expression vector pFGC5941-IbGER5 interferes with the protein IbGER5 shown in SEQ ID No.1 in the expression sequence list.

The recombinant vector pFGC5941-IbGER5 has an expression cassette, and the nucleotide sequence of the expression cassette has the CaMV35S promoter, the coding gene fragment of the IbGER5 protein and the OCS polyA terminator.

2. Transformation of plant expression vector into Agrobacterium

(1) Thaw the prepared Agrobacterium EHA105 competent cells on ice, add 2 μg of the extracted pCAMBI A1300-GFP-IbGER5 or pFGC5941-IbGER5 plasmid, flick the tube wall to mix, and ice-bath for 10 minutes;

(2) Liquid nitrogen quick freezing for 5 minutes, 37°C water bath for 10 minutes, and ice bath for 5 minutes;

(3) Add 600 μL of liquid LB medium, culture at 28°C, 200 rpm for 5 hours;

(4) Spread 200 μL of bacterial liquid on the LB solid medium containing 100ug/mL kanamycin and 100ug/mL rifampicin;

(5) Invert and dark culture at 28°C for 2 days, take an appropriate amount of Agrobacterium and culture it with liquid LB medium for later use, and obtain the Agrobacterium liquid introduced into the pCAMBIA1300-GFP-IbGER5 or pFGC5941-IbGER5 vector, and name the recombinant Agrobacterium EHA105/ pCAMBIA1300-GFP-IbGER5 or EHA105/pFGC5941-IbGER5.

3. Genetic transformation and regeneration of sweet potato

A. Transformation and regeneration of sweet potato overexpression transgene positive plants

EHA105/pCAMBIA1300-GFP-IbGER5 was introduced into sweet potato variety Lizixiang by Agrobacterium-mediated method. The specific method is as follows:

(1) Stripping the shoot apical meristem of the sweet potato variety Lizixiang, placing it on MS solid medium containing 2.0mg/L 2,4-D, and culturing it at 27±1°C for 8 weeks to obtain embryogenic callus;

(2) Put the embryogenic callus into MS liquid medium containing 2.0mg/L 2,4-D, place it on a shaker and culture it horizontally for 8 weeks, and obtain embryogenic cell clusters with a diameter of 0.7-1.3mm ;

(3) Screen the embryogenic cell mass through a 20-mesh mesh sieve, transfer the larger cell mass to a 30-mesh mesh sieve, grind gently, so that the embryogenic cell mass has a wound, and shake the ground larger embryogenic cell mass Cultivate for 3 days;

(4) EHA105/pCAMBIA1300-GFP-IbGER5 was transformed into embryogenic cell mass by Agrobacterium-mediated method, and then placed in co-culture medium (MS solid culture containing 30mg/L AS, 2.0mg/L 2,4-D base), cultured in the dark at 28°C for 3 days;

(5) The embryogenic cell mass was washed once in MS liquid medium containing 400 mg/L cefotaxime sodium (cefotaxime sodium, CS) and 2.0 mg/L 2,4-D, and then washed in MS liquid medium containing 2.0 mg/L 2, Shaking culture in 4-D MS liquid medium for 1 week;

(6) Place the embryogenic cell mass on the screening medium (MS solid medium containing 100mg/L CS, 5mg/L hygromycin (Hyg), 2,4-D), culture in dark at 28°C for 10-12 week, wherein the culture medium was replaced every two weeks;

(7) Put the embryogenic cell mass on the somatic embryo induction medium (MS solid medium containing 100mg/L CS, 1.0mg/L ABA), and culture in alternating light and dark at 28°C for 2-4 weeks to acquire resistance callus;

(8) Place the resistant callus on MS solid medium, and culture in alternating light and dark at 28°C for 4-8 weeks, and then 5 overexpressed transgenic plants to be identified (i.e. proposed overexpressed transgenic plants) can be obtained, and named respectively OE-G1, OE-G2, OE-G4, OE-G34, OE-G53.

(9) Extract the genomic DNA of the leaves of the overexpressed transgenic plants (OE-G1, OE-G2, OE-G4, OE-G34, OE-G53) with the CTAB method, and use the extracted genomic DNA as a template, water and contrast chestnut Fragrant plant is negative control, and plasmid pCAMBIA1300-GFP-IbGER5 is positive control, carries out PCR amplification with 35S-F and IbGER5-R as primer, obtains PCR amplification product; If contain the band of about 1090bp in the PCR amplification product, The corresponding sweet potato transgenic plants to be identified are sweet potato overexpression transgene positive plants. Primer 35S-F and IbGER5-R sequences are as follows:

35S-F: 5'-TGACGCACAATCCCACTATCCT-3'

IbGER5-R: 5'-GTTAGCAGTAGCAGGTTGTGACA-3'

Electrophoresis detection amplification results are shown in A in Fig. 1 (A in Fig. 1, swimming lane M shows as Maker band, and swimming lane W shows as the band of negative control (water); Swimming lane P shows as positive control (recombinant plasmid pCAMBIA1300-GFP- IbGER5) band; Swimming lane WT shows the band of sweet potato Chestnut fragrant plant; Swimming lane OE-G1, OE-G2, OE-G4, OE-G34, OE-G53 shows that the sweet potato that transforms pCAMBIA1300-GFP-IbGER5 is simulated The bands of expressing transgenic plants can be seen from A in Figure 1, and the target bands of 852bp are all amplified in the swimming lanes OE-G1, OE-G2, OE-G4, OE-G34, OE-G53 and the positive control, indicating that the IbGER5 gene Be integrated in the genome of sweet potato Lizixiang, and prove that these regenerated plants are over-expression transgenic plants.The sweet potato plants that are identified as over-expression transgenes are propagated by vegetative propagation (the plants obtained by the propagation of a transgenic seedling are used as A strain) for the identification of salt tolerance and drought resistance.

B. Transformation and regeneration of sweet potato RNAi positive plants

EHA105/pFGC5941-IbGER5 was introduced into sweet potato variety Lizixiang by Agrobacterium-mediated method.

(1) According to the method of steps (1) to (8) in A, replace EHA105/pCAMBIA1300-GFP-IbGER5 with EHA105/pFGC5941-IbGER5, and replace the screening medium with 100mg/L CS, 0.3mg/L herbicide (PPT), 2,4-D MS solid medium, and other steps were kept unchanged, and two sweet potato interference transgenic plants to be identified (ie pseudo-RNAi plants) were obtained, which were named Ri-G6 and Ri-G7 in turn.

(2) Extract genomic DNA from leaves of pseudo-RNAi plants (Ri-G6, Ri-G7), use the extracted genomic DNA as a template, water and contrast Lizixiang plants as negative controls, plasmid pFGC5941-IbGER5 as positive control, and int-F (5'-CAACCACAAAAGTATCTATGAGCCT-3') and int-R(5'-TTCACATGTCAGAAACATTCTGATG-3') are primers for PCR amplification to obtain PCR amplification products; if the PCR amplification products contain a band of about 888bp, the corresponding The sweet potato transgenic plants to be identified are sweet potato RNAi positive plants.

Electrophoresis detection amplification results are shown in B in Figure 1 (in B in Figure 1, swimming lane M shows as Maker band, and swimming lane W shows as the band of negative control (water); Swimming lane P shows as positive control (recombinant plasmid pFGC5941-IbGER5) The band of Swimming Lane WT shows the band of sweet potato chestnut fragrance plant; Swimming lane Ri-G6, Ri-G7 shows the band of the sweet potato quasi-RNAi plant transformed pFGC5941-IbGER5, as can be seen from B in Fig. 1, swimming lane Ri-G6 , Ri-G7 and the positive control all amplified the 888bp target band, indicating that the pFGC5941-IbGER5 vector had been transferred into the genome of sweet potato Lizixiang, and proved that these regenerated plants were RNAi plants. The sweet potato plants identified as RNAi The method of vegetative propagation is adopted for propagation (the plant obtained from the propagation of a transgenic seedling is regarded as a strain), and the resistance identification of salt tolerance and drought resistance is carried out.

4. Salt tolerance identification of transgenic plants

Tested plants: the sweet potato variety Lizixiang was used as a control plant (WT); the strain numbers were respectively OE-G1, OE-G2, OE-G4, OE-G34, OE-G53, Ri-G6, Ri-G7 plants, 3 plants per line.

The IbGER5 transgenic lines (strain numbers are OE-G1, OE-G2, OE-G4, OE-G34, OE-G53, Ri-G6, Ri-G7) plants and Lizixiang control plants (WT) in normal After 4 weeks of stress culture on the MS medium and the MS medium containing 150mM NaCl, observe the growth status of the plant, and measure the root length of the plant, the results are shown in A, B, D and E in Fig. 2 (A in Fig. 2 For the growth status of sweet potato plants under normal growth conditions, B in Figure 2 is the growth status of sweet potato plants under salt stress, D in Figure 2 is the statistical result of the root length of sweet potato plants under normal growth conditions, and E in Figure 2 is the root length of sweet potato plants under salt stress long statistics). It can be found that after salt stress treatment (150mM NaCl), the root length of the overexpression transgenic plants (OE-G1, OE-G2, OE-G4, OE-G34, OE-G53) was significantly lower than that of the contrast Lizixiang (WT), However, the root length of RNAi plants (Ri-G6, Ri-G7) was higher than that of the control Lizixiang (WT). The results of in vitro identification showed that the salt tolerance of overexpressed transgenic plants was significantly reduced, and the salt tolerance of RNAi plants was improved. IbGER5 gene Or the protein IbGER5 can regulate the salt tolerance of plants.

It was further verified whether the overexpression of IbGER5 gene could improve the salt tolerance of transgenic sweet potato under hydroponic conditions. Get back the stems of the plants of the transgenic lines (strain numbers are OE-G1, OE-G2, OE-G4, OE-G34, OE-G53) overexpressing the IbGER5 gene and the control Lizixiang plant (WT) from the isolated field Each section was cut into 25cm, and a stem node was guaranteed to be placed in normal Hoagland's solution and Hoagland's solution containing 150mM NaCl respectively. The stress treatment was carried out for 4 weeks, and the medium was replaced every 7 days to observe the growth status of the plants. , the root length results of the measurement plant are shown in A, B, D and E in Fig. 3 (in Fig. 3, A, B, the left picture is the growth status of the sweet potato plant in Hoagland's solution, and the right picture is taken out from the solution after washing. sweet potato plant, A in Figure 3 is the growth status of sweet potato plants under normal growth conditions, B in Figure 3 is the growth status of sweet potato plants under salt stress, D in Figure 3 is the statistical result of root length of sweet potato plants under normal growth conditions, in Figure 3 E is the statistical result of root length of sweet potato plants under salt stress). It can be found that after salt stress treatment (150mM NaCl), the root length of overexpressed transgenic plants is significantly shorter than that of contrast Lizixiang (WT). Significantly reduce the salt tolerance of the plant, and the IbGER5 gene or protein IbGER5 has the function of regulating the salt tolerance of the plant.

5. Identification of drought resistance of transgenic plants

The plants of the IbGER5 transgenic lines (strain numbers are OE-G1, OE-G2, OE-G4, OE-G34, OE-G53, Ri-G6, Ri-G7) and the control Lizixiang plant (WT) in the containing After 4 weeks of stress cultivation on the MS medium of the simulated drought stress of 20% PEG6000, observe the growth status of the plant, and measure the root length of the plant, the results are shown in Fig. 2 in C and in Fig. 2 F (in Fig. 2, C is The growth status of sweet potato plants under drought stress, F in Fig. 2 is the statistical result of root length of sweet potato plants under drought stress). It can be found that after drought stress treatment (20% PEG6000), the root length of the overexpression transgenic plants was significantly lower than that of the control Lizixiang (WT), and the growth state was significantly worse than that of the control plants, while the RNAi plants (Ri-G6, Ri-G7 ) root length was higher than that of the control Lizixiang (WT), and the growth state was good. The results of in vitro identification showed that the drought resistance of overexpressed transgenic plants was significantly reduced, and the drought resistance of RNAi plants was improved. Overexpression of IbGER5 gene can significantly reduce the drought resistance of plants , Interfering with the expression of the IbGER5 gene can improve the drought resistance of the plant, and the IbGER5 gene or the protein IbGER5 can regulate the drought resistance of the plant.

It was further verified whether the overexpression of IbGER5 gene could improve the drought resistance of transgenic sweet potato under hydroponic conditions. Get back the stems of the plants of the transgenic lines (strain numbers are OE-G1, OE-G2, OE-G4, OE-G34, OE-G53) overexpressing the IbGER5 gene and the control Lizixiang plant (WT) from the isolated field Each section was cut into 25cm, and one stem node was placed in Hoagland's solution containing 20% PEG6000. After 2 weeks of simulated drought stress treatment, rehydration was carried out for 2 weeks. The medium was replaced every 7 days, and the growth status of the plants was observed. , measure the root length of the plant, the results are shown in C in Fig. 3 and F in Fig. 3 (the left picture in C in Fig. 3 is the growth status of the sweet potato plant under drought stress, and the right picture is the sweet potato plant taken out from the solution after washing , F in Fig. 3 is the statistical result of root length of sweet potato plants under drought stress). It can be found that after the drought stress treatment (20% PEG6000), the root length of the transgenic plants was significantly shorter than that of the control Lizixiang (WT), and the growth state was significantly worse than that of the control plants, showing more serious wilting than the control. It shows that the drought resistance of the transgenic plants is significantly reduced, and the overexpression of the IbGER5 gene can significantly reduce the drought resistance of the plants, and the IbGER5 gene or protein IbGER5 has the function of regulating the drought resistance of the plants.

It was further verified whether the overexpression of IbGER5 gene could improve the drought resistance of transgenic sweet potato under soil cultivation conditions. Get back the stem sections of the plants of the transgenic lines (strain numbers are OE-G1, OE-G2, OE-G4) and the contrast chestnut fragrant plant (WT) overexpressing the IbGER5 gene from the isolated field, and each section is cut into 20cm , ensure that a stem node is inserted into the soil, water for 2 weeks until the stem section grows normally, and carry out drought treatment (that is, do not water for 8 consecutive weeks), observe the growth state of the plant after 8 weeks, measure the root length and root number of the plant, the results are shown in Fig. 4 (A in Fig. 4 is the growth status of sweet potato plants in dry ponds, B in Fig. 4 is the sweet potato plants after taking out from the soil, and C in Fig. 4 is the sweet potato plant phenotype index of statistics). It can be found that after the drought stress treatment, the root length of the transgenic plants was significantly shorter than that of the control Lizixiang (WT), the number of roots was less, and the growth state was obviously worse than that of the control plants, showing more serious wilting than the control plants. The results of the dry pond showed that The drought resistance of the transgenic plant is significantly reduced, and the overexpression of the IbGER5 gene can significantly reduce the drought resistance of the plant, and the IbGER5 gene or protein IbGER5 has the function of regulating the drought resistance of the plant.

6. Determination of physiological and biochemical indicators

Tested plants: sweet potato variety Lizixiang as control plant (WT); plants with line numbers OE-G1, OE-G2, OE-G4, Ri-G6, Ri-G7, 3 plants for each line.

(1) Determination of H ₂ O ₂ content

H ₂ O ₂ is the most common reactive oxygen molecule in plants, which can directly or indirectly oxidize biological macromolecules such as nucleic acid and protein in cells, causing damage to cell membranes, thereby accelerating cell aging and disintegration. Therefore, the higher the H ₂ O ₂ content, the greater the degree of stress damage to plants.

References (Zhang H, Gao X, Zhi Y, et al. A non-tandem CCCH-type zinc-fingerprotein, IbC3H18, functions as a nuclear transcriptional activator and enhancesabiotic stress tolerance in sweet potato[J]. New Phytologist, 2019, 223:1918-1936) DAB and NBT staining method, DAB, NBT staining were carried out to sweet potato leaf respectively. The hydrogen peroxide content (H ₂ O ₂ ) kit of Suzhou Keming Biotechnology Co., Ltd. was used to detect the H ₂ O ₂ content in sweet potato plants. The dyed sweet potato leaves are the materials that have been treated for 10 days in vitro for salt tolerance and drought resistance, and the plants for content determination are the plants that have been treated for 4 weeks in vitro for identification of salt tolerance and drought resistance. The experiment was repeated 3 times, and the results were averaged.

The results are shown in A, B, C, D and E in Figure 5 (A in Figure 5 is the DAB staining result, and B in Figure 5 is the relative intensity of the DAB staining spots using the imageJ software statistics, compared with the normal MS medium in the control chestnut incense The spot intensity of the plant is taken as 100%. C in Figure 5 is the NBT staining result, and D in Figure 5 is the relative intensity of the NBT staining spots counted by imageJ software, taking the spot intensity of the control Lizixiang plant in the normal MS medium as 100% , E in Fig. 5 is the statistical result of H ₂ O ₂ content). It can be found that after the salt stress treatment of 150mM NaCl and the simulated drought stress treatment of 20% PEG6000, the _{H2O2 content} of the transgenic plants (strain numbers OE-G1, OE-G2, OE-G4) overexpressing the IbGER5 gene significantly higher than that of the control Lizixiang plants, while the H ₂ O ₂ content of the RNAi plants (strain numbers: Ri-G6 and Ri-G7) was significantly lower than that of the control Lizixiang plants.

(2) Determination of malondialdehyde (MDA) content

When plant organs age or suffer damage under adversity, oxygen free radicals often act on unsaturated fatty acids in lipids to generate lipid peroxides, and MDA is the final decomposition product of lipid peroxides, and its content can reflect the damage of plants to adversity Degree. Therefore, the higher the MDA content, the greater the degree to which the plant was subjected to stress.

The malondialdehyde (MDA) content kit of Suzhou Keming Biotechnology Co., Ltd. was used to detect the content of MDA in sweet potato plants. The sweet potato plants were plants after 4 weeks of in vitro identification treatment for salt tolerance and drought resistance. The experiment was repeated 3 times, and the results were averaged.

The results are shown in F in Figure 5. It can be found that after the salt stress treatment of 150mM NaCl and the simulated drought stress treatment of 20% PEG6000, the MDA content of the transgenic plants (strain number is OE-G1, OE-G2, OE-G4) overexpressing the IbGER5 gene was significantly higher than The MDA content of the RNAi plants (strain numbers Ri-G6 and Ri-G7) was significantly lower than that of the control chestnut fragrance plants.

(3) Determination of proline (Pro) content

Proline widely exists in plants. Under stress conditions, the proline content in plants increases significantly, and the increase reflects stress resistance to a certain extent. Therefore, proline can be used as a biochemical indicator of plant stress resistance.

The Proline (PRO) Content Determination Kit of Suzhou Keming Biotechnology Co., Ltd. was used to detect the proline content in sweet potato plants. The sweet potato plants were plants after 4 weeks of in vitro identification treatment for salt tolerance and drought resistance. The experiment was repeated 3 times, and the results were averaged.

The results are shown in G in Figure 5. It can be found that after the salt stress treatment of 150mM NaCl and the simulated drought stress treatment of 20% PEG6000, the proline content of the transgenic plants (strain numbers being OE-G1, OE-G2, OE-G4) overexpressing the IbGER5 gene was significantly The content of proline in RNAi plants (strain numbers Ri-G6 and Ri-G7) was significantly higher than that of the control Lizixiang plants.

(4) Determination of peroxidase (Peroxidase, POD) activity

POD (EC 1.11.1.7) widely exists in plants and can be used as a biochemical indicator of plant stress resistance. The lower the activity of POD, the greater the degree of stress damage to plants.

The POD content in sweet potato plants was detected using the peroxidase (Peroxidase, POD) kit of Suzhou Keming Biotechnology Co., Ltd. The sweet potato plants were plants after 4 weeks of in vitro identification treatment for salt tolerance and drought resistance. The experiment was repeated 3 times, and the results were averaged.

The results are shown in Figure 5 H. It can be found that after 150mM NaCl salt stress treatment and 20% PEG6000 simulated drought stress treatment, the POD activity of the transgenic plants (strain numbers being OE-G1, OE-G2, OE-G4) overexpressing the IbGER5 gene was significantly lower than that of the control Chestnut fragrance plants, and the POD activity of RNAi plants (strain numbers Ri-G6, Ri-G7) was significantly higher than that of the control chestnut fragrance plants.

In conclusion, under the salt stress treatment of 150mM NaCl and the simulated drought stress treatment of 20% PEG6000, the overexpression of IbGER5 gene significantly increased H ₂ O ₂ and MDA content, decreased proline (Pro) content and POD activity, and resisted The activity of oxidases was reduced, thereby increasing the oxidative damage caused by salt stress and drought stress to transgenic plants, and greatly reducing the tolerance of overexpressed transgenic plants to adversity stress, while interfering with IbGER5 gene expression significantly reduced H ₂ O ₂ and MDA content, increased proline (Pro) content and POD activity, and the activity of antioxidant enzymes increased, thereby reducing the oxidative damage caused by salt stress and drought stress to transgenic plants, and greatly improving the resistance of RNAi plants to adversity stress tolerance.

The above results indicated that the overexpression of IbGER5 gene in sweet potato would significantly reduce the salt and drought tolerance of sweet potato plants, and the interference expression of IbGER5 gene would significantly improve the salt and drought tolerance of sweet potato plants. The IbGER5 protein of the present invention and its coding gene IbGER5 can regulate the stress resistance (such as salt tolerance and/or drought resistance) of plants, by increasing the content and/or activity of the IbGER5 protein in the target plant (such as overexpressing the IbGER5 gene) can Remarkably reduce the stress resistance of the target plant, and can significantly improve the stress resistance of the target plant by reducing the content and/or activity of the IbGER5 protein in the target plant (such as interfering with the expression of the IbGER5 gene).

Embodiment 2, the acquisition of the protein IbGER5 and its coding gene regulating the stress resistance of sweet potato

Experimental material: The sweet potato line ND98 was used as the experimental material.

1. Sweet potato total RNA extraction: Take 1 g of young leaves of sweet potato line ND98 and grind them into powder in liquid nitrogen, add them to a 2 mL centrifuge tube, and use TransZol method to extract sweet potato total RNA, using PrimeScript ^TM RT reagent Kit with gDNA Eraser reagent The first-strand cDNA was reverse-transcribed using a cassette (product of Kangwei Century Biotechnology (Beijing) Co., Ltd.).

2. From the published sweet potato salt-tolerant transcriptome, obtain the EST sequence shown in SEQ ID No.3 in the sequence list, and compare it with the Sweetpotato Garden library to obtain the EST sequence shown in SEQ ID No.4 in the sequence list homologous sequences. According to the nucleotide sequence (SEQ ID No.4) obtained by homology alignment, design and artificially synthesize primers IbGER5-F and IbGER5-R, the sequences are:

IbGER5-F: 5'-ATGGAGCCAAAAGGAGAAGAAT-3'

IbGER5-R: 5'-GTTAGCAGTAGCAGGTTGTGACA-3'

3. Using the cDNA obtained in step 1 as a template and the IbGER5-F and IbGER5-R synthesized in step 2 as primers, perform PCR amplification to obtain a PCR amplified fragment product of about 852 bp and sequence it.

The results show that the nucleotide sequence of the PCR amplification product obtained in step 3 is as shown in SEQ ID No.2 in the sequence table, the gene shown in the sequence is named IbGER5 gene, and the protein encoded by it is named IbGER5 protein or protein The amino acid sequence of IbGER5 is shown in SEQ ID No.1 in the sequence listing.

The present invention has been described in detail above. For those skilled in the art, without departing from the spirit and scope of the present invention, and without unnecessary experiments, the present invention can be practiced in a wider range under equivalent parameters, concentrations and conditions. While specific embodiments of the invention have been shown, it should be understood that the invention can be further modified. In a word, according to the principles of the present invention, this application intends to include any changes, uses or improvements to the present invention, including changes made by using conventional techniques known in the art and departing from the disclosed scope of this application. Applications of some of the essential features are possible within the scope of the appended claims below.

SEQUENCE LISTING

<110> China Agricultural University

<120> Protein IbGER5 regulating plant stress resistance and its coding gene and application

<160> 4

<170> PatentIn version 3.5

<210> 1

<211> 283

<212> PRT

<213> Sweet potato (Ipomoea batatas (L.) Lam.)

<400> 1

Met Glu Pro Lys Gly Glu Glu Ser Glu Pro His Lys Pro Leu Ser Ser

1 5 10 15

Ser Ser Ser Glu Ser Gln Ala Pro Ala Glu Met Asp Pro Gln Lys Trp

20 25 30

Gly Thr His Val Met Gly Arg Pro Ala Val Pro Thr Thr His Pro Asp

35 40 45

Asn Gln Lys Ala Ala Leu Trp Arg Ser Glu Asp Gln His Gln Asp Phe

50 55 60

His Pro Gln Pro Tyr Val Val Tyr Ser Pro Val Asp Arg Pro Pro Ser

65 70 75 80

Asn Asn Pro Phe Glu Ser Val Cys His Met Phe Asn Ser Trp Ser His

85 90 95

Arg Ala Glu Thr Val Ala Arg Asn Val Trp His Asn Leu Lys Thr Ala

100 105 110

Pro Ser Val Ser Glu Ala Ala Trp Gly Lys Leu Asn Met Thr Ala Lys

115 120 125

Ala Ile Thr Glu Gly Gly Phe Glu Ala Phe Tyr Lys Gln Ile Phe Ala

130 135 140

Thr Asp Pro Tyr Glu Lys Leu Lys Lys Thr Tyr Ala Cys Tyr Leu Ser

145 150 155 160

Thr Thr Thr Gly Pro Val Ala Gly Thr Leu Tyr Leu Ser Thr Thr Lys

165 170 175

Val Ala Phe Cys Ser Asp Arg Pro Leu Thr Phe Thr Ala Pro Ser Gly

180 185 190

Gln Glu Ala Trp Ser Tyr Tyr Lys Ile Ala Val Pro Leu Ala Asn Ile

195 200 205

Ala Ala Val Asn Pro Val Val Met Arg Glu Asn Pro Gln Glu Lys Tyr

210 215 220

Ile Gln Leu Val Thr Val Asp Gly His Asp Phe Trp Phe Met Gly Phe

225 230 235 240

Val Asn Phe Glu Lys Ala Thr His Asn Leu Leu Asp Gly Leu Ser Ser

245 250 255

Phe Arg Ala Tyr Gly Asn Asn Asn Ala Ala Gly Gln Ser Val Ser Gly

260 265 270

His Ala Asn Val Ser Gln Pro Ala Thr Ala Asn

275 280

<210> 2

<211> 852

<212> DNA

<213> Sweet potato (Ipomoea batatas (L.) Lam.)

<400> 2

atggagccaa aaggagaaga atcagagccc cataaaccct tgtcatcatc ttcttctgaa 60

tcccaagcac ctgcagaaat ggaccctcag aaatggggaa ctcatgtgat gggtcgtcct 120

gcagtcccca ccaccacccc ggataaccag aaggcggcct tgtggaggtc tgaagatcag 180

caccaagatt tccaccctca acctacgtc gtctattctc ccgtggatcg gccccccagc 240

aacaacccct ttgaatcagt gtgccacatg ttcaattctt ggagccatag agctgagact 300

gtagctcgta acgtatggca caacttgaaa actgcgccat ctgtatcaga agctgcttgg 360

ggaaagctga acatgactgc caaggcaata acagaaggtg gatttgaggc attttacaag 420

caaatctttg caaccgatcc ttatgagaag ctaaagaaga cgtatgcttg ttacctttca 480

acaacaaccg gccctgttgc cgggaccctc tatttgtcaa ccaccaaggt tgctttctgc 540

agtgatcgcc ctttgacctt cacagctcct tctggccagg aggcttggag ctactacaag 600

attgcagtac ctttggcaaa tatagcagca gtgaatccgg tggtaatgag agagaatcca 660

caagagaagt aattcagtt agtgacagtt gatggtcatg acttctggtt catggggttt 720

gtgaattttg agaaagcaac ccataatctc ctagacggct tgtcaagttt tagagcatat 780

ggaaataaca atgcagcagg gcagtctgtt agtggacatg cgaatgtgtc acaacctgct 840

actgctaact ag 852

<210> 3

<211> 1736

<212> DNA

<213> Sweet potato (Ipomoea batatas (L.) Lam.)

<400> 3

taattaataa ttaaaattga tatatgtatc ttggataaat acgaagtgga tggccggaaa 60

acccaacacct ggaaacttcg ctatcatcta ttcaacggct ccacttgttg acacataaat 120

taagaaacaa ttactagcaa taataaatta gtaatataac gacgaacctt gttacttcta 180

cgcgtaatct cacgcttgtt ctagaacgat gctaaggttt ctattctgat cttaccagat 240

ttcttttccg gcccaattaa ccttcatctc aaagaaacca tcttcaatta atcctataaa 300

ttgcagtgtt ccgatcccca tctctatcta tctcaccact tctttaccaa agtatattct 360

tgccttcatt ccaattcaga aagatattcc cataagtccc caaataatgg agccaaaagg 420

agaagaatca gagccccata aacccttgtc atcatcttct tctgaatccc aagcacctgc 480

agaaatggac cctcagaaat ggggaactca tgtgatgggt cgtcctgcag tccccaccac 540

ccaccccagat aaccagaagg ctgccttgtg gaggtctgag gatcattacc aagagttcca 600

ccctcaacct tacgtcgtct attctcccgt ggatcggccc cccagcaaca acccctttga 660

atcagtgtgc cacatgttca attcttggag ccatagagct gagactgtag ctcgtaacgt 720

atggcacaac ttgaaaactg cgccatctgt atcagaagct gcttggggaa agctgaacat 780

gactgccaag gcaataacag aaggtggatt tgaggcatat tacaagcaaa tctttgcaac 840

cgatccttat gagaagctaa agaagacgta tgcttgttac ctttcaacaa caaccggccc 900

tgttgccggg accctctatt tgtcaaccac caaggttgct ttctgcagtg atcgcccttt 960

gaccttcaca gctccttctg gccaggaggc ttggagctac tacaagattg cagtaccttt 1020

ggcaaatata gcagcagtga atccggtggt aatgagagag aatccacaag agaagtacat 1080

tcagttagtg acagttgatg gtcatgactt ctggttcatg gggtttgtga attttgagaa 1140

agcaacccat aatctcctag acggcttgtc aataaatttt agagcatatg gaaataataa 1200

tgcagcaggg cagtctgtga gtggacatgg gaatgtgtca caacttgcta gtgctaacta 1260

gcaataaagt agtatttcac cattcaactc ctcttacttg agttgtcttt ctgccatata 1320

cacagttat gtattacttt gtgtgcttaa ttgcttggag tgtatccatg tatattgtta 1380

gttgttgttg ttggatgcat tatagatttt tcccttctgg gtatcgagga tgaggttgta 1440

ggcaatctgt gtagagaatt ttcaatataa actaaggggg tttcgtacat attctttagc 1500

ttaataatta cattgtttaa ttcgaagaaa ttacaattcc acggaattgt aacttcttct 1560

ttttgttgta ttgaaattca tactttcata tgaattctaa acaatgaagc atggaaaaga 1620

aaaagagaag agaaagattt gtttatcaat gaagcatgga aaagaaaaag agaagagaaa 1680

gatttgttta tcaatgaagc atggaaaaga aaaagagaag agaaagattt gtttat 1736

<210> 4

<211> 918

<212> DNA

<213> Sweet potato (Ipomoea batatas (L.) Lam.)

<220>

<221> misc_feature

<222> (555)..(555)

<223> n is a, c, g, or t

<400> 4

atggagccaa aaggagaaga atcagagccc cataaaccct tgtcagaaat ggaccctcag 60

aaatggggaa ctcatgtgat gggtcgtcct gcagtcccca ccaccccaccc ggataaccag 120

aaggctgcct tgtggaggtc tgaggatcag caccaagatt tccaccctca accttacgtc 180

gtctattctc ccgtggatcg gccccccagc aacaacccct ttgaatcagt gtgccacatg 240

ttcaattctt ggagccatag agctgagacc gtagctcgta acgtatggca caactcacaa 300

gagtcaatat tgactccact aaggctcgaa accacaccc ctcttgaagc acaagagtcg 360

gtattgtctt cactgaggct ccacctcccg tataaaggaa aggtgaaaac tgcgccatcc 420

gtatcagaag ctgcttgggg aaagctgaac atgactgcca aggcaataac agaaggtgga 480

tttgaggcat attacaagca aatctttgca accgatccta atgagaagct aaagaagacc 540

tccttctggc caggnggccc tgttgccggg accctctatt tgtcaaccac caaggttgct 600

ttctgcagtg atcgcccttt gaccttcaca gctccttctg gccaggaggc ttggagctac 660

tacaagattg cagtaccttt ggcaaatata gcagcagtga atccggtggt aatgagagag 720

aatccacaag agaagtacat tcagatagtg acagttgatg gtcatgactt ctggttcatg 780

gggtttgtga attttgataa agcaacccat aatctcctag acggcttgtc aaattttaga 840

gcatatggaa ataataatgc agcagggcat tctgtgagtg gacatgcgaa tgtgtcacaa 900

cctgctactg ctaactag 918

Claims (5)

1. Use of a protein, characterized in that the use is any of the following:

d1 Application in improving salt tolerance or drought resistance of plants;

D2 Use in the cultivation of salt-tolerant or drought-tolerant plants;

d3 Application in plant salt tolerance or drought resistance breeding;

the protein is any one of the following:

a1 A protein having an amino acid sequence of SEQ ID No. 1;

a2 A fusion protein with the same function obtained by connecting a label to the N end and/or the C end of A1);

the plant is a dicotyledon;

wherein the use described in D1) to D3) is achieved by reducing the expression of the protein.

2. The use according to claim 1, wherein the protein is derived from sweet potato.

3. A method of growing a transgenic plant, said method comprising reducing the amount of a protein according to claim 1 or 2 in a plant of interest to obtain said transgenic plant; said reducing the amount of the protein of claim 1 or 2 in a plant of interest is achieved by reducing the expression level of a gene encoding the protein in the plant of interest; the plant is a dicotyledonous plant.

4. The protein according to claim 1 or 2, wherein the amino acid sequence of the protein is the sequence shown in SEQ ID No. 1.

5. A nucleic acid molecule encoding a protein according to claim 4, wherein the sequence of said nucleic acid is SEQ ID No.2.

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