CN103667278B - The nucleotide sequence of mediating plant male fertility and use its method - Google Patents

Abstract

The present invention relates to a nucleotide sequence that mediates male fertility in plants and a method for using it. The nucleotide sequence encodes the following RNA molecule, which inhibits plant MS26 gene expression, including SEQ? ID? At least 19 consecutive nucleotides shown in NO:1 and/or its complementary sequence. The present invention uses RNAi technology to construct the MS26 male sterile line for the first time, the MS26 male sterile line inhibits the expression of the MS26 gene in corn, and can obtain completely male sterile plants, which can not only shorten the cycle of hybrid breeding, but also be more targeted It solves the current situation of lack of male sterile line resources and low purity of hybrid seed production, and saves the step of castration in the process of hybrid seed production, reduces the mechanical damage and yield impact of mechanical castration on corn plants, and ensures The purity and yield of hybrid germplasm, so as to achieve the greatest economic benefit.

Description

Nucleotide sequences mediating male fertility in plants and methods of using same

technical field

The present invention relates to a nucleotide sequence that mediates the male fertility of plants and a method for using it, in particular to a genetic method for producing male sterile plants by using RNAi technology to specifically delete or close the expression of fertility genes.

Background technique

Heterosis refers to the performance of F1 hybrids that are superior to those of the F1-producing parents. Heterosis in crop plants is often manifested in production as larger plants, stress tolerance, disease resistance, uniformity and increased yield, collectively referred to as hybrid vigor.

Heterosis has been observed and documented in many important crop species, and the development of hybrids has become routine by plant breeders. However, hybrids can only be used in such plants when an effective and economical method of pollination control exists in the plant to ensure cross-pollination and prevent self-pollination. Mechanisms of pollination control include mechanical, chemical and genetic methods.

Mechanical methods of hybrid plant production can be applied if the plants have been spatially separated from male and female flowers or if male and female plants have been separated. For example, maize plants have male pollen-producing flowers in the apical inflorescence and female flowers along the leaf rachis along the stem. Outcrossing in maize is ensured by mechanical emasculation of the female parent to prevent selfing. Although detasseling is currently used in hybrid seed production of plants such as maize, the above process is labor intensive and expensive in terms of actual detasseling costs and yield losses of the female parent. Also, most crop plants have functional male and female organs in the same flower, so detasseling is not a simple process. Although pollen-forming organs can be removed by hand prior to pollination, the method of hybrid production described above is extremely labor-intensive and expensive.

Chemical methods of producing hybrid plants involve the use of chemicals to kill or prevent the formation of fertile pollen. These chemicals are known as gameticides and are used to impart temporary male sterility. Commercial production of hybrid plants using gametocides is limited due to the cost and availability of the chemicals, as well as the duration and reliability of application. A serious limitation of gametocides is their phytotoxic effects, the severity of which depends on the genotype. Other limitations include that these chemicals may not be effective on crops in extended flowering, as new flowers growing may not be affected. Therefore, it is necessary to repeatedly apply the above-mentioned chemicals.

In the 1990s, Mariani et al. created a new way to artificially prepare male sterile lines. They used the specific promoter (TA29) and ribonuclease gene (Barnase) from tobacco anther tapetum to construct an expression cassette to transform tobacco and rapeseed, resulting in the destruction of the anther tapetum of the transgenic plants and the formation of male sterile lines, but did not affect other traits of the transgenic plants. In addition to using Barnase to construct male sterile lines, many current studies have shown that other functional genes can also be widely used in genetic engineering methods to construct transgenic male sterile plants.

Van der Meer et al. used RNA interference to inhibit the expression of chalcone synthase (CHS), a gene related to pollen development, and achieved male sterility in petunias; Kitashiba et al. combined antisense alternative oxidase (alternative oxidase) with TA29 Connected to form a chimeric gene to transform tobacco, resulting in abortion of part of the pollen of the transgenic tobacco. Since the MS26 gene was cloned, the gene was mainly used to restore the fertility of the MS26 mutants, and so far there has been no report on the construction of MS26 male sterile lines using RNAi technology.

Contents of the invention

The purpose of the present invention is to provide a nucleotide sequence that mediates male fertility in plants and a method for using it, that is, using RNAi technology to obtain a new MS26 male sterile line.

To achieve the above object, the present invention provides a nucleotide sequence, characterized in that it encodes the following RNA molecule, which inhibits plant MS26 gene expression, including at least 19 consecutive sequences shown in SEQ ID NO:1 Nucleotides and/or their complements.

Further, the nucleotide sequence includes at least 19 consecutive nucleotides shown in positions 500-1000 of SEQ ID NO: 1 and/or its complementary sequence.

Further, the nucleotide sequence includes SEQ ID NO: 2 and/or its complementary sequence.

Preferably, the nucleotide sequence is SEQ ID NO:3.

To achieve the above object, the present invention also provides an expression cassette, comprising the nucleotide sequence under the control of an operably linked regulatory sequence.

To achieve the above purpose, the present invention also provides a recombinant vector comprising the nucleotide sequence or the expression cassette.

To achieve the above object, the present invention also provides a ribonucleotide sequence, which inhibits the expression of plant MS26 gene, and is transcribed from the following DNA sequence, the DNA sequence including the nucleotide sequence.

To achieve the above object, the present invention also provides a method for inhibiting the expression of plant MS26 gene, comprising using the ribonucleotide sequence to inhibit the expression of plant MS26 gene.

To achieve the above object, the present invention also provides a method for inducing male sterility in plants, comprising using the ribonucleotide sequence to inhibit the expression of plant MS26 gene.

Further, the ribonucleotide sequence includes a first ribonucleotide sequence and a second ribonucleotide sequence, the first ribonucleotide sequence inhibits the expression of the plant MS26 gene, and the second ribonucleotide sequence The acid sequence is complementary to the first ribonucleotide sequence.

Furthermore, the ribonucleotide sequence also includes a spacer sequence.

Preferably, the ribonucleotide sequence is SEQ ID NO:3.

To achieve the above object, the present invention also provides a method for producing male sterile plants, comprising introducing the expression cassette into plant cells to produce transgenic plants.

Preferably, the plant is soybean, wheat, barley, corn, tobacco, rice, rapeseed or sunflower.

The genome of a plant, plant tissue or plant cell in the present invention refers to any genetic material in a plant, plant tissue or plant cell, and includes nucleus, plastid and mitochondrial genome.

A preferred method for post-transcriptional gene suppression in plants uses sense- and antisense-oriented, stably transcribed RNA, eg, as hairpin and stem-loop structures. A preferred DNA construct for post-transcriptional gene suppression is one in which the first segment encodes an RNA exhibiting an antisense orientation that exhibits a high degree of identity to the target gene segment to be suppressed, The first segment is connected to the second segment, and the second segment encodes an RNA exhibiting high complementarity with the first segment. Such constructs are expected to form a stem-loop structure (which is formed by the hybridization of the first segment to the second segment) and a loop structure from connecting the two.

"Nucleic acid" in the present invention refers to a single or double-stranded polymer of deoxyribonucleic acid or ribonucleic acid bases read from the 5' to the 3' end. Alternatively, a "nucleic acid" may also contain non-naturally occurring or altered bases that allow correct reading by polymerases without reducing expression of the polypeptide encoded by the nucleic acid. "Nucleotide sequence"refers to the sense and antisense strands of a nucleic acid existing as separate single strands or in duplexes. "Ribose nucleic acid" (RNA) includes RNAi (RNA interference), dsRNA (double-stranded RNA), siRNA (small interfering RNA), mRNA (messenger RNA), miRNA (microRNA), tRNA (transfer RNA, acylated amino acids plus charges), as well as cDNA and genomic DNA and DNA-RNA hybrids. "Nucleic acid fragment", "nucleic acid sequence fragment" or more commonly "fragment" will be understood by those skilled in the art to include genomic sequences, ribosomal RNA sequences, transfer RNA sequences, messenger RNA sequences, operator sequences and smaller An engineered nucleotide sequence that expresses or can be engineered to express a protein, polypeptide or peptide.

"Expression" in the present invention refers to the transcription and stable accumulation of sense or antisense RNA obtained from the nucleic acid disclosed in the present invention. Expression can also refer to the translation of mRNA into a polypeptide or protein. "Sense" RNA in the present invention refers to RNA transcripts corresponding to sequences or fragments that exist in the form of mRNA that can be translated into protein by plant cells. In the present invention, "antisense" RNA refers to RNA that is complementary to all or a part of mRNA normally produced in plants. Complementation of antisense RNA can be directed to any part of a particular gene transcript, i.e. 5' non-coding sequence, 3' non-coding sequence, intron or coding sequence. "RNA transcript" in the present invention refers to the product obtained by RNA polymerase-catalyzed transcription of a DNA sequence. When an RNA transcript is a perfectly complementary copy of a DNA sequence, it is called a primary transcript, or it may be the RNA obtained by post-transcriptional processing of a primary transcript, which is called a mature RNA.

DNA fragments useful in the present invention are at least about 19 to about 23 nucleotides in length, or about 23 to about 100 nucleotides in length, but less than about 2000 nucleotides in length.

"Inhibition of gene expression" in the present invention refers to the absence (or observable decrease) of the protein and/or mRNA product level of the target gene. Specificity refers to the ability to inhibit a gene of interest without affecting other genes in the cell and without affecting any gene in the cell that produces the dsRNA molecule.

The dsRNA or siRNA molecules of the present invention comprise double-stranded polymerized ribonucleotides, which may include modifications to the phosphate sugar backbone or nucleosides. Modifications in the RNA structure can be tailed to allow specific genetic inhibition.

In the present invention, dsRNA molecules can be modified by enzymatic methods, and siRNA can be produced. siRNA can effectively mediate down-regulation of target genes. This enzymatic approach can be accomplished by utilizing the RNAse III enzyme or the DICER enzyme present in plant cells in the eukaryotic RNAi pathway. The method can also be carried out using recombinant DICER or RNAse III introduced into plant cells by recombinant DNA techniques, as is readily known to those skilled in the art. DICER enzyme or RNAse III, naturally present in plant cells or produced by recombinant DNA technology, cleaves larger dsRNA strands into smaller oligonucleotides. DICER enzyme can specifically cut dsRNA molecules into siRNA fragments, wherein each siRNA fragment is about 19-25 nucleotides long, while RNAseⅢ enzyme usually cuts dsRNA molecules into siRNAs of 12-15 base pairs. siRNA molecules produced by any of the above enzymes have a 3' overhang of 2-3 nucleotides and a 5' phosphate and 3' hydroxyl terminus. siRNAs produced by RNAse III enzymes are identical to those produced by DICER in the eukaryotic RNAi pathway, and thus can be targeted and then loosened and then degraded by the intrinsic intracellular RNA degradation machinery, separated into single-stranded RNA, and transcribed with the target gene RNA-seq hybridization. This method can effectively degrade or remove the RNA sequence encoded by the nucleotide sequence of the target gene. The result is silencing of the target gene.

dsRNA molecules can be synthesized in vivo or in vitro. A dsRNA can be formed from a single self-complementary RNA strand, or it can be formed from two complementary RNA strands. The cell's endogenous RNA polymerase can mediate transcription in vivo, or cloned RNA polymers can be used for transcription in vivo or in vitro. Inhibition can be targeted by: specific transcription in an organ, tissue, or cell type; stimulation of environmental conditions (e.g., infection, stress, temperature, chemical inducers); and/or engineering at developmental stages sexual transcription. The RNA strand may or may not be polyadenylated; the RNA strand may or may not be translated into a polypeptide by the cell's translation machinery.

The RNA, dsRNA, siRNA or miRNA of the present invention is produced by a person skilled in the art chemically or enzymatically, by manual or automatic reactions or in another organism. RNA can also be produced by partial or complete organic synthesis; any modified ribonucleotides can be introduced by in vitro enzymatic synthesis or organic synthesis. RNA can be synthesized by intracellular RNA polymerase or bacteriophage RNA polymerase (eg T3, T7, SP6). The use and manufacture of expression constructs is known in the art. If synthesized chemically or enzymatically in vitro, the RNA may be purified prior to introduction into the cell. RNA can be purified from the mixture, for example, by extraction with solvents or resins, precipitation, electrophoresis, chromatography, or combinations thereof. Alternatively, RNA can be used with no or minimal purification to avoid losses due to sample processing. RNA can be dried for storage, or dissolved in an aqueous solution. The solution may contain buffers and salts to facilitate stabilization and/or annealing of the dimer chains.

The regulatory sequences in the present invention include but not limited to promoters, transit peptides, terminators, enhancers, leader sequences, introns and other regulatory sequences operably linked to the target gene.

The promoter is a promoter that can be expressed in plants, and the "promoter that can be expressed in plants" refers to a promoter that ensures the expression of the coding sequence linked to it in plant cells. A promoter expressible in plants may be a constitutive promoter. Examples of promoters directing constitutive expression in plants include, but are not limited to, 35S promoter derived from cauliflower mosaic virus, maize ubi promoter, rice GOS2 gene promoter and the like. Alternatively, the promoter expressible in plants may be a tissue-specific promoter, i.e., the promoter directs expression of the coding sequence at higher levels in some tissues of the plant, such as in green tissues, than in other tissues of the plant (which can be determined by routine RNA assays), such as the PEP carboxylase promoter. Alternatively, the promoter expressible in plants may be a wound-inducible promoter. A wound-inducible promoter or a promoter directing a wound-induced expression pattern means that when a plant is subjected to mechanical or insect-induced wounds, the expression of the coding sequence under the regulation of the promoter is significantly increased compared with normal growth conditions. Examples of wound-inducible promoters include, but are not limited to, the promoters of the potato and tomato protease inhibitors (pinI and pinII) and the maize proteinase inhibitor (MPI).

The transit peptide (also called secretory signal sequence or targeting sequence) directs the transgene product to a specific organelle or cellular compartment. The transit peptide can be heterologous to the receptor protein, for example, using a gene encoding a chloroplast transport The peptide sequences target the chloroplast, or the endoplasmic reticulum using the 'KDEL' retention sequence, or the vacuole using the CTPP of the barley lectin gene.

The leader sequence includes, but is not limited to, a picornavirus leader sequence, such as an EMCV leader sequence (5' non-coding region of encephalomyocarditis virus); a potyvirus leader sequence, such as a MDMV (maize dwarf mosaic virus) leader sequence; Human immunoglobulin heavy chain binding protein (BiP); untranslated leader of coat protein mRNA of alfalfa mosaic virus (AMV RNA4); tobacco mosaic virus (TMV) leader.

The enhancers include, but are not limited to, cauliflower mosaic virus (CaMV) enhancers, Scrophulariaceae mosaic virus (FMV) enhancers, carnation weathering ring virus (CERV) enhancers, cassava vein mosaic virus (CsVMV) enhancers , Mirabilis Mosaic Virus (MMV) Enhancer, Night Scent Yellow Leaf Curl Virus (CmYLCV) Enhancer, Multan Cotton Leaf Curl Virus (CLCuMV), Commelina Yellow Mottle Virus (CoYMV) and Peanut Chlorotic Streak Flower Leaf virus (PCLSV) enhancer.

For monocot applications, the introns include, but are not limited to, the maize hsp70 intron, the maize ubiquitin intron, the Adh intron 1, the sucrose synthase intron, or the rice Act1 intron. For dicot applications, such introns include, but are not limited to, the CAT-1 intron, the pKANNIBAL intron, the PIV2 intron, and the "super ubiquitin" intron.

The terminator may be a suitable polyadenylation signal sequence that functions in plants, including but not limited to, the polyadenylation signal sequence derived from the nopaline synthase (NOS) gene of Agrobacterium tumefaciens , the polyadenylation signal sequence from the protease inhibitor Ⅱ (pinⅡ) gene, the polyadenylation signal sequence from the pea ssRUBISCO E9 gene and the polyadenylation signal sequence from the α-tubulin gene Polyadenylation signal sequence.

The nucleotide sequences of the invention may comprise inverted repeats separated by "spacers". A spacer sequence may be a region comprising any nucleotide sequence that, if desired, facilitates secondary structure formation between each repeat. A spacer sequence is part of the sense or antisense coding sequence for an mRNA. Alternatively, the spacer sequence may comprise any combination of nucleotides or homologues thereof that can be covalently linked to the nucleic acid molecule. The spacer sequence may comprise a nucleotide sequence of at least about 10-100 nucleotides in length, or at least about 100-200 nucleotides in length, at least about 200-400 nucleotides in length, or at least about 200-400 nucleotides in length, or at least About 400-500 nucleotides.

The "operably linked" in the present invention refers to the linkage of nucleic acid sequences, which allows one sequence to provide the required function for the linked sequence. The "operably linked" in the present invention can be linking a promoter with a sequence of interest, so that the transcription of the sequence of interest is controlled and regulated by the promoter. "Operably linked" when a sequence of interest encodes a protein and expression of that protein is desired means that a promoter is linked to said sequence in such a way that the resulting transcript is efficiently translated. If the junction of the promoter and coding sequence is a transcript fusion and expression of the encoded protein is desired, the junction is made such that the first translation initiation codon in the resulting transcript is that of the coding sequence. Alternatively, if the junction of the promoter and coding sequence is a translational fusion and expression of the encoded protein is to be achieved, the junction is made such that the first translation initiation codon contained in the 5' untranslated sequence is fused with the promoter linked in such a way that the resulting translation product is in-frame with the translational open reading frame encoding the desired protein. Nucleic acid sequences that may be "operably linked" include, but are not limited to: sequences that provide gene expression function (i.e., gene expression elements such as promoters, 5' untranslated regions, introns, protein coding regions, 3' untranslated regions, polynucleotides adenylation sites and/or transcription terminators), sequences that provide DNA transfer and/or integration functions (i.e. T-DNA border sequences, site-specific recombinase recognition sites, integrase recognition sites), provide selection Sequences for sexual function (i.e., antibiotic resistance markers, biosynthetic genes), sequences that provide scoreable marker function, sequences that facilitate sequence manipulation in vitro or in vivo (i.e., polylinker sequences, site-specific recombination sequences), and sequences that provide Sequences that are functional for replication (i.e., bacterial origins of replication, autonomously replicating sequences, centromere sequences).

Nucleic acid molecules or fragments thereof are capable of specifically hybridizing to other nucleic acid molecules under certain circumstances. In the present invention, if two nucleic acid molecules can form an antiparallel double-stranded nucleic acid structure, it can be said that the two nucleic acid molecules can specifically hybridize to each other. A nucleic acid molecule is said to be the "complement" of another nucleic acid molecule if two nucleic acid molecules exhibit perfect complementarity. In the present invention, two nucleic acid molecules are said to exhibit "complete complementarity" when every nucleotide of one nucleic acid molecule is complementary to the corresponding nucleotide of the other nucleic acid molecule. Two nucleic acid molecules are said to be "minimally complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under at least conventional "low stringency" conditions. Similarly, two nucleic acid molecules are said to be "complementary" if they are capable of hybridizing to each other with sufficient stability such that they anneal and bind to each other under conventional "high stringency" conditions. Deviations from perfect complementarity are permissible as long as the deviation does not completely prevent the two molecules from forming a double-stranded structure. In order for a nucleic acid molecule to serve as a primer or probe, it only needs to be sufficiently complementary in sequence to form a stable double-stranded structure under the particular solvent and salt concentration employed.

In the present invention, a substantially homologous sequence is a nucleic acid molecule that can specifically hybridize to a complementary strand of another matched nucleic acid molecule under highly stringent conditions. Appropriate stringent conditions to promote DNA hybridization, for example, treatment with 6.0X sodium chloride/sodium citrate (SSC) at approximately 45°C, followed by washing with 2.0X SSC at 50°C, are known to those skilled in the art. is well known. For example, the salt concentration in the washing step can be selected from about 2.0×SSC, 50°C for low stringency conditions to about 0.2×SSC, 50°C for high stringency conditions. In addition, the temperature conditions in the washing step can be increased from about 22°C at room temperature for low stringency conditions to about 65°C for high stringency conditions. Both the temperature condition and the salt concentration can be changed, or one can be kept constant while the other variable is changed.

To introduce the construct or the recombinant vector in the present invention into plants, conventional transformation methods include, but are not limited to, Agrobacterium-mediated transformation, microprojection bombardment, direct DNA intake into protoplasts, electroporation or whisker silicon-mediated transformation. Guided DNA introduction.

When "introducing" a nucleotide sequence into a plant as described in the present invention, it means that it can occur by direct transformation methods, such as Agrobacterium-mediated transformation of plant tissues, particle emission bombardment, electroporation, etc.; Alternatively, it can be done by crossing a plant with a heterologous nucleotide sequence to another plant so that the offspring have the nucleotide sequence incorporated into their genome. Such breeding techniques are well known to those skilled in the art.

The present invention provides a nucleotide sequence that mediates male fertility in plants and a method for using it, which has the following advantages:

1. The present invention utilizes RNAi technology to construct MS26 male sterile line for the first time.

2. Complete abortion. The MS26 male sterile line constructed by the RNAi technology in the invention inhibits the expression of the maize MS26 gene, and can obtain completely male sterile plants.

3. Good benefit and high purity. The MS26 male sterile line constructed by the RNAi technology of the present invention can not only shorten the cycle of hybrid breeding, but also more targetedly solve the current situation of lack of male sterile line resources and low purity of hybrid seed production; and eliminate the need for hybrid seed production The detasseling step in the process reduces the mechanical damage and yield impact of mechanical detasseling on corn plants, ensures the purity and yield of hybrid germplasm, and thus achieves the greatest economic benefits.

The technical solutions of the present invention will be described in further detail below with reference to the accompanying drawings and embodiments.

Description of drawings

Fig. 1 is the construction flowchart of the recombinant cloning vector DBN01-T of the present invention mediating the nucleotide sequence of plant male fertility and the method for using it;

Fig. 2 is a construction flow diagram of the recombinant expression vector DBN100349 of the present invention mediating the nucleotide sequence of plant male fertility and the method for using it;

Fig. 3 is the pollen and anther development diagram of the transgenic maize plant T ₀ generation of the nucleotide sequence of the present invention mediating plant male fertility and the method for using it;

Fig. 4 is _a map of the pollen and anther development of the T1 generation of transgenic maize plants of the nucleotide sequence mediating plant male fertility and the method for using it according to the present invention.

Detailed ways

The technical scheme of the nucleotide sequence for mediating plant male fertility and the method for using it will be further illustrated below through specific examples.

The first embodiment, the synthesis of rZmMS26 nucleotide sequence

According to the known ZmMS26 nucleotide sequence (as shown in SEQ ID NO:1 in the sequence listing), the RZmMS26 nucleotide sequence of the present invention is obtained as shown in SEQ ID NO:2 in the sequence listing; the rZmMS26 nucleotide sequence is as shown in As shown in SEQ ID NO:3 in the sequence listing, the rZmMS26 nucleotide sequence was synthesized by Nanjing GenScript Biotechnology Co., Ltd.; the 5' end of the synthesized rZmMS26 nucleotide sequence (SEQ ID NO:3) was also A RsrII restriction site is connected, and the 3' end of the rZmMS26 nucleotide sequence (SEQ ID NO: 3) is also connected with a BsaI restriction site.

The second embodiment, construction of recombinant expression vector and transformation of recombinant expression vector into Agrobacterium

1. Construction of recombinant cloning vector DBN01-T containing rZmMS26 nucleotide sequence

The synthetic rZmMS26 nucleotide sequence was ligated into the cloning vector pGEM-T (Promega, Madison, USA, CAT: A3600), and the operation steps were carried out according to the instructions of the pGEM-T vector produced by Promega Company to obtain the recombinant cloning vector DBN01-T, which The construction process is shown in Figure 1 (wherein, Amp represents the ampicillin resistance gene; f1 represents the replication origin of phage f1; LacZ is the LacZ start codon; SP6 is the SP6 RNA polymerase promoter; T7 is the T7 RNA polymerase promoter; rZmMS26 is the nucleotide sequence of rZmMS26 (SEQ ID NO: 3); MCS is the multiple cloning site).

Then, the recombinant cloning vector DBN01-T was transformed into Escherichia coli T1 competent cells (Transgen, Beijing, China, CAT: CD501) by heat shock method. The heat shock conditions were: 50 μl E. coli T1 competent cells, 10 μl plasmid DNA (recombinant Cloning vector DBN01-T), 42°C water bath for 30 seconds; 37°C shaking culture for 1 hour (shaking at 100rpm), the surface was coated with IPTG (isopropylthio-β-D-galactoside) and X -gal (5-bromo-4-chloro-3-indole-β-D-galactoside) ampicillin (100 mg/L) on LB plates (tryptone 10 g/L, yeast extract 5 g/L, NaCl 10g/L, agar 15g/L, adjust the pH to 7.5 with NaOH) and grow overnight. Pick white colonies and culture them overnight at 37°C in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, ampicillin 100mg/L, adjust pH to 7.5 with NaOH) . Extract the plasmid by alkaline method: centrifuge the bacterial solution at 12000rpm for 1min, remove the supernatant, and use 100μl ice-precooled solution I (25mM Tris-HCl, 10mM EDTA (ethylenediaminetetraacetic acid), 50mM glucose , pH8.0) suspension; add 150μl freshly prepared solution II (0.2M NaOH, 1% SDS (sodium dodecyl sulfate)), invert the tube 4 times, mix, put on ice for 3-5min; add 150μl ice cold Mix solution III (4M potassium acetate, 2M acetic acid) immediately and thoroughly, place on ice for 5-10min; centrifuge at 4°C and 12000rpm for 5min, add 2 times the volume of absolute ethanol to the supernatant, and mix After uniformity, place at room temperature for 5 minutes; centrifuge at 4°C and 12,000 rpm for 5 minutes, discard the supernatant, wash the precipitate with ethanol with a concentration (V/V) of 70%, and dry it; add 30 μl containing RNase (20 μg/ml) TE (10mM Tris-HCl, 1mM EDTA, pH8.0) to dissolve the precipitate; bathe in water at 37°C for 30min to digest RNA; store at -20°C for later use.

After the extracted plasmid was identified by RsrII and BsaI digestion, the positive clones were sequenced and verified, and the results showed that the rZmMS26 nucleotide sequence inserted in the recombinant cloning vector DBN01-T was the nucleotide sequence shown in SEQID NO:3 in the sequence table The acid sequence, that is, the rZmMS26 nucleotide sequence was inserted correctly.

2. Construction of recombinant expression vector DBN100349 containing rZmMS26 nucleotide sequence

The recombinant cloning vector DBN01-T and the expression vector DBNBC-01 (vector backbone: pCAMBIA2301 (available from CAMBIA institutions)) were digested with restriction endonucleases RsrII and BsaI respectively, and the excised rZmMS26 nucleotide sequence fragment was inserted into the expression vector Between the RsrII and BsaI sites of the vector DBNBC-01, it is well known to those skilled in the art to construct the vector by using the conventional enzyme digestion method, and construct the recombinant expression vector DBN100349, and its construction process is shown in Figure 2 (Kan: Kana Mycin gene; RB: right border; Ubi: maize Ubiquitin (ubiquitin) gene promoter (SEQ ID NO:4); rZmMS26: rZmMS26 nucleotide sequence (SEQ ID NO:3); Nos: nopaline synthase gene terminator (SEQ ID NO:5); PMI: phosphomannose isomerase gene (SEQ ID NO:6); LB: left border).

The recombinant expression vector DBN100349 was transformed into Escherichia coli T1 competent cells by heat shock method, and the heat shock conditions were: 50 μl Escherichia coli T1 competent cells, 10 μl plasmid DNA (recombinant expression vector DBN100349), 42°C water bath for 30 seconds; 37°C shaking Cultivate for 1 hour (shaking on a shaker at 100 rpm); L, adjust the pH to 7.5 with NaOH) and culture at 37°C for 12 hours, pick white colonies, and in LB liquid medium (tryptone 10g/L, yeast extract 5g/L, NaCl 10g/L, kana Mycin 50 mg/L, adjust the pH to 7.5 with NaOH) and cultivate overnight at 37°C. The plasmid was extracted by alkaline method. The extracted plasmid was identified after digestion with restriction endonucleases RsrII and BsaI, and the positive clones were sequenced and identified. The results showed that the nucleotide sequence between the RsrII and BsaI sites of the recombinant expression vector DBN100349 was SEQ ID in the sequence table The nucleotide sequence shown in NO:3 is the rZmMS26 nucleotide sequence.

3. Transformation of recombinant expression vector into Agrobacterium

The constructed recombinant expression vector DBN100349 was transformed into Agrobacterium LBA4404 (Invitrgen, Chicago, USA, CAT: 18313-015) by liquid nitrogen method, and the transformation conditions were: 100 μL Agrobacterium LBA4404, 3 μL plasmid DNA (recombinant expression Carrier); put in liquid nitrogen for 10 minutes, and warm water bath at 37°C for 10 minutes; inoculate the transformed Agrobacterium LBA4404 in LB test tubes and cultivate them for 2 hours at a temperature of 28°C and a rotation speed of 200rpm, and apply to the Rifampicin (Rifampicin) and 100mg/L Kanamycin (Kanamycin) on the LB plate until a positive single clone grows, pick the single clone culture and extract its plasmid, and use restriction endonuclease AhdI and AatII to Recombinant expression vector DBN100349 was digested and verified by enzyme digestion, and the results showed that the structure of recombinant expression vector DBN100349 was completely correct.

The third embodiment, the acquisition and verification of maize plants transferred to the rZmMS26 nucleotide sequence

1. Obtaining maize plants transferred to the rZmMS26 nucleotide sequence

According to the commonly used Agrobacterium infection method, the immature embryos of the aseptically cultured maize variety Zong 31 (Z31) were co-cultured with the Agrobacterium described in 3 in the second example, so that the The T-DNA in the recombinant expression vector DBN100349 (including the rZmMS26 nucleotide sequence, the promoter sequence of the maize Ubiquitin gene, the PMI gene and the Nos terminator sequence) was transferred into the maize genome, and the rZmMS26 nucleotide sequence was obtained maize plants; meanwhile, wild-type maize plants were used as controls.

For Agrobacterium-mediated transformation of maize, briefly, immature immature embryos are isolated from maize and the immature embryos are contacted with a suspension of Agrobacterium capable of delivering the rZmMS26 nucleotide sequence to at least one of the immature embryos Cells (Step 1: Infection Step). In this step, the immature embryos are preferably immersed in the Agrobacterium suspension (OD660=0.4-0.6, infection medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 68.5g/L, glucose 36g /L, acetosyringone (AS) 40mg/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/L, pH5.3)) to initiate inoculation. The immature embryos were co-cultured with Agrobacterium for a period of time (3 days) (step 2: co-cultivation step). Preferably, immature embryos are cultured on solid medium (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 20g/L, glucose 10g/L, acetosyringone (AS) 100mg/L after the infection step , 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH5.8). After this co-cultivation phase, there can be an optional "recovery" step. In the "recovery" step, recovery medium (MS salts 4.3g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, 2,4-dichlorophenoxyacetic acid (2,4-D) 1mg/ L, agar 8g/L, pH 5.8), there is at least one antibiotic (cephalosporin) known to inhibit the growth of Agrobacterium, and no selection agent for plant transformants is added (step 3: recovery step). Preferably, immature embryos are cultured on solid medium with antibiotics but no selection agent to eliminate Agrobacterium and provide a recovery period for infected cells. Next, the inoculated immature embryos are cultured on a medium containing a selection agent (mannose) and selected for growing transformed calli (step 4: selection step). Preferably, the immature embryos are cultured on a selective solid medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 5g/L, mannose 12.5g/L, 2,4-dichlorobenzene Oxyacetic acid (2,4-D) 1mg/L, agar 8g/L, pH 5.8) resulted in selective growth of transformed cells. Then, the callus regenerates into plants (step 5: regeneration step), preferably, the callus grown on the medium containing the selection agent is cultured on solid medium (MS differentiation medium and MS rooting medium) to regenerated plants.

The resistant callus obtained by screening was transferred to the MS differentiation medium (MS salt 4.3g/L, MS vitamin, casein 300mg/L, sucrose 30g/L, 6-benzyl adenine 2mg/L, mannose 5g/L, agar 8g/L, pH5.8), cultured and differentiated at 25°C. Differentiated seedlings were transferred to the MS rooting medium (MS salt 2.15g/L, MS vitamins, casein 300mg/L, sucrose 30g/L, indole-3-acetic acid 1mg/L, agar 8g/L, pH5 .8) above, cultivate at 25°C to a height of about 10cm, and move to the greenhouse to cultivate until firm. In the greenhouse, culture was carried out at 28°C for 16 hours and at 20°C for 8 hours every day.

2. Use TaqMan to verify the maize plants transferred to the rZmMS26 nucleotide sequence

About 100 mg of the leaves of maize plants transferred to the rZmMS26 nucleotide sequence were taken as a sample, and the genomic DNA was extracted with Qiagen's DNeasy Plant Maxi Kit, and the copy number of the rZmMS26 nucleotide sequence was detected by the Taqman probe fluorescence quantitative PCR method. At the same time, wild-type maize plants were used as a control, and detection and analysis were carried out according to the above method. The experiment was repeated 3 times, and the average value was taken.

The specific method for detecting the copy number of the rZmMS26 nucleotide sequence is as follows:

Step 11. Take 100 mg of leaves of corn plants and wild-type corn plants with rZmMS26 nucleotide sequence, respectively, grind them into homogenate with liquid nitrogen in a mortar, and take 3 replicates for each sample;

Step 12, use Qiagen's DNeasy Plant Mini Kit to extract the genomic DNA of the above sample, and refer to its product manual for specific methods;

Step 13, using NanoDrop2000 (Thermo Scientific) to measure the genomic DNA concentration of the above sample;

Step 14, adjusting the genomic DNA concentration of the above samples to the same concentration value, the concentration value ranges from 80-100ng/μl;

Step 15, using the Taqman probe fluorescent quantitative PCR method to identify the copy number of the sample, using the sample with known copy number after identification as a standard, and using the sample of the wild-type corn plant as a control, each sample was repeated 3 times, and the average Value; Fluorescence quantitative PCR primer and probe sequences are respectively:

The following primers and probes were used to detect the rZmMS26 nucleotide sequence:

Primer 1 (MF1): CATCGTCCAGCACTGCTATTACC as shown in SEQ ID NO: 7 in the sequence listing;

Primer 2 (MR1): TTTGCTCCATAGATCGTATGATGTG as shown in SEQ ID NO: 8 in the sequence listing;

Probe 1 (MP1): CAGCCAGACTGTCGGATGGACCACAC as shown in SEQID NO: 9 in the sequence listing;

The PCR reaction system is:

The 50X primer/probe mix contained 45 μl of each primer at a concentration of 1 mM, 50 μl of probe at a concentration of 100 μM and 860 μl of 1X TE buffer, and was stored in amber tubes at 4°C.

The PCR reaction conditions are:

Data were analyzed using SDS2.3 software (Applied Biosystems).

The experimental results show that the rZmMS26 nucleotide sequence has been integrated into the genome of the detected maize plant, and the maize plant transformed with the rZmMS26 nucleotide sequence has obtained a transgenic maize plant containing a single copy of the rZmMS26 nucleotide sequence.

The fourth embodiment, analysis of transgenic corn plants

Eighteen maize plants (T ₀ ) with a single copy of the rZmMS26 nucleotide sequence in the third embodiment were evaluated for overall plant morphology, and analyzed for anthers and pollen. Apart from the degree of male fertility, no other morphological differences were observed between the rZmMS26 nucleotide sequence-transferred maize plants and wild-type maize control plants. The results showed that the maize plants (T ₀ ) with a single copy of the rZmMS26 nucleotide sequence were male sterile in different degrees (partial-complete), and 12 plants showed complete male sterile (Fig. 3 , the anthers shriveled and turned yellow, without pollen staining), accounting for 66.7% of the tested transgenic plants. The wild-type maize control plants were completely male-fertile (Fig. 3, the anthers were plump and green, and the pollen stained normally). T ₁ generation seeds were obtained from the completely male sterile maize plants transferred with the rZmMS26 nucleotide sequence, and T ₁ generation plants were grown after sowing, and all T ₁ generation plants had no pollen formation ( FIG. 4 ).

It is thus proved that the maize plant transformed with the rZmMS26 nucleotide sequence constructed by the RNAi technology inhibits the expression of the maize MS26 gene, and can obtain completely male sterile plants.

In summary, the present invention utilizes RNAi technology to construct MS26 male sterile line for the first time, and described MS26 male sterile line suppresses the expression of maize MS26 gene, can obtain the plant of complete male sterile; Said MS26 male sterile line It can not only shorten the cycle of hybrid breeding, but also more targetedly solve the current situation of lack of male sterile line resources and low purity of hybrid seed production. The mechanical damage and yield impact on corn plants ensure the purity and yield of the hybrid germplasm, thereby achieving the greatest economic benefit.

Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention without limitation. Although the present invention has been described in detail with reference to the preferred embodiments, those of ordinary skill in the art should understand that the technical solutions of the present invention can be The scheme shall be modified or equivalently replaced without departing from the spirit and scope of the technical scheme of the present invention.

Claims (11)

1. A nucleotide sequence, characterized in that, it encodes the following RNA molecule, said RNA molecule suppresses plant MS26 gene expression, said nucleotide sequence consists of SEQ ID NO:2, spacer sequence and SEQ IDNO:2 Complementary sequence composition. 2. nucleotide sequence according to claim 1, is characterized in that, described nucleotide sequence is SEQID NO:3. 3. An expression cassette, characterized in that it comprises the nucleotide sequence of claim 1 or 2 under the control of an operably linked regulatory sequence. 4. A recombinant vector comprising the nucleotide sequence of claim 1 or 2 or the expression cassette of claim 3. 5. A ribonucleotide sequence, characterized in that it inhibits plant MS26 gene expression and is transcribed from the following DNA sequence, which is the nucleotide sequence according to claim 1 or 2. 6. A method for inhibiting the expression of plant MS26 gene, characterized in that it comprises using the ribonucleotide sequence according to claim 5 to inhibit the expression of plant MS26 gene, and the plant is maize. 7. A method for inducing male sterility in plants, comprising using the ribonucleotide sequence according to claim 5 to suppress the expression of the plant MS26 gene, the plant being maize. 8. The method for inducing plant male sterility according to claim 7, wherein said ribonucleotide sequence comprises a first ribonucleotide sequence and a second ribonucleotide sequence, and said first ribonucleotide sequence The nucleotide sequence inhibits the expression of plant MS26 gene, and the second ribonucleotide sequence is complementary to the first ribonucleotide sequence. 9. The method for inducing male sterility in plants according to claim 8, wherein the ribonucleotide sequence further comprises a spacer sequence. 10. the method for inducing plant male sterility according to claim 9, is characterized in that, described ribonucleotide sequence is transcribed by SEQ ID NO:3. 11. A method for producing male sterile plants, comprising introducing the expression cassette according to claim 3 into plant cells to produce transgenic plants, the plants being maize.