CN113215172A - Male sterile gene MsJMT and application thereof - Google Patents
Male sterile gene MsJMT and application thereof Download PDFInfo
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- CN113215172A CN113215172A CN202110471073.9A CN202110471073A CN113215172A CN 113215172 A CN113215172 A CN 113215172A CN 202110471073 A CN202110471073 A CN 202110471073A CN 113215172 A CN113215172 A CN 113215172A
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- C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
- C07K14/415—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from plants
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8287—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for fertility modification, e.g. apomixis
- C12N15/8289—Male sterility
Abstract
The invention discloses application of a male sterility gene MsJMT and a method for recovering male sterility of alfalfa, belonging 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
Technical Field
The invention relates to an alfalfa strain creating method in the technical field of bioengineering, in particular to a male sterile gene MsJMT and application thereof.
Background
Alfalfa (Medicago sativa L.) is a high-quality perennial legume forage, known as the king of forage, and has a cultivation history in China for many years. As a typical cross-pollinated plant of a male and female isogamy, the hybrid has self-incompatibility and needs to be produced in a hybrid mode. Compared with the common alfalfa varieties, the hybrid alfalfa cultivated by the three-line method has the advantages of high plant size, strong stress resistance, high leaf proportion and the like. Because of the obvious heterosis, the seed production by utilizing the heterosis is mainly divided into two ways of artificial emasculation hybrid seed production and hybrid seed production by utilizing a nucleoplasm interaction male sterile line in practical application, and the latter can not only reduce the labor for emasculation and reduce the seed production cost, but also is proved to be capable of effectively cultivating high-quality alfalfa hybrid varieties. In order to improve the hybrid vigor of the alfalfa and reduce the time and economic cost caused by artificial emasculation, the development of an excellent alfalfa male sterile line is the key point for completing the matching of the alfalfa three lines at present; however, the three-line method requires the relationship between a restorer line and a maintainer line to be limited, and the selection of new genotypes of the sterile line is difficult, so that the selection efficiency of superior varieties is low, and the yield is difficult to improve, therefore, the selection and cultivation of the new genotypes of the sterile line, the cytoplasm background is expanded, and a foundation is laid for the heterosis utilization of perennial pasture.
The research of alfalfa in breeding is always carried out, but the screening of high-quality male sterile line and matched maintenance line materials is difficult in the breeding process of hybrid seeds, and meanwhile, as tetraploid plants, the factors of relatively complex genome, long growth period and the like limit the progress of related research of hybridization and hybrid vigor utilization in fertility genetic basis, genetic pattern and the like.
Disclosure of Invention
The invention aims to provide an application of an MsJMT gene and a method for restoring male sterility of alfalfa caused by MsJMT gene deletion, wherein an alfalfa male sterile line is obtained by over-expressing the MsJMT gene, and the male sterility of the alfalfa is restored by inhibiting the expression of the MsJMT gene.
The invention is realized by the following technical scheme:
in one aspect, the invention provides a male sterile gene MsJMT, wherein the MsJMT gene is a nucleotide sequence shown as SEQ ID No. 1.
In another aspect, the present invention provides an application of a male sterility gene MsJMT, wherein the application comprises: and (3) over-expressing the MsJMT gene to obtain a medicago sativa male sterile line, and producing seeds by using the medicago sativa male sterile line.
Finally, the invention also provides a method for restoring the male sterility of the alfalfa caused by the MsJMT gene deletion, and the fertility and the wild type phenotype of the alfalfa male sterile line can be restored by constructing an interference expression vector and transforming the bred alfalfa male sterile line by a genetic transformation means.
Preferably, the method comprises the steps of:
transferring agrobacterium complementarily constructed by MsJMT into the alfalfa male sterile line, and culturing to obtain the alfalfa male sterile line; wherein the MsJMT complementary construction vector contains nucleotide sequences shown as SEQ ID NO.3 (forward fragment) and SEQ ID NO.4 (reverse fragment).
Preferably, the method specifically comprises the following steps:
(a) providing agrobacterium LBA4404 carrying an interference expression vector constructed by MsJMT complementation;
taking alfalfa cDNA as a template and using primers
MsJMT-RNAi (sense primer) ACTGACGTAAGGGATGACGCAC and
MsJMT-RNAi(Anti-sense primer)GATTTGTAGAGAGAGACTGGT
a specific fragment of 569bp from 195 th to 764 th of the sequence of the coding region, so that the product contains attB sites at both ends; this fragment was inserted into pENTR-MsJMT vector using BP Clonase in forward and reverse directions, respectively; sequencing verified correctly and ligated into pRNAi vector with LR clone. Checking whether the nucleotide sequence is correct by sequencing again, successfully constructing a pRNAi-MsJMT interference expression vector, and introducing the obtained pRNAi-MsJMT interference expression vector into agrobacterium;
(b) contacting the sterile line cells or tissues of the alfalfa with the agrobacterium-infected cells in the step (a), so that the nucleotide sequences shown as SEQ ID NO.3 and SEQ ID NO.4 are transferred into the sterile line cells of the alfalfa and are integrated on the chromosome of the sterile line cells of the alfalfa;
(c) and (3) selecting the alfalfa cells or tissues transferred with the nucleotide, and regenerating to obtain alfalfa plants.
The invention has the beneficial effects that:
the invention clones a brand new gene from the anther of leguminous plant, namely the alfalfa jasmonate methyl synthetase MsJMT gene for the first time, obtains a variant of alfalfa male reproductive development by controlling the alfalfa jasmonate methyl synthetase MsJMT gene and a coding protein thereof, and realizes the control of alfalfa reproductive process; the alfalfa sterile line plant obtained by the invention has no obvious difference with the original parent in the vegetative stage, the male reproductive development is abnormal after the plant enters the reproductive growth stage, the pollen is aborted, and the completely sterile plant is obtained.
Drawings
FIG. 1 is a schematic diagram of the construction of a pBI121-MsJMT overexpression vector in example 1 provided by the present invention;
FIG. 2 is a schematic diagram of the construction of pRNAi-MsJMT interference expression vector in example 2 provided by the present invention;
FIG. 3 is a schematic diagram of morphological observation of a sterile line provided by the present invention;
FIG. 4 is a representation mirror image of a plant with restored fertility provided by the invention.
Detailed Description
The invention is further illustrated by the following examples. These examples are intended to illustrate the invention and are not intended to limit the scope of the invention. Experimental procedures without specific conditions noted in the following examples are generally performed according to conventional conditions.
Example 1 alfalfa male sterile line creation method
1.1 cloning of MsJMT gene for fertility control of alfalfa
By using the conventional alfalfa variety gong nong No.1 as a material (hereinafter, referred to as alfalfa), a specific primer is designed according to the full-length sequence of the MsJMT gene
MsJMT-25(Sense primer) ATGGTACAGAAAAAGGTTCTTTCTT and
MsJMT-26(Anti-sense primer)TCACACTTTTTTGGTCATTGATACGC
extracting anther total RNA, synthesizing cDNA, amplifying MsJMT gene cDNA full length by PCR, sequencing to identify cDNA sequence full length 1047bp, nucleotide sequence shown as SEQ ID No.1, and encoding alfalfa male reproductive development control protein with 348 amino acids in full length, wherein the sequence is shown as SEQ ID No. 2.
1.2 increasing the expression level of MsJMT in alfalfa by means of overexpression
In order to apply the MsJMT protein, an overexpression vector pBI121-MsJMT of the MsJMT gene is constructed, and a wild plant is transformed, so that the expression of the MsJMT is improved, and the aim of changing the fertility of alfalfa is fulfilled.
Primers from alfalfa cDNA clone (MsJMT-ESTclone)
MsJMT-25(Sense primer) ATGGTACAGAAAAAGGTTCTTTCTT and
MsJMT-26(Anti-sense primer)TCACACTTTTTTGGTCATTGATACGC
the total sequence of the MsJMT gene is 1047bp, and is inoculated into a cloning vector pMD20T-MsJMT to complete the construction of the cloning vector. The cloning vector is used as a template, SacI and XbaI enzyme cutting sites are respectively added to the 3 'end of the 5' end to design primers, and the sequences of the primers are as follows.
MsJMT-JM(Senseprimer)GCTCTAGAATGGTACAGAAAAAGGTTCTTTCTTTG
MsJMT-JM(Anti-sense primer)CGAGCTCTCACACTTTTTTGGTCATTGATACG
And performing double enzyme digestion on the amplified fragment and the unloaded pBI121, connecting, sequencing again to check whether the nucleotide sequence is correct, and successfully constructing a pBI121-MsJMT plasmid.
(1) Agrobacterium transformation
Firstly, agrobacterium competent LBA4404 (Alternaria sinensis) is melted on ice.
② adding pRNAi-MsJMT into two tubes of competent cells respectively.
③ standing on ice for 5min, liquid nitrogen for 5min, water bath at 28 ℃ for 5min, ice-bath for 5min, and then adding 700 mul of non-antibiotic YEP culture medium.
And fourthly, shaking and culturing for 3 hours at the temperature of 28 ℃ and the speed of 180 rpm.
Fifthly, 10000xg is centrifuged for 1min for bacteria collection, 600 mul of supernatant is discarded, the bacteria block is re-suspended by the residual culture medium, and the whole is coated on a solid selection culture medium of 90mm YEP, 25 mg.L < -1 > Rif and 50 mg.L < -1 > Spe and is inversely cultured for 48h at the temperature of 28 ℃.
Sixthly, selecting a yellow-white single colony, inoculating the yellow-white single colony into a liquid selection culture medium of 5ml YEP, 25 mg.L-1 Rif and 100 mg.L-1 Spe, and performing shake culture at 28 ℃ and 180rpm for 24 hours.
Seventhly, inoculating the culture medium into 50ml of the same YEP culture medium at the concentration of 1:100, and performing shake culture at the temperature of 28 ℃ and the rpm of 180 until the OD value is 0.6-0.8.
Eighty 10000xg of the centrifugal bacterial liquid, abandoning the supernatant, and suspending the bacterial block by 20ml of MS liquid culture medium.
(2) Infested alfalfa leaves
Firstly, cutting 30d wild alfalfa sterile seedling leaves into square small blocks with the side length of 1cm, and placing the square small blocks in MS liquid culture in which the agrobacterium is resuspended.
② shaking and culturing at 140rpm for 15min to ensure that the agrobacterium is fully infected on the leaves.
And thirdly, pouring the leaves on sterile filter paper, sucking water, placing on a 90mm non-resistance MS solid culture medium, and culturing for 3d in dark.
Fourthly, the leaves of the alfalfa after the co-culture are washed for 3 times by sterile water containing 500 mg.L < -1 > Cef, and the leaves are embedded into a 90mm alfalfa differentiation medium (MS +1 mg.L) after water is absorbed-16-BA+0.1mg·L-1IAA+200mg·L-1Kan+500mg·L-1Cef+100mg·L-1Tim). Setting parameters of the artificial climate box: constant temperature of 28 ℃, humidity of 10%, illumination of 80% for 16 hours, and illumination of 0% for 8 hours.
After 10 days, the reverse side of the leaves is replaced by a new alfalfa differentiation culture medium, so that the alfalfa differentiation culture medium can be fully bacteriostatic.
(3) Tissue culture of alfalfa
Breaking off the differentiation buds growing to 0.3-1 cm, inserting the differentiation buds into a new alfalfa differentiation culture medium with the diameter of 90mm for centralized culture, and transferring the buds into a 650ml culture bottle containing an alfalfa rooting culture medium (1/2MS +200 mg. L-1Kan) after 7 d.
And moving the rooted alfalfa into the non-resistance MS after 15d to continue growing.
(4) Detection of Positive plants
Plant DNA Mini Kit (OMEGA) was used for total DNA extraction.
And after the eluted DNA is obtained, PCR detection is carried out on the extracted total DNA by using corresponding primers, and the band is checked after electrophoresis. And (3) analyzing the expression level of the MsJMT gene in the positive plant by RT-PCR, and determining that the expression level is reduced to below 20% of the wild type as an effective RNA interference plant. And hardening the seedling of the screened effective plant.
(5) Hardening off seedlings
At room temperature, the sterile bottle cap with the positive plants is opened, the sterile bottle cap is placed in a shady and cool ventilated place, and the leaves are sprayed with mist to be wet once in the morning and at night. And 3d, pulling out the plants, washing the residual culture medium at the roots, planting the plants into a 10cm seedling culture pot, and culturing by natural illumination.
1.3 loss of MsJMT protein Activity or expression levels leading to alfalfa Male dysplasia
As shown in figure 3, compared with the phenotype of wild alfalfa, the phenotype of the male sterile line is that the plant shape of the sterile line is compact, the plant is small, anthers are dry and do not crack, the plant shape of the wild type plant is loose, the plant is high, the anthers are large and full, and the cracking and the pollen scattering (A, B and C) show that the dystapetum cells of the pollen in the tetrad period cause microspore development defects by adopting a potassium iodide dyeing method, so that the pollen is expressed to release a small amount of pollen grains or can not normally release the pollen grains, the pollen of the alfalfa is aborted, and a new alfalfa male sterile line is created.
1.4 application of the above-created male sterile line in alfalfa seed production
And hybridizing the MsJMT sterile line serving as a male parent with the sterile parent in the three-line or two-line hybrid combination to obtain the Fl generation. Plants with both male sterility and sterility characteristics were selected in generation F2 and crossed with the maintainer line corresponding to the original sterile parent. And then, plants with the characteristics of male sterility and sterility are screened in the F2 generation to be hybridized with the maintainer line, and a new male sterile line is obtained after multi-generation hybridization screening and is suitable to be used as a female parent in a hybridization combination.
Example 2 method for restoring alfalfa male sterility due to overexpression of MsJMT gene
2.1 restoration of fertility of New Male sterile line by inhibition of MsJMT Gene
The nucleotide sequence of the MsJMT gene is subjected to silent expression through RNAi technology, and a successfully constructed pRNAi-MsJMT vector is transferred into a alfalfa sterile line plant, so that the alfalfa sterile line plant can be recovered to a wild type phenotype, wherein the pRNAi-MsJMT vector contains nucleotide sequences shown as SEQ ID No.3 (a forward fragment) and SEQ ID No.4 (a reverse fragment).
Taking alfalfa cDNA as a template and using primers
MsJMT-RNAi (sense primer) ACTGACGTAAGGGATGACGCAC and
MsJMT-RNAi(Anti-sense primer)GATTTGTAGAGAGAGACTGGT
a specific fragment of 569bp from 195 th to 764 th of the sequence of the coding region, so that the product contains attB sites at both ends; this fragment was inserted into pENTR-MsJMT vector using BP Clonase in forward and reverse directions, respectively; sequencing verified correctly and ligated into pRNAi vector with LR clone. Checking whether the nucleotide sequence is correct by sequencing again, successfully constructing a pRNAi-MsJMT plasmid, introducing the silencing vector into the created new male sterile line plant, and performing positive identification and pollen fertility observation through the culture processes of co-culture, screening, differentiation, root induction and the like, wherein as shown in a mirror image figure 4, the male sterile line plant has a small number of microspores in a visual field, is different in size, is partially unsaturated in appearance, is yellow-green to transparent after dyeing and is poor in activity; the microspores contained in the wild type visual field are uniform in size, are purple gray after being dyed, and are strong in vitality, most of the shapes are full, and the content is large; the number of microspores contained in a plant field obtained by transforming a male sterile line plant through an RNAi technology is far more than that of the sterile line, and the pollen of a new male sterile line of the alfalfa is changed from yellow brown to blue by adopting I2-KI solution dyeing for fertility determination in a flowering period, so that the fertility of the new male sterile line of the alfalfa is restored, and the phenotype of the alfalfa is basically the same as that of the wild alfalfa.
Sequence listing
<110> Jilin agriculture university, Jilin province academy of agriculture science
<120> male sterility gene MsJMT and application thereof
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agtagtttat ctaaaccact cattgacgag gcatacaacg tcacaaaatg catgagggct 900
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Claims (5)
1. A male sterility gene MsJMT is characterized in that the MsJMT gene is a nucleotide sequence shown as SEQ ID NO. 1.
2. The application of a male sterility gene MsJMT is characterized in that the application comprises the following steps: and (3) over-expressing the MsJMT gene to obtain a medicago sativa male sterile line, and producing seeds by using the medicago sativa male sterile line.
3. A method for restoring the male sterility of alfalfa caused by MsJMT gene deletion is characterized in that an interference expression vector is constructed, and the bred alfalfa male sterile line is transformed by a genetic transformation means, so that the fertility and the wild phenotype can be restored.
4. The method according to claim 3, wherein the method comprises the following steps:
transferring agrobacterium complementarily constructed by MsJMT into the alfalfa male sterile line, and culturing to obtain the alfalfa male sterile line; wherein the MsJMT complementary construction vector contains nucleotide sequences shown as SEQ ID NO.3 (forward segment) and SEQ ID NO.4 (reverse segment).
5. The method for restoring male sterility in alfalfa due to deletion of the MsJMT gene as claimed in claim 4, comprising the following steps:
(a) providing agrobacterium LBA4404 carrying an interference expression vector constructed by MsJMT complementation;
taking alfalfa cDNA as a template and using primers
MsJMT-RNAi (sense primer) ACTGACGTAAGGGATGACGCAC and
MsJMT-RNAi(Anti-sense primer)GATTTGTAGAGAGAGACTGGT
a specific fragment of 569bp from 195 th to 764 th of the sequence of the coding region, so that the product contains attB sites at both ends; this fragment was inserted into pENTR-MsJMT vector using BP Clonase in forward and reverse directions, respectively; sequencing verified correctly and ligated into pRNAi vector with LR clone. Checking whether the nucleotide sequence is correct by sequencing again, successfully constructing a pRNAi-MsJMT interference expression vector, and introducing the obtained pRNAi-MsJMT interference expression vector into agrobacterium;
(b) contacting the sterile line cells or tissues of the alfalfa with the agrobacterium-infected cells in the step (a), so that the nucleotide sequences shown as SEQ ID No.3 and SEQ ID No.4 are transferred into the sterile line cells of the alfalfa and are integrated on the chromosome of the sterile line cells of the alfalfa;
(c) and (3) selecting the alfalfa cells or tissues transferred with the nucleotide, and regenerating to obtain alfalfa plants.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN113046377A (en) * | 2021-04-28 | 2021-06-29 | 吉林农业大学 | Male sterile gene MsGAL and application thereof |
| CN116790536A (en) * | 2022-07-13 | 2023-09-22 | 中国科学院昆明植物研究所 | Bud ketomethyltransferases EnEMT1 and EnEMT2 from lithocarpus erythropolis, and genes and applications thereof |
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| CN113046377A (en) * | 2021-04-28 | 2021-06-29 | 吉林农业大学 | Male sterile gene MsGAL and application thereof |
| CN116790536A (en) * | 2022-07-13 | 2023-09-22 | 中国科学院昆明植物研究所 | Bud ketomethyltransferases EnEMT1 and EnEMT2 from lithocarpus erythropolis, and genes and applications thereof |
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