CN113215172B - Male sterile gene MsJMT and application thereof - Google Patents

Abstract

The invention discloses application of a male sterility gene MsJMT and a method for restoring male sterility of alfalfa, and belongs to the technical field of bioengineering. The invention provides an application of an MsJMT gene, wherein the MsJMT gene has a nucleotide sequence shown in SEQ ID NO.1, and the application comprises the following steps: obtaining a medicago sativa male sterile line by overexpressing an MsJMT gene and producing seeds by using the medicago sativa male sterile line; the invention also provides a method for restoring the alfalfa male sterility caused by the overexpression of the MsJMT gene, which is characterized in that an RNAi expression vector is constructed, and the bred alfalfa male sterility line is transformed by a genetic transformation means so as to restore the fertility and the wild phenotype; the alfalfa male sterile line created by the invention and the provided method for restoring the fertility of the male sterile line have very important application in construction of hybrid alfalfa and agricultural production.

Description

Male sterility gene MsJMT and its application

technical field

The invention relates to a method for creating alfalfa strains in the technical field of bioengineering, in particular to a male sterility gene MsJMT and its application.

Background technique

Alfalfa (Medicago sativa L.) is a high-quality perennial leguminous forage, known as the "king of forages", and has a long history of cultivation in my country. As a typical monoecious cross-pollination plant, it has self-incompatibility and needs to be hybridized for seed production. Compared with ordinary alfalfa varieties, the hybrid alfalfa cultivated by the "three-line method" has the advantages of taller plants, stronger stress resistance and higher proportion of leaves. Due to the obvious heterosis, in practical application, the use of this advantage in seed production is mainly divided into two ways: artificial emasculation hybrid seed production and hybrid seed production using nucleoplasmic interaction male sterile lines. The latter can not only reduce emasculation. labor, reducing seed production costs and proven effective in breeding high-quality alfalfa hybrids. In order to improve the heterosis of alfalfa and reduce the time and economic cost caused by artificial emasculation, the development of high-quality alfalfa male sterile lines is the focus of completing the "three-line" matching of alfalfa; however, the "three-line" method requires the relationship between restorer lines and maintainer lines. Therefore, screening and breeding new genotype sterile lines, expanding the cytoplasmic background, and laying the foundation for the utilization of heterosis of perennial forages.

Although research on alfalfa breeding has been ongoing, it is difficult to screen high-quality male sterile lines and supporting maintainer materials in the process of hybrid breeding. At the same time, as a tetraploid plant, the genome is relatively complex and the growth cycle is long. It also limits the progress of related researches such as hybridization, heterosis utilization, fertility genetic basis and inheritance pattern.

SUMMARY OF THE INVENTION

The purpose of the present invention is to provide a method for the application of the MsJMT gene and restore the male sterility of alfalfa caused by the deletion of the MsJMT gene, to obtain a male sterile line of alfalfa by overexpressing the MsJMT gene, and to restore the alfalfa by inhibiting the expression of the MsJMT gene. of male sterility.

The present invention is achieved through the following technical solutions:

In one aspect, the present invention provides a male sterility gene MsJMT, wherein the MsJMT gene is the nucleotide sequence shown in SEQ ID NO.1.

In another aspect, the present invention provides an application of the male sterile gene MsJMT, the application is: obtaining a male sterile line of alfalfa by overexpressing the MsJMT gene, and using the male sterile line of alfalfa to generate Produce seeds.

Finally, the present invention also provides a method for restoring the male sterility of alfalfa caused by the deletion of the MsJMT gene. By constructing an interference expression vector containing the MsJMT gene, and transforming the above-bred male sterile line of alfalfa by means of genetic transformation, the It restores fertility and wild-type phenotype.

Preferably, the method comprises the steps of:

The Agrobacterium of the complementary construction of MsJMT is transferred into the male sterile line of alfalfa, cultivated, and obtained; wherein the complementary construction vector of MsJMT contains as SEQ ID NO.3 (forward fragment) and SEQ ID NO.4 (reverse fragment) Nucleotide sequences shown.

Preferably, it specifically includes the following steps:

(a) provide Agrobacterium LBA4404 carrying the complementary construct interfering expression vector expressing MsJMT;

Using alfalfa cDNA as a template, use primers:

MsJMT-RNAi (Sense primer) ACTGACGTAAGGGATGACGCAC and

MsJMT-RNAi (Anti-sense primer) GATTTGTAGAGAGAGACTGGT

The 195th to 764th position of the coding region sequence is a specific fragment of 569bp in total, so that the product contains attB sites at both ends; this fragment was inserted into the pENTR-MsJMT vector using BP Clonase in the forward and reverse directions respectively; Ligated into pRNAi vector with LR Clonase. Sequencing again to check whether the nucleotide sequence is correct, the pRNAi-MsJMT interference expression vector was successfully constructed, and the obtained pRNAi-MsJMT interference expression vector was introduced into Agrobacterium;

(b) contacting the sterile alfalfa cell or tissue with the competent Agrobacterium cells in step (a), so that the nucleotide sequences encoding the nucleotide sequences shown in SEQ ID NO. 3 and SEQ ID NO. 4 are transformed into alfalfa sterile line cells, and integrated into the chromosome of alfalfa sterile line cells;

(c) selecting alfalfa cells or tissues into which the nucleotides have been transferred, and regenerating to obtain alfalfa plants.

Beneficial effects of the present invention:

The invention clones a new gene from the anthers of leguminous plants for the first time, namely the alfalfa jasmonate methyl synthase MsJMT gene, and obtains the variation of alfalfa male reproductive development by controlling the alfalfa jasmonate methyl synthase MsJMT gene and its encoded protein The sterile alfalfa plant obtained by the present invention has no obvious difference from the source parent in the vegetative period, and after entering the reproductive growth stage, the male reproductive development is abnormal, the pollen is aborted, and a completely sterile plant is obtained, At the same time, the male sterile lines can be restored to fertility by inhibiting the expression of the gene MsJMT, which has a very important application in the construction of hybrid alfalfa and agricultural production.

Description of drawings

1 is a schematic diagram of the construction of the pBI121-MsJMT overexpression vector in Example 1 provided by the present invention;

2 is a schematic diagram of the construction of pRNAi-MsJMT interference expression vector in Example 2 provided by the present invention;

3 is a schematic diagram of the morphological observation of the sterile line provided by the present invention;

Figure 4 is a mirror image of the characterization of the restored fertility plants provided by the present invention.

Detailed ways

Below in conjunction with embodiment, the present invention is further elaborated. These examples are only intended to illustrate the present invention and not to limit the scope of the present invention. The experimental steps that do not specify specific conditions in the following examples are generally in accordance with conventional conditions.

Embodiment 1, the method for creating male sterile lines of alfalfa

1.1 Cloning of the MsJMT gene for fertility control in alfalfa

Using the conventional alfalfa variety Gongnong No. 1 as the material (hereinafter referred to as: alfalfa), specific primers were designed according to the full-length sequence of the MsJMT gene

MsJMT-25 (Sense primer) ATGGTACAGAAAAAGGTTCTTTCTT and

MsJMT-26 (Anti-sense primer) TCACACTTTTTTGGTCATTGATACGC

The total RNA of the anthers was extracted in turn, cDNA was synthesized and the full-length cDNA of the MsJMT gene was amplified by PCR, and the full-length cDNA sequence was identified by sequencing as 1047bp, as shown in SEQ ID NO. Alfalfa male reproductive development control protein, its sequence is shown in SEQ ID NO.2.

1.2 Increase the expression level of MsJMT in alfalfa by overexpression

In order to apply the MsJMT protein, the overexpression vector pBI121-MsJMT of the MsJMT gene was constructed, and the wild-type plants were transformed to increase the expression of MsJMT, thereby achieving the purpose of changing the fertility of alfalfa.

Using primers from alfalfa cDNA clone (MsJMT-EST clone)

MsJMT-25 (Sense primer) ATGGTACAGAAAAAGGTTCTTTCTT and

MsJMT-26 (Anti-sense primer) TCACACTTTTTTGGTCATTGATACGC

The full-length sequence of the MsJMT gene was amplified with a total of 1047 bp, and was inserted into the cloning vector pMD20T-MsJMT to complete the construction of the cloning vector. Using the cloning vector as the template, SacI and XbaI restriction sites were added to the 5' and 3' ends to design primers. The primer sequences are as follows.

MsJMT-JM(Senseprimer)GCTCTAGAATGGTACAGAAAAAGGTTCTTTCTTTG

MsJMT-JM (Anti-sense primer) CGAGCTCTCACACTTTTTTGGTCATTGATACG

The amplified fragment was double digested with the empty vector pBI121 and then ligated. The nucleotide sequence was re-sequenced to check whether the nucleotide sequence was correct. The pBI121-MsJMT plasmid was successfully constructed.

(1) Agrobacterium transformation

① Melt Agrobacterium-competent LBA4404 (Angyu Bio) on ice.

②Add pRNAi-MsJMT into two tubes of competent cells respectively.

③ After standing on ice for 5 minutes, liquid nitrogen for 5 minutes, water bath at 28°C for 5 minutes, and ice bath for 5 minutes, 700 μl of anti-YEP medium was added.

④ Shake culture at 180 rpm at 28°C for 3 hours.

⑤ Collect bacteria by centrifugation at 10,000×g for 1 min, discard 600 μl of supernatant, resuspend the bacterial block with the remaining medium, and apply all of them to solid selective medium of 90mmYEP+25mg·L-1Rif+50mg·L-1Spe, and invert at 28°C for 48h.

⑥ Pick a single yellow-white colony and inoculate it into the liquid selective medium of 5ml YEP+25mg·L-1Rif+100mg·L-1Spe, and cultivate with shaking at 180rpm at 28°C for 24h.

⑦Inoculate it into 50ml of the same YEP medium at a concentration of 1:100, and shake it at 28°C at 180rpm until the OD value is 0.6-0.8.

⑧ Centrifuge the bacterial solution at 10000×g, discard the supernatant, and resuspend the bacterial block in 20 ml of MS liquid medium.

(2) Infection of alfalfa leaves

① Cut the leaves of 30-day sterile wild alfalfa seedlings into small square pieces with a side length of 1 cm, and place them in the MS liquid culture with resuspended Agrobacterium.

② 140rpm shaking culture for 15min, so that Agrobacterium can fully infect the leaves.

③ Pour the leaves onto sterile filter paper, absorb the water and place them on 90mm non-anti-MS solid medium, and cultivate in the dark for 3 days.

④ Rinse the co-cultured alfalfa leaves with sterile water containing 500mg·L-1Cef for 3 times, absorb the water and embed in 90mm alfalfa differentiation medium (MS+1mg·L ^{- 1} 6-BA+0.1mg·L- ¹ IAA+200 mg·L ⁻¹ Kan+500 mg·L ⁻¹ Cef+100 mg·L ⁻¹ Tim). Artificial climate box setting parameters: 28 ℃ constant temperature, humidity 10%, 80% light for 16 hours, 0% light for 8 hours.

⑤ After 10 days, the reverse side of the leaves was changed to a new alfalfa differentiation medium to make it fully inhibited.

(3) Tissue culture of alfalfa

①Break off the differentiated buds that grow to 0.3-1cm, insert them into a new 90mm alfalfa differentiation medium for concentrated culture, and transfer them to a 650ml culture bottle containing alfalfa rooting medium (1/2MS+200mg·L-1Kan) after 7 days .

②After 15 days, the rooted alfalfa was moved into MS without resistance to continue to grow.

(4) Detection of positive plants

Total plant DNA was extracted using Plant DNA Mini Kit (OMEGA).

After obtaining the eluted DNA, use the corresponding primers to perform PCR detection on the extracted total DNA, and check the bands after electrophoresis. The expression level of MsJMT gene in the positive plants was analyzed by RT-PCR, and the expression level decreased to less than 20% of the wild type, which was an effective RNA interference plant. The effective plants after screening were hardened.

(5) Refining seedlings

At room temperature, open the sterile bottle cap with positive plants, put it in a cool and ventilated place, and spray the leaves with spray, once in the morning and once in the evening. After 3 days, the plants were pulled out, the residual medium at the roots was rinsed, and the plants were planted in 10cm seedling pots and cultivated in natural light.

1.3MsJMT protein activity loss or expression level leads to abnormal male development in alfalfa

As shown in Figure 3, compared with the phenotype of the wild-type alfalfa, the male-sterile line has a more compact plant shape, smaller plants, withered anthers and no cracking, while the wild-type plant shape is looser and the plants are taller. , the anthers were large and plump, with dehiscence and loose powder (A, B, C). After potassium iodide staining assay, it showed that the abnormal tapetum cells of the pollen in the tetrad stage resulted in defects in the development of microspores, which in turn showed that the number of released pollen grains was low or Unable to release pollen grains normally, resulting in abortion of alfalfa pollen, creating a new male sterile line of alfalfa.

1.4 Use of the male sterile line created above in alfalfa seed production

The MsJMT sterile line was crossed as the male parent with the sterile parent in a three-line or two-line cross combination to obtain the F1 generation. Plants with both male-sterile and sterile characteristics are selected in the F2 generation, and the plants are crossed with the maintainer line corresponding to the original sterile parent. The plants with both male sterility and sterility are screened again in the F2 generation and crossed with the maintainer line, and a new male sterile line is obtained after multi-generation cross screening, which is suitable as the female parent in the hybrid combination.

Example 2. Method for restoring the overexpression of MsJMT gene to cause male sterility in alfalfa

2.1 Restoring the fertility of new male sterile lines by suppressing the MsJMT gene

The nucleotide sequence encoding the MsJMT gene is silenced and expressed by RNAi technology, and the successfully constructed pRNAi-MsJMT vector is transferred into the sterile alfalfa plant, which can restore the sterile alfalfa plant to the wild-type phenotype, wherein The pRNAi-MsJMT vector contains the nucleotide sequences shown in SEQ ID NO. 3 (forward fragment) and SEQ ID NO. 4 (reverse fragment).

Using alfalfa cDNA as a template and using primers

MsJMT-RNAi (Sense primer) ACTGACGTAAGGGATGACGCAC and

MsJMT-RNAi (Anti-sense primer) GATTTGTAGAGAGAGACTGGT

The 195th to 764th position of the coding region sequence is a specific fragment of 569bp in total, so that the product contains attB sites at both ends; this fragment was inserted into the pENTR-MsJMT vector using BP Clonase in the forward and reverse directions respectively; Ligated into pRNAi vector with LR Clonase. Sequencing again to check whether the nucleotide sequence is correct, the pRNAi-MsJMT plasmid was successfully constructed, the silencing vector was introduced into the new male sterile line created above, and positive identification was carried out through co-cultivation, screening, differentiation and induction of rooting and other culture processes And pollen fertility observation, as shown in the mirror image Figure 4, the male sterile line plant field contains a small number of microspores with different sizes, some of which are not full in shape, yellow-green to transparent color after staining, poor vitality; wild-type field of view The microspores contained are uniform in size, purple-gray after staining, with strong vitality, most of which are full in shape and have a lot of content; the number of microspores in the field of view of the plants transformed by the RNAi technology of the male sterile lines is much higher than that of the sterile lines, and in the Fertility was measured by I2-KI solution staining at flowering stage. The pollen of the new male sterile line of alfalfa changed from yellow-brown to blue, that is, the fertility of the new male sterile line was restored. The phenotype is basically the same.

sequence listing

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Claims (5)

1. A male sterility gene MsJMT, wherein the MsJMT gene is the nucleotide sequence shown in SEQ ID NO.1. 2. the application of a male sterile gene MsJMT, it is characterized in that, described application is: thereby obtain alfalfa male sterile strain by overexpressing MsJMT gene, and produce seed with described alfalfa male sterile strain . 3. a method for restoring the male sterility of MsJMT alfalfa, it is characterized in that, by constructing interference expression vector, utilize genetic transformation means to transform the male sterile line of alfalfa in claim 2, can make it restore fertility and wild type phenotype. 4. a kind of method of restoring the male sterility of MsJMT alfalfa according to claim 3, is characterized in that, described method comprises the steps: The Agrobacterium constructed by MsJMT complementation is transformed into the male sterile line of alfalfa, and then it is obtained; wherein the MsJMT complementation construct vector contains the nucleotide sequences shown in SEQ ID NO.3 and SEQ ID NO.4. 5. a kind of method of restoring the male sterility of MsJMT alfalfa according to claim 4, is characterized in that, specifically comprises the following steps: (a) provide Agrobacterium LBA4404 carrying the complementary construct interfering expression vector expressing MsJMT; Using alfalfa cDNA as a template, use primers: MsJMT-RNAi Sense primer ACTGACGTAAGGGATGACGCAC and MsJMT-RNAi Anti-sense primer GATTTGTAGAGAGAGACTGGT The 195th to 764th position of the coding region sequence is a specific fragment of 569bp in total, so that the product contains attB sites at both ends; this fragment was inserted into the pENTR-MsJMT vector using BP Clonase in the forward and reverse directions respectively; Linked into pRNAi vector with LRClonase; sequenced again to check whether the nucleotide sequence was correct, successfully constructed pRNAi-MsJMT interference expression vector, and introduced the obtained pRNAi-MsJMT interference expression vector into Agrobacterium; (b) contacting the sterile alfalfa cell or tissue with the competent Agrobacterium cells in step (a), so that the nucleotide sequences encoding SEQ ID NO. alfalfa sterile line cells, and integrated into the chromosome of alfalfa sterile line cells; (c) selecting alfalfa cells or tissues into which the nucleotides have been transferred, and regenerating to obtain alfalfa plants.

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