CN116024226B

CN116024226B - A cassava MeGRXC3 gene for improving resistance and its application

Info

Publication number: CN116024226B
Application number: CN202211009155.2A
Authority: CN
Inventors: 阮孟斌; 于晓玲; 赵平娟; 邹良平; 彭明
Original assignee: Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Current assignee: Institute of Tropical Bioscience and Biotechnology Chinese Academy of Tropical Agricultural Sciences
Priority date: 2022-08-22
Filing date: 2022-08-22
Publication date: 2024-12-06
Anticipated expiration: 2042-08-22
Also published as: CN116024226A

Abstract

The present invention provides a cassava gene, which is a cassava MeGRXC3 gene, and the nucleotide sequence is shown in SEQ ID NO: 1. The present invention also provides a silencing fragment screened from the cassava MeGRXC3 gene and an application thereof. The specific silencing fragment of the cassava MeGRXC3 gene of the present invention is used to construct a silencing system, which can effectively silence the target gene MeGRXC3, and can effectively improve the resistance of transgenic cassava to low temperature stress, drought stress, osmotic stress, etc., and provides a research idea and technical basis for improving the resistance of other plants to low temperature stress, drought stress, and osmotic stress, and has good application prospects.

Description

Cassava MeGRXC gene for improving resistance and application thereof

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a cassava MeGRXC gene for improving resistance and application thereof.

Background

Cassava (Manihot esculenta Crantz, cassava) is a perennial plant of the genus cassava of the family Euphorbiaceae, and is an important raw material for the current production of starch and bioenergy together with sweet potato and potato, and is also a main grain for about 10 hundred million people worldwide. In China, cassava is used for processing modified starch or other food raw materials, so that the requirements of mass living diversity of people in China can be met, and the grain consumption in food processing production can be reduced. Therefore, the stability and the increase of the cassava yield are important components of national grain safety guarantee. Cassava can be planted in barren mountainous areas and its aerial parts can be used for the production of feed.

The cassava is planted in 2-4 months in a growth period of 10 months, is in dry seasons in tropical and subtropical areas in a seedling stage (60 days after germination) and a root tuber forming stage (60-90 days after germination), and is easily damaged by low temperature and drought stress. The low temperature and drought stress seriously affect the plant morphology, photosynthetic efficiency and root starch accumulation of cassava, thereby causing the reduction of the yield and quality of cassava. Therefore, the stress resistance of the cassava in the seedling stage is an important precondition for guaranteeing the yield and the quality of the cassava. The important genes are utilized to excavate and cultivate new varieties of the stress-resistant high-yield cassava, which is helpful for the development of the cassava industry in China and the income of peasants.

Based on the result of gene expression analysis, the applicant verifies the stress resistance function of MeGRXC gene in transgenic cassava, and accordingly, the application of the patent is proposed.

Disclosure of Invention

The invention aims to overcome the defects in the prior art and provides a cassava MeGRXC gene for improving resistance and application thereof.

The first aspect of the invention provides a cassava gene which is a cassava MeGRXC gene and has a nucleotide sequence shown as SEQ ID NO. 1.

In a second aspect, the invention provides a protein encoded by the cassava gene according to the first aspect of the invention, and the amino acid sequence of the protein is shown as SEQ ID NO. 2.

In a third aspect, the invention provides a silencing fragment which is a specific fragment of the cassava MeGRXC gene sequence, comprising 269bp from the start codon ATG (as shown in SEQ ID NO: 3).

In a fourth aspect, the invention provides a recombinant vector or host bacterium comprising a cassava gene as described in the first aspect, or a silencing fragment as described in the third aspect of the invention.

As the original vector for constructing the recombinant vector, vectors commonly used in the field of gene recombination, such as viruses, plasmids, and the like, can be used. The invention is not limited in this regard. In one embodiment of the invention, the original vector is a pLB background rapid cloning vector, a p18T-RNAi intermediate vector, or a p35S-RNAi plant expression vector, although it is understood that other plasmids, viruses, etc. may be used in the present invention.

In a fifth aspect, the invention provides a recombinant expression vector or host bacterium comprising a sense sequence (i.e., the sequence of SEQ ID NO:3, a fragment from 9bp to 277bp in the sequence of SEQ ID NO: 4) and an antisense sequence of the fragment from 9bp to 277bp in the sequence of SEQ ID NO:5, comprising the silencing fragment of the third aspect of the invention.

The original vector for constructing the recombinant expression vector may be a vector commonly used in the field of gene recombination, such as a virus, a plasmid, etc. The invention is not limited in this regard. In one embodiment of the invention, the original vector is a pLB background rapid cloning vector, a p18T-RNAi intermediate vector, or a p35S-RNAi plant expression vector, although it is understood that other plasmids, viruses, etc. may be used in the present invention.

The p18TRNAi intermediate vector is modified based on pMD18-T, the hairpin RNA structure required by RNAi is inserted into the multiple cloning site of the pMD18-T vector through two cleavage sites of KpnI and BamHI, and the vector is mainly used for subcloning the RNAi structure, and is reported in Ruan et al, journal of Experimental Botany,2017,68:3657-3672.

The p35S-RNAi plant expression vector (abbreviated as "p35S-RNAi vector") is modified based on a plant expression vector pCAMBIA1301, a hairpin RNA structure required by RNAi is inserted into a multiple cloning site of the pCAMBIA1301 vector through two enzyme cutting sites of KpnI and BamHI, and the vector is mainly used for stably expressing hairpin RNA in plants so as to inhibit the expression of a target gene through RNAi effect in transgenic plants, and is reported in Ruan et al, journal of Experimental Botany,2017,68:3657-3672.

Preferably, the recombinant expression vector is a p35S-RNAi plant expression vector comprising the sense and antisense sequences of the silencing fragment of the third aspect of the present invention.

In a sixth aspect, the invention provides the use of a cassava gene as described in the first aspect of the invention, or a silencing fragment as described in the third aspect of the invention, or a recombinant vector or host bacterium as described in the fourth aspect of the invention, or a recombinant expression vector or host bacterium as described in the fifth aspect of the invention, for silencing MeGRXC genes.

In a seventh aspect, the invention provides the use of a cassava gene as described in the first aspect, or a silencing fragment as described in the third aspect, or a recombinant vector or host bacterium as described in the fourth aspect, or a recombinant expression vector or host bacterium as described in the fifth aspect, for increasing the low temperature stress resistance, and/or drought stress resistance, and/or osmotic stress resistance of a plant.

Preferably, osmotic stress is mannitol.

The specific silencing segment of the cassava MeGRXC gene is adopted to construct a silencing system, the target gene MeGRXC3 can be effectively silenced, and the low-temperature stress resistance, drought stress resistance, permeation stress resistance and the like of plants can be effectively improved, for example, after the sense sequence and the antisense sequence of the silencing segment are converted into cassava, the expression of the MeGRXC gene is inhibited, the transgenic cassava grows well under low-temperature stress, the ion permeability is obviously reduced, the low-temperature stress resistance is obviously improved, only a few leaves are wilted under drought stress, basically no leaves die and fall off, the chlorophyll content is reduced and the drought stress resistance is obviously improved, and under mannitol stress, the number of main roots and biomass are obviously higher than that of wild type, and the permeation stress resistance is obviously improved. The invention provides a research thought and a technical basis for improving the low temperature stress resistance, drought stress resistance and permeation stress resistance of other plants, and has good application prospect.

Drawings

FIG. 1 is a plasmid map of a p18T-RNAi intermediate vector. The figure shows schematic diagrams of endonuclease sites for constructing RNAi structures and general sequencing primers that can be used.

FIG. 2 is a plasmid map of a p35S-RNAi plant expression vector. The figure shows the location of the RNAi hairpin structure and the corresponding endonuclease site.

FIG. 3 shows the results of RT-PCR analysis of the molecular characterization of transgenic cassava Southernblot (upper panel) and of the gene expression of MeGRXC3 (lower panel). Different numbers and letters above the upper graph and the lower graph represent different transgenic lines, black bands in the upper graph represent copy numbers of transgene insertion, the upper part of the lower graph represents the expression level of MeGRXC in the leaf stalk of the transgenic cassava, and the lower part represents the expression level of the internal reference gene Actin 1.

FIG. 4 is a photograph of transgenic cassava after low temperature treatment.

FIG. 5 shows the results of leaf ion permeability analysis after low temperature treatment of transgenic cassava.

FIG. 6 is a photograph of transgenic cassava after drought treatment.

FIG. 7 shows the variation of leaf chlorophyll relative content after drought treatment of transgenic cassava.

FIG. 8 is the results after stress treatment of transgenic cassava mannitol.

Detailed Description

The invention will be further described with reference to specific embodiments in order to provide a better understanding of the invention. The specific techniques or conditions are not identified in the examples and are performed according to techniques or conditions described in the literature in this field or according to the product specifications. The reagents or apparatus used were conventional products commercially available without the manufacturer's attention.

1. MeGRXC3 Gene acquisition

As a tropical crop, cassava is relatively sensitive to low temperatures, and air temperatures below 14 ℃ have significant damage to the cassava plants. By transcriptome and gene expression analysis, we found that tapioca MeGRXC gene expression was significantly responsive to low temperature treatment. Since the gene has no intron, we extracted genomic DNA of the leaves of cassava cultivar cv.60444, and used genomic DNA of cassava cultivar cv.60444 as a template, the following primers were used:

The PCR amplification reaction was carried out in a manner that the template DNA0.1ng, 1. Mu.L of each of the gene-specific primers P1 and P2, 25. Mu.L of a2 XTaq PCR reaction premix system, and 50. Mu.L of the reaction system was supplemented with ddH ₂ O. The PCR amplification procedure was 94℃for 5min, 94℃for 30s,55℃for 30s,72℃for 40s, 30 cycles total, and 72℃for 7min. And (3) carrying out electrophoresis detection and gel cutting recovery on the PCR product, connecting the recovered product to a pLB zero background rapid cloning vector (MeGRXC 3-pLB plasmid) according to a pLB zero background rapid cloning kit, carrying out sequencing by Shanghai engineering after positive cloning is detected by transformation, and the sequence of the MeGRXC3 gene is shown as SEQ ID No. 1 (without enzyme cutting site sequence).

MeGRXC3 is a gene of a subfamily of glutaredoxin CC, the gene has no intron, the length of the coding sequence is 315bp, and the coding sequence codes 104 amino acids of the protein. The conserved domain of the protein comprises a CC active center, a P-G glutathione binding domain and L-LL and ALWL domains related to protein C-terminal interaction. Such proteins are proteins specific to terrestrial plants.

2. Construction of recombinant vectors

1) The MeGRXC gene sequence is selected to comprise a 269bp specific fragment (shown as SEQ ID NO: 3) from the start codon ATG as a silencing fragment, and a specific primer is designed as follows:

2) The MeGRXC-pLB plasmid in the 'obtaining of the first and MeGRXC genes' is used as a template, a primer MeGRXC-Sense-KpnI with a cleavage site KpnI added at the 5 'end and a primer MeGRXC-Sense-ClaI with a cleavage site ClaI added at the 5' end are used, a Sense fragment (shown as SEQ ID No. 4) containing a Sense sequence of a silent fragment is obtained through amplification by a PCR method, the PCR reaction system is that 0.1ng of the template MeGRXC3-pLB plasmid, each 1 mu L of Sense fragment specific primer, 25 mu L of 2 xTaq PCR reaction premix system is adopted, and a ddH ₂ O-supplemented 50 mu L reaction system is added. The PCR amplification procedure was 94℃for 5min, 94℃for 30s,55℃for 30s,72℃for 40s, 30 cycles total, and 72℃for 7min.

The MeGRXC-pLB plasmid in the 'obtaining of the first and MeGRXC3 genes' is used as a template, a primer MeGRXC3-Anti-BamHI with an enzyme cutting site BamHI at the 5 'end and a primer MeGRXC-Anti-XhoI with an enzyme cutting site XhoI at the 5' end are used, an antisense fragment containing a silent fragment antisense sequence (shown as SEQ ID No. 5) is obtained through amplification by a PCR method, the PCR reaction system is that 0.1ng of the template MeGRXC-pLB plasmid, each 1 mu L of the antisense fragment specific primer, 25 mu L of the 2 xTaq PCR reaction premix system and 50 mu L of the ddH ₂ O supplement are added. The PCR amplification procedure was 94℃for 5min, 94℃for 30s,55℃for 30s,72℃for 40s, 30 cycles total, and 72℃for 7min.

3) The sense fragment and the antisense fragment were inserted into an intermediate vector p18TRNAi (the vector was modified based on pMD18-T, the hairpin RNA structure required for RNAi was inserted into the multiple cloning site of the pMD18-T vector through two cleavage sites of KpnI and BamHI, the vector was mainly used for subcloning the RNAi structure, see the report Ruan et al, journal ofExperimental Botany,2017,68:3657-3672, before we were presented) by cleavage, recovery and ligation, respectively, and then the MeGRXC-RNAi subcloning vector was formed in the vector p 18-T. Subcloning vector is transferred into colibacillus, and positive cloning is determined by PCR and then sequencing is carried out.

4) Double-restriction of a subclone vector plasmid containing MeGRXC-RNAi structure determined by sequencing with KpnI and BamHI, recovering a fragment containing MeGRXC-RNAi structure (a fragment containing a sense fragment and an antisense fragment), simultaneously, linearizing a p35S-RNAi vector (the vector is modified based on a plant expression vector pCAMBIA1301, the hairpin RNA structure required by RNAi is inserted into a multiple cloning site of the pCAMBIA1301 vector through two restriction sites of KpnI and BamHI, the vector is mainly used for stably expressing hairpin RNA in plants so as to inhibit the expression of a target gene by RNAi effect in transgenic plants, see Ruan et al, journal ofExperimental Botany,2017,68:3657-3672, reported earlier, and recovering the vector (FIG. 2);

5) The MeGRXC-RNAi fragment was ligated to a p35S-RNAi vector (p 35S-MeGRXC3-RNAi vector plasmid) to transform E.coli. Selecting monoclonal, extracting plasmid, enzyme cutting and sequencing and verifying.

3. P35S-MeGRXC-RNAi vector plasmid transformed cassava

The correct p 35S-MeGRXC-RNAi vector plasmid was verified to transform Agrobacterium LBA4404, and positive clones were identified by PCR for cassava genetic transformation after screening. Cassava genetic transformation is described primarily with reference to the method of Zainuddin et al (Zainuddin et al 2012), and is performed as follows:

(1) The identified agrobacterium single colony is selected and cultured in 5mL LB liquid medium containing three antibodies (Kan 50mg/L, rif25 mg/L and Str 50 mg/L) at 28 ℃ and 220r/min overnight;

(2) Taking 1mL of bacterial liquid cultured overnight in 50mL of LB liquid medium containing the three antibodies, wherein the temperature is 28 ℃, and the culture value OD ₆₀₀ per minute is 0.6-0.8;

(3) Centrifuging at 4deg.C and 6000r/min for 10min to collect thallus, re-suspending thallus in 50mLMS liquid culture medium (pH5.3), centrifuging at 4deg.C and 6000r/min for 10min to collect thallus;

(4) Adding a proper amount of MS+AS (final concentration of 200 mM) liquid culture medium to resuspend the thalli, and standing for 4-8 hours or overnight at room temperature in a dark place;

(5) Thoroughly scattering the cassava friable callus suspension cells (FEC) to be transformed in an MS liquid culture medium, and removing floating suspension cells and MS;

(6) Adding the MS bacterial liquid heavy suspension after standing into suspension cells, and carrying out shaking culture for 45min at the temperature of 30 ℃;

(7) After infection, transferring the suspension cells onto a sterile nylon filter membrane, carefully transferring the filter membrane onto sterile filter paper by using forceps, and airing for 1min;

(8) After air drying, the filter membrane is transferred to a solid culture medium of SH (pH5.6) +100mMAS, and co-culture is carried out for 72 hours at 21 ℃ in a dark place (the co-culture can be stopped at any time to prevent the pollution of agrobacterium by observing the growth condition of agrobacterium on the culture medium during the process);

(9) After the co-culture is finished, suspending cells from a filter membrane are hung in a culture bottle, 40mL SH (pH5.8) +5mg/mL hygromycin+500mg/mL Carb liquid culture medium is added, and shaking and washing are carried out twice;

(10) 50mL SH (pH 5.8) +5mg/mL hygromycin+500 mg/mL Carb liquid medium is added, and shaking culture is carried out for 24 hours under the illumination condition of 30 ℃;

(11) 50mL SH (pH 5.8) +5mg/mL hygromycin+500 mg/mL Carb liquid medium is added, and shaking culture is carried out for 3d under the illumination condition of 30 ℃ to replace the medium once;

(12) After shaking culture for 2-3 weeks, transferring the suspension cells to a sterile nylon membrane filter membrane, transferring to MSN+5mg/mL hygromycin+500mg/mL Carb solid medium, and culturing at 26 ℃ in light/dark (16 h/8 h);

(13) Selecting and screening resistant somatic embryos appearing on MSN culture medium, transferring the resistant somatic embryos to CMM+5mg/mL hygromycin+500 mg/mL Carb solid culture medium, and culturing the resistant somatic embryos in light/dark (16 h/8 h) at a temperature of 26 ℃ until buds grow;

(14) The sprouts grown on CMM medium were transferred to CBM+10mg/mL hygromycin+500 mg/mL Carb solid medium and incubated at 26℃in light/dark (16 h/8 h) until rooting.

4. Identification of transgenic cassava molecule and MeGRXC gene expression analysis

1) Identification of transgenic cassava molecules

The transgenic cassava molecular identification mainly identifies transgenic plants through Southern blot, and the specific operation is as follows:

(1) The PCR is used to identify the selected cassava resistant seedlings, the primers used are MeGRXC-sense-KpnI and MeGRXC-sense-ClaI, the PCR reaction system is that the cassava resistant seedlings genome DNA0.1ng, the specific primers P1 and P2 are respectively 1 mu L, the 2 xTaq PCR reaction premix system is 12.5 mu L, and the ddH ₂ O is added to supplement 25 mu L reaction system. The PCR amplification procedure was 94℃for 5min, 94℃for 30s,55℃for 30s,72℃for 40s, 30 cycles total, and 72℃for 7min.

(2) Extracting DNA of cassava resistant seedlings which are identified as positive by PCR, and adjusting the concentration to 1 mug/mu L for later use;

(3) Southern blot experiments were performed according to the kit from Roche (Roche) and the full length of the coding region of the hygromycin resistance gene hptII on the p35S-RNAi vector was used as a probe;

(4) Digestion of 70. Mu.g of positive cassava resistant seedling DNA with EcoRI, precipitation and recovery of the digested product, constant volume of 50. Mu.L, and separation of digested DNA fragments by agarose gel electrophoresis with 0.8% (w/v);

(5) Southern blot experimental procedures were performed, and after transfer, cross-linking, hybridization, washing and development, images of Southern blot results were taken using ImageQuant LAS4000mini (GE Healthcare Life Science). As shown in the upper graph of FIG. 3, the numbers and letter combinations on lanes correspond to different transgenic lines, the black bar on each lane indicates the copy number of the target gene (hptII) inserted into the transgenic cassava genome, transgenic line #1A copy number is 2, transgenic line #1 copy number is 1, transgenic line #17 copy number is 1, transgenic line #2 copy number is 1, transgenic line #24 copy number is 1, transgenic line #26 copy number is 2, and transgenic line #9 copy number is 1.

2) MeGRXC3 Gene expression analysis

Extracting total RNA of a transgenic cassava petiole obtained by Southern blot screening, carrying out reverse transcription to obtain a first strand of cDNA, and determining the inhibition effect of target gene expression in the transgenic cassava by RT-PCR, wherein the used primers are as follows:

MeGRXC3-RT-pF	ACGCAGTGACAAGAATGGTT
		MeGRXC3-RT-pR	CAGTGTCTTAATGGAGTGGC

The PCR reaction system is 1 mu L of cassava resistant seedling cDNA, 1 mu L of each of the specific primers P1 and P2, 12.5 mu L of a2 xTaq PCR reaction premix system, and a reaction system of adding ddH ₂ O and supplementing 25 mu L. The PCR amplification procedure was 94℃for 5min, 94℃for 30s,55℃for 30s,72℃for 40s, 30 cycles total, and 72℃for 7min.

As shown in the lower graph of FIG. 3, the upper part of the graph shows the expression level of MeGRXC in the leaf stalk of the transgenic cassava, the band representing the relative expression level of MeGRXC gene is obvious in Wild Type (WT) cassava, but the condition representing the relative expression level of MeGRXC gene is not visible or obvious in 4 different transgenic cassava strains, which shows that the expression of the gene is obviously inhibited in the transgenic cassava, and the lower part of the graph shows the expression level of the internal gene, namely, actin1, which is used for controlling experimental errors among different samples.

5. Low temperature stress test

We selected 2 MeGRXC3-RNAi transgenic cassava lines (# 1, # 17) for the low temperature treatment experiments to determine the function of this gene in cassava in relation to low temperature stress. We treated transgenic cassava for 60 days at low temperature of 7 ℃ for low temperature stress treatment and recovered growth under normal temperature conditions after 24 hours of treatment.

After 40 days of transplanting, wild Type (WT) and MeGRXC-RNAi transgenic cassava pot seedlings with good growth vigor are treated in an illumination incubator with the temperature of 7 ℃ for 24 hours and then taken out, more than 80% of wild type cassava leaves are found to be wilted, and only about 20% -40% of transgenic cassava leaves are wilted (figure 4), which shows that compared with the wild type cassava, the tolerance of MeGRXC-RNAi transgenic cassava to low temperature is obviously improved.

The Ion permeability (Ion leakage) of mature leaves was measured, and the Ion permeability of transgenic cassava was found to be significantly lower than that of wild type (fig. 5), indicating that MeGRXC-RNAi transgenic cassava leaf cell membranes were not significantly damaged after low temperature treatment compared with wild type cassava, indicating that the tolerance of transgenic cassava to low temperature stress was significantly improved.

After the potted cassava seedlings subjected to low-temperature treatment are grown for 21 days under normal temperature conditions, the mortality rate is counted, and the low-temperature mortality rate of wild cassava is found to be 100%, while the low-temperature mortality rate of MeGRXC-RNAi transgenic cassava is found to be 0.

6. Drought stress experiments

Drought stress experiments were performed with cassava pot seedlings 90 days after germination of the stem sections. After water is supplied at the same time, the relative water content of the soil is measured, potted seedlings with the relative water content of the soil being about 30% are selected, drought treatment is carried out in a mode of stopping water supply, the day of water supply is 0day (0 day) of drought treatment, the growth condition of the potted cassava seedlings is observed after 10 days (10 day) of drought treatment, the result is that all leaves of wild cassava (WT) plants are wilted and 30% -40% of the leaves die and fall off, and only few leaves of transgenic cassava have wilting and basically no leaves die and fall off. The experimental result shows that the drought resistance of the transgenic cassava is obviously improved.

The chlorophyll content meter is used for measuring the chlorophyll relative content index of the cassava leaves in the drought treatment process, the result is shown in figure 7, the chlorophyll relative content index of the wild cassava leaves in the drought treatment process is reduced from the 3 rd day of the drought treatment, the reduction trend is obviously enhanced along with the drought treatment time, and the chlorophyll relative content index of the transgenic cassava leaves is also reduced from the 3 rd day of the drought treatment, but the reduction trend is relatively mild. The experimental result also shows that the drought resistance of MeGRXC-RNAi transgenic cassava is obviously improved. And when drought treatment is carried out for 20 days, normal water supply of each treated plant is recovered, the death rate of the plants is counted after 7 days, the death rate of wild cassava is found to be higher than 90%, and the death rate of transgenic cassava is found to be below 10%.

7. Osmotic stress (mannitol) assay

The tolerance of transgenic cassava to osmotic stress was analyzed on media containing mannitol (D-mannitol) at different concentrations. The Wild Type (WT) and MeGRXC-RNAi transgenic cassava terminal buds with the same length are respectively inoculated on CBM culture medium containing 0mM,200mM,300mM D-mannitol, and cultured for 50 days under the same conditions (26 ℃ and 12 hours of light/12 hours of darkness), and the growth condition of cassava group cultured seedlings is observed and recorded.

As a result, as shown in FIG. 8, the major roots and biomass of transgenic cassava were not significantly different from those of wild-type cassava under 200mM mannitol treatment, whereas all the buds of wild-type cassava died under 300mM mannitol treatment, while the buds of transgenic cassava continued to survive and the major roots and biomass were significantly higher than that of wild-type cassava. The above results demonstrate that MeGRXC-RNAi transgenic cassava has significantly improved tolerance to osmotic stress (mannitol).

The above description of the specific embodiments of the present invention has been given by way of example only, and the present invention is not limited to the above described specific embodiments. Any equivalent modifications and substitutions for this practical use will also occur to those skilled in the art, and are within the scope of the present invention. Accordingly, equivalent changes and modifications are intended to be included within the scope of the present invention without departing from the spirit and scope thereof.

Claims

1. A silent fragment, characterized in that it is a cassava MeGRXC3 gene sequence from the start codon ATG

The initial fragment includes a 269 bp specific fragment, and the nucleotide sequence is shown in SEQ ID NO.3.

2. A recombinant vector or host bacteria containing the silencing fragment according to claim 1.

3. A recombinant vector or host bacteria containing the sense sequence and antisense sequence of the silencing fragment of claim 1, wherein the antisense sequence is a fragment from 9 bp to 277 bp in the sequence shown in SEQ ID NO: 5.

4. The recombinant vector or host bacterium according to claim 2, characterized in that the recombinant vector is a p35S-RNAi plant vector containing the sense sequence and antisense sequence of the silencing fragment according to claim 1.

5. Use of the silencing fragment according to claim 1, or the recombinant vector or host bacteria according to claim 2, or the recombinant vector or host bacteria according to claim 3 or 4 in silencing the MeGRXC3 gene, wherein the nucleotide sequence of the MeGRXC3 gene is shown in SEQ ID NO.1.

6. Use of the silencing fragment according to claim 1, or the recombinant vector or host bacteria according to claim 2, or the recombinant vector or host bacteria according to claim 3 or 4 in improving the resistance of plants to low temperature stress, wherein the plant is cassava cultivar cv. 60444.

7. Use of the silencing fragment according to claim 1, or the recombinant vector or host bacteria according to claim 2, or the recombinant vector or host bacteria according to claim 3 or 4 for improving the resistance of plants to low temperature stress and osmotic stress, wherein the plant is cassava cultivar cv. 60444.

8. Use of the silencing fragment according to claim 1, or the recombinant vector or host bacteria according to claim 2, or the recombinant vector or host bacteria according to claim 3 or 4 in improving the resistance of plants to low temperature stress and drought stress, wherein the plant is cassava cultivar cv. 60444.

9. Use of the silent fragment according to claim 1, or the recombinant vector or host bacteria according to claim 2, or the recombinant vector or host bacteria according to claim 3 or 4 in improving the ability of plants to resist low temperature stress, and simultaneously improving the ability of plants to resist drought stress and osmotic stress, wherein the plant is cassava cultivar cv.60444.

10. The use according to claim 7 or 9, characterized in that mannitol is used as the osmotic stress.