CN113046377B - Male sterile gene MsGAL and application thereof - Google Patents
Male sterile gene MsGAL and application thereof Download PDFInfo
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Abstract
The invention discloses application of a male sterility gene MsGAL and a method for recovering male sterility of alfalfa, belonging to the technical field of bioengineering. The invention provides an application of an MsGAL gene, wherein the MsGAL 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 inhibiting the expression of the MsGAL gene and producing seeds by using the medicago sativa male sterile line; the invention also provides a method for restoring the male sterility of the alfalfa caused by the MsGAL gene deletion, which comprises the steps of constructing an overexpression vector, and transforming the bred male sterile line of the alfalfa by using a genetic transformation means 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 sterility gene MsGAL and application thereof.
Background
Alfalfa (Medicago sativa L.) is the preferred forage grass resource for the development of the forage and livestock industry, and is widely planted worldwide. 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. 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. With the increasing demand of high-quality pasture in various countries, the male sterility mechanism of alfalfa is comprehensively disclosed as early as possible, and the maximum exertion of the heterosis effect is an important task.
Male sterility is a trait which is not beneficial to propagation for plants, but is one of survival modes which are evolved in nature, and the utilization of the trait for carrying out important genetic improvement is a research hotspot about alfalfa breeding at present. In higher plants, pollen development is a complex process during which expression and regulation of a large number of genes is involved. Pollen, as a male gametophyte, contains all the genetic information that completes the fertilization process in conjunction with a female gametophyte, plays an important role in the sexual reproduction process of flowering plants, and any process disruption during pollen development may lead to pollen abortion and the manifestation of male sterility in plants. The occurrence of male sterility of the alfalfa is sometimes empty of specificity, a complex regulation network path of a male sterility related gene of the alfalfa is explored and clarified, and a theoretical basis is provided for better utilizing the male sterility line of the alfalfa and heterosis to culture high-quality hybrid seeds and expand the alfalfa industry in production practice.
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 MsGAL gene and a method for restoring alfalfa male sterility caused by MsGAL gene deletion aiming at the defects and shortcomings in the prior art, utilizes the characteristic that the MsGAL gene and protein thereof participate in regulating alfalfa pollen abortion, utilizes a transgenic technology to control alfalfa male reproductive development, generates a new alfalfa male sterile line by inhibiting the protein, and restores alfalfa male sterility by over-expressing the protein.
The invention is realized by the following technical scheme:
in one aspect, the invention provides a male-sterile gene MsGAL, and the nucleotide sequence of the gene MsGAL is shown in SEQ ID No. 1.
In another aspect, the present invention provides an application of a male sterile MsGAL gene, wherein the application is: obtaining a medicago sativa male sterile line by inhibiting the expression of the MsGAL gene, and producing seeds by using the medicago sativa male sterile line.
Finally, the invention provides a method for restoring male sterility of alfalfa caused by MsGAL gene deletion, which comprises the steps of constructing an overexpression vector containing the MsGAL gene, and transforming the bred alfalfa male sterile line by using a genetic transformation means, so that the fertility and the wild type phenotype of the alfalfa male sterile line can be restored;
preferably, the method comprises the steps of:
transferring agrobacterium subjected to MsGAL complementary construction into the alfalfa male sterile line, and culturing to obtain the alfalfa male sterile line; wherein the MsGAL complementary construction vector contains a nucleotide sequence shown as SEQ ID No. 1;
preferably, the method specifically comprises the following steps:
(a) providing agrobacterium LBA4404 carrying a complementary construction vector for expressing Msgal;
XbaI and SacI enzyme cutting sites are added at two ends of the full length of the MsGAL gene, a specific primer MsGAL-F-pBI/MsGAL-G-pBI is designed, and PCR amplification is carried out by using high-fidelity enzyme;
MsGAL-F-pBI: 5'-cgagctctccacacttcacttggattgca-3' and
MsGAL-R-pBI：5’-gctctagaatggaattacttaaattaattaatatgtctttaactct-3’
carrying out enzyme digestion on a pBI-121 expression vector at 37 ℃ for 3h by using the same double enzymes, connecting the target fragment with a linear long vector with GUS removed, transferring the target fragment into an escherichia coli competent cell to culture on an LB/Kan solid culture medium, selecting a carrier primer M13-F/R for single cloning to carry out PCR detection and sequencing;
M13-F: 5'-cgccagggttttcccagtcacgac-3' and
M13-R：5’-cacacaggaaacagctatgac-3’
expanding propagation of the bacterial liquid in LB/Kan liquid culture solution, successfully constructing a pBI121-MsGAL overexpression vector after correct sequencing, and pouring the obtained pBI121-MsGAL overexpression vector into agrobacterium;
(b) contacting the sterile line cell or tissue of the alfalfa with the agrobacterium-infected cell in the step (a), so that the nucleotide sequence shown as SEQ ID No.1 is transferred into the sterile line cell of the alfalfa and is integrated on the chromosome of the sterile line cell 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, namely the alfalfa beta-galactosidase gene (MsGAL), from the anther of leguminous plants for the first time, obtains a variant of alfalfa male reproductive development by controlling the alfalfa beta-galactosidase MsGAL gene and a coding protein thereof, and realizes the control of alfalfa pollen fertility; the inventive alfalfa male sterile line has no obvious difference in vegetative stage and yield and quality character of the parent, and male reproductive dysplasia is obtained after entering into reproductive growth stage, and pollen abortion plant is obtained.
Drawings
FIG. 1 is a schematic diagram of the construction of pRNAi-MsgAL interference expression vector in example 1 provided by the present invention;
FIG. 2 is a schematic diagram of the pBI121-Msgal overexpression vector construction 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 MsGAL gene cloning for alfalfa fertility control
A specific primer MsGAL-F/R is designed according to the full-length sequence of the MsGAL gene by using the wild alfalfa gong-nong No.1 material, the total RNA of the anther is extracted in sequence, cDNA is synthesized, and the cDNA full length of the MsGAL gene is amplified by PCR.
MsGAL-F: 5'-atggaattacttaaattaattaatatgtct-3' and
MsGAL-R：5’-tcatccacacttcacttggattg-3’
the full length of 2499bp cDNA sequence is identified by sequencing, and the cDNA sequence comprises 832 amino acids and a nucleotide sequence shown as SEQ ID No. 1; the secreted protein encoded by the gene comprises a typical glycoside hydrolase 35 family domain, and a galectin domain is present at the C-terminal.
1.2 reduction of expression level of Msgal Gene in alfalfa by RNAi
In order to inhibit the expression of MsGAL genes, pRNAi-MsGAL interference expression vectors are constructed and wild alfalfa is transformed so as to regulate fertility and obtain alfalfa male sterile lines, a recombinant vector construction map is shown in figure 1, and the vectors comprise nucleotide sequences shown in SEQ ID NO.3 (forward fragment) and SEQ ID NO.4 (reverse fragment).
Based on homologous recombination technology, a conserved fragment 537bp in length is selected from the sequence, a specific primer MsGALI-F/R is designed, and PCR amplification is carried out by adopting high-fidelity enzyme.
Msgali-F: 5'-GGGGACAAGTTTGTACAAAAAAGCAGGCTCAatggaattacttaaattaatt-3' and
MsGALi-R：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAttaaaaggttcattgttaga-3’
constructing RNAi expression vector through BP recombination and LR reaction, picking single colony on LB/Spe resistance plate, shaking bacteria overnight, and performing PCR identification and sequencing verification by using primer RNAi-F/R.
RNAi-F: 5'-actgacgtaagggatgacgcac-3' and
RNAi-R：5’-gatttgtagagagagactggt-3’
after the sequencing is correct, the pRNAi-MsgAL recombinant vector plasmid is transformed into agrobacterium competent cell LBA4404, the resuspended cells are evenly spread on YEP/Spe solid culture medium (containing 20mg/Lrif), and the inverted culture is carried out for 48h at 28 ℃. Selecting a single bacterial colony to a corresponding liquid culture medium for expanding propagation of the bacterial colony, sucking 100 mu L of bacterial colony the next day, taking bacterial colony according to the proportion of 1/50, transferring the bacterial colony to a YEP liquid culture medium (without any antibiotics) for expanding propagation and activation, centrifuging at 4000xg for 10min, collecting thalli, re-suspending the thalli by using an MS liquid culture medium (4.43g/L MS +30g/L sucrose), and adjusting the OD600 value of the bacterial colony to 0.6-0.8, wherein the bacterial colony can be used for genetic transformation of the alfalfa.
Selecting a wild alfalfa aseptic seedling with good growth vigor, cutting leaves into small pieces of 1cm2 with wounds on four sides as explants, oscillating and infecting the small pieces of the alfalfa aseptic seedling in agrobacterium tumefaciens resuspension for 15min, arranging 9 pieces/dish on an MS solid culture medium (MS5194.43g/L + sucrose 30g/L + agar powder 6.8g/L) in a mode that the upper surface of each leaf faces downwards after no residual bacteria liquid exists, and culturing the leaves in a dark place at 28 ℃ for 3 d.
After co-culture, the explants are washed by sterile water (containing 500mg/LCef), transferred to an MS1 culture medium (MS +2, 4-D2 mg/L + KT 0.25mg/L + Kan25mg/L + AHC 2000mg/L) to induce callus growth, cut off when the callus growth reaches 1cm or more, inoculated to an MS2 culture medium (MS + KT 1.5mg/L + Kan25mg/L + AHC 2000mg/L) to perform further differentiation culture, transferred to an MS3 culture medium (1/2MS + Kan25mg/L + AHC 2000mg/L) to induce rooting after about 30 days, and refined and transplanted to a test field for culture after a large number of adventitious roots grow.
Performing molecular detection on the transplanted survival regenerated seedlings, extracting genome DNA of leaves to perform PCR positive identification, performing RT-PCR analysis on the expression level of MsGAL genes in the positive seedlings, and comprehensively selecting effective RNA interference plants.
1.3 deletion of the MsGAL Gene leads to abnormal pollen development of alfalfa
As shown in FIG. 3, compared with the wild-type alfalfa phenotype, the positive plants with suppressed MsGAL gene expression have shrunken pollen grains, small number, small size and deletion (B, C); microscopic examination is carried out after a potassium iodide staining determination method is adopted, and microscopic examination results show that the pollen causes microspore development defects due to tapetum cell abnormality in a tetrad period, and further shows that the pollen grains released are few or can not normally release the pollen grains (A), so that alfalfa pollen abortion is caused, 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 MsGAL 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 male sterility of Medicago sativa due to Msgal gene deletion
2.1 restoration of fertility of New Male sterile line by overexpression of MsGAL Gene
In order to restore the fertility of the new male sterile line, a pBI121-MsGAL overexpression vector is constructed and transferred into the created new alfalfa male sterile line so as to restore the fertility, a recombinant vector construction map is shown in figure 2, and the vector comprises a nucleotide sequence shown in SEQ ID NO. 1.
XbaI and SacI enzyme cutting sites are added at both ends of the full length of the MsGAL gene, a specific primer MsGAL-F-pBI/MsGAL-G-pBI is designed, and PCR amplification is carried out by using high fidelity enzyme.
MsGAL-F-pBI: 5'-cgagctctccacacttcacttggattgca-3' and
MsGAL-R-pBI：5’-gctctagaatggaattacttaaattaattaatatgtctttaactct-3’
the pBI-121 expression vector is digested by the same double enzymes at 37 ℃ for 3h, the target fragment is connected with the linear long vector with GUS removed, the vector is transferred into an escherichia coli competent cell to be cultured on an LB/Kan solid culture medium, and a carrier primer M13-F/R for single cloning is selected for PCR detection and sequencing.
M13-F: 5'-cgccagggttttcccagtcacgac-3' and
M13-R：5’-cacacaggaaacagctatgac-3’
after the bacterial liquid is propagated in LB/Kan liquid culture solution and sequenced correctly, the successfully constructed pBI121-MsGAL overexpression vector is introduced into the created new male sterile line plant by utilizing an agrobacterium-mediated alfalfa genetic transformation system, and the positive identification and pollen fertility observation are carried out through the culture processes of co-culture, screening, differentiation, induced rooting and the like, as shown in figure 4, the number of microspores in a mirror image of a recovered plant obtained by transforming the male sterile line is far more than that of the sterile line, and the fertility determination is carried out by adopting I2-KI solution dyeing in a flowering period, so that the pollen of the alfalfa new male sterile line is changed from yellow brown to blue, namely the fertility of the new male sterile line is recovered, and the phenotype of the alfalfa new male sterile line is basically the same as that of wild alfalfa.
Sequence listing
<110> Jilin agriculture university, Jilin province academy of agriculture science
<120> male sterility gene MsGAL and application thereof
<141> 2021-04-28
<160> 5
<170> SIPOSequenceListing 1.0
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atggaattac ttaaattaat taatatgtct ttaactctca aacttctctc tattaccttc 60
ctttccatca tcagcattgt ttgtgctcaa aataccgcgc aaactattac ttatgatggt 120
cgctcactcc ttcttgatgg aaaacgagaa cttttcttct ccggttccat ccattatcca 180
cgaagcaccc ctgatatgtg gccagatatt cttgataaag caagacgtgg aggactaaac 240
gtaatccaaa catatgtgtt ttggaatggt catgaacctg agaaagacaa ggtgaatttc 300
gaaggccggt atgatttggt aaaattcctt aagcttgtac aagagaaagg aatgtatgtt 360
acccttagaa ttggcccttt catccaagct gaatggaatc acggaggact tccatattgg 420
ctaagagagg tccctgacat catattccgt tctaacaatg aaccttttaa gaaatacatg 480
aaagaatatg tatcaattgt tataaataga atgaaagaag aaaaactctt tgctcctcaa 540
ggaggcccta tcatcttggc tcagattgag aatgagtaca accacatcca acttgcttat 600
gaagctgatg gagataatta tgttcagtgg gctgcaaaaa tggcagtttc actatataat 660
ggagttccat gggtcatgtg caagcagaag gatgctcctg atccagttat taatgcatgc 720
aatggaaggc actgtggtga tacctttaca ggtccaaaca aaccatacaa acctttcata 780
tggactgaaa actggactgc tcagtacaga gtatttggag atccaccatc tcaaagatct 840
gcagaagaca ttgccttctc agttgctcgc ttcttctcta agcatggatc tttagtcaat 900
tactatatgt atcacggtgg aacaaatttt ggtagaacaa cctctgcctt tacaacaaca 960
cgttactacg acgaagctcc tcttgatgag tttggcctac aaagagaacc aaaatggagt 1020
cacctaaggg atgtgcacaa ggctgtcaac ctatgtaaga agcctctact caatggtgag 1080
cctaccacac aaaaaattag ccagtatcat gaggttatag tctatgaaaa gaaaggaagt 1140
aatttatgtg ctgctttcat cactaacaac cacacgcaaa caccaaaaac aataagtttc 1200
agaggttcag actattattt gccaccacgt tccatcagca ttcttcctga ttgcaagact 1260
gtggtcttca acactcaaaa tattgcttca caacacagtt caagacactt tgagaaatca 1320
aagactgcaa acaatttcaa gtgggaggtg ttttctgagc ctataccaag tgccaaggaa 1380
ttgccatcca aacaaaaact tcctgcagaa ctttacagct tgcttaagga caaaactgat 1440
tatggatggt acaccacaag tgtggagttg ggtccagaag acataccaaa gaagactgat 1500
gtagcacccg ttcttcgtat tctgagtctt ggacattcat tgcaggcctt tgtaaacgga 1560
gaatacatcg gatccaaaca tggtagccat gaagagaaag gttttgaatt ccagaaacct 1620
gtaagcttca aggttggagt taaccagata gctatcttgg ctaatttagt aggactacct 1680
gatagtggag catacatgga acacagatat gctggaccta agactataac catccttggt 1740
ttgaattctg gaacaattga cctcacttcc aatggttggg gtcatcaggt tggtctccaa 1800
ggtgagacgg atagcatttt cactgagaag ggatcaaaga aagtagaatg gaaagatggc 1860
aagggaaaag gatcaactct ctcctggtac aagacaaatt ttgatgcacc agaaggaaca 1920
aacccagttg ccatcggaat ggaaggtatg gcaaaaggaa tgatttgggt caatggtgaa 1980
agcattggtc gtcactggat gagctacctg tctcctcttg gaaagcctac tcaatcagag 2040
taccacattc caagatcttt ccttaaacca aaagacaact tgcttgtaat atttgaagaa 2100
gaggcagtaa gtccagacaa gattgctata ctaactgtca acagagatac aatatgcagt 2160
ctcatcacag agaatcaccc tcctaatatt aagtcatttg caagtaagaa ccaaaaactc 2220
gaaatggggg agaatctgac tccagaagct tttatcacgt gtccggacca gaaaaaaatt 2280
acggctgttg agtttgcaag ctttggtgat ccttcaggtt tctgtggaaa ctttactatg 2340
ggaaaatgta atgcaccttc ctccaagaag attgttgagc agctatgctt aggaaaagca 2400
acttgttcgg ttccaatggt caaagcaact ttcaccggtg gcaatgatgg ttgtccagat 2460
gttacgaaga cgcttgcaat ccaagtgaag tgtggatga 2499
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<213> Alfalfa mosaic virus
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Ala Gln Thr Ile Thr Tyr Asp Gly Arg Ser Leu Leu Leu Asp Gly Lys
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Arg Glu Leu Phe Phe Ser Gly Ser Ile His Tyr Pro Arg Ser Thr Pro
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Asp Met Trp Pro Asp Ile Leu Asp Lys Ala Arg Arg Gly Gly Leu Asn
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Val Ile Gln Thr Tyr Val Phe Trp Asn Gly His Glu Pro Glu Lys Asp
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Val Gln Glu Lys Gly Met Tyr Val Thr Leu Arg Ile Gly Pro Phe Ile
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Gln Ala Glu Trp Asn His Gly Gly Leu Pro Tyr Trp Leu Arg Glu Val
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Pro Asp Ile Ile Phe Arg Ser Asn Asn Glu Pro Phe Lys Lys Tyr Met
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Lys Glu Tyr Val Ser Ile Val Ile Asn Arg Met Lys Glu Glu Lys Leu
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Phe Ala Pro Gln Gly Gly Pro Ile Ile Leu Ala Gln Ile Glu Asn Glu
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Tyr Asn His Ile Gln Leu Ala Tyr Glu Ala Asp Gly Asp Asn Tyr Val
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Gln Trp Ala Ala Lys Met Ala Val Ser Leu Tyr Asn Gly Val Pro Trp
210 215 220
Val Met Cys Lys Gln Lys Asp Ala Pro Asp Pro Val Ile Asn Ala Cys
225 230 235 240
Asn Gly Arg His Cys Gly Asp Thr Phe Thr Gly Pro Asn Lys Pro Tyr
245 250 255
Lys Pro Phe Ile Trp Thr Glu Asn Trp Thr Ala Gln Tyr Arg Val Phe
260 265 270
Gly Asp Pro Pro Ser Gln Arg Ser Ala Glu Asp Ile Ala Phe Ser Val
275 280 285
Ala Arg Phe Phe Ser Lys His Gly Ser Leu Val Asn Tyr Tyr Met Tyr
290 295 300
His Gly Gly Thr Asn Phe Gly Arg Thr Thr Ser Ala Phe Thr Thr Thr
305 310 315 320
Arg Tyr Tyr Asp Glu Ala Pro Leu Asp Glu Phe Gly Leu Gln Arg Glu
325 330 335
Pro Lys Trp Ser His Leu Arg Asp Val His Lys Ala Val Asn Leu Cys
340 345 350
Lys Lys Pro Leu Leu Asn Gly Glu Pro Thr Thr Gln Lys Ile Ser Gln
355 360 365
Tyr His Glu Val Ile Val Tyr Glu Lys Lys Gly Ser Asn Leu Cys Ala
370 375 380
Ala Phe Ile Thr Asn Asn His Thr Gln Thr Pro Lys Thr Ile Ser Phe
385 390 395 400
Arg Gly Ser Asp Tyr Tyr Leu Pro Pro Arg Ser Ile Ser Ile Leu Pro
405 410 415
Asp Cys Lys Thr Val Val Phe Asn Thr Gln Asn Ile Ala Ser Gln His
420 425 430
Ser Ser Arg His Phe Glu Lys Ser Lys Thr Ala Asn Asn Phe Lys Trp
435 440 445
Glu Val Phe Ser Glu Pro Ile Pro Ser Ala Lys Glu Leu Pro Ser Lys
450 455 460
Gln Lys Leu Pro Ala Glu Leu Tyr Ser Leu Leu Lys Asp Lys Thr Asp
465 470 475 480
Tyr Gly Trp Tyr Thr Thr Ser Val Glu Leu Gly Pro Glu Asp Ile Pro
485 490 495
Lys Lys Thr Asp Val Ala Pro Val Leu Arg Ile Leu Ser Leu Gly His
500 505 510
Ser Leu Gln Ala Phe Val Asn Gly Glu Tyr Ile Gly Ser Lys His Gly
515 520 525
Ser His Glu Glu Lys Gly Phe Glu Phe Gln Lys Pro Val Ser Phe Lys
530 535 540
Val Gly Val Asn Gln Ile Ala Ile Leu Ala Asn Leu Val Gly Leu Pro
545 550 555 560
Asp Ser Gly Ala Tyr Met Glu His Arg Tyr Ala Gly Pro Lys Thr Ile
565 570 575
Thr Ile Leu Gly Leu Asn Ser Gly Thr Ile Asp Leu Thr Ser Asn Gly
580 585 590
Trp Gly His Gln Val Gly Leu Gln Gly Glu Thr Asp Ser Ile Phe Thr
595 600 605
Glu Lys Gly Ser Lys Lys Val Glu Trp Lys Asp Gly Lys Gly Lys Gly
610 615 620
Ser Thr Leu Ser Trp Tyr Lys Thr Asn Phe Asp Ala Pro Glu Gly Thr
625 630 635 640
Asn Pro Val Ala Ile Gly Met Glu Gly Met Ala Lys Gly Met Ile Trp
645 650 655
Val Asn Gly Glu Ser Ile Gly Arg His Trp Met Ser Tyr Leu Ser Pro
660 665 670
Leu Gly Lys Pro Thr Gln Ser Glu Tyr His Ile Pro Arg Ser Phe Leu
675 680 685
Lys Pro Lys Asp Asn Leu Leu Val Ile Phe Glu Glu Glu Ala Val Ser
690 695 700
Pro Asp Lys Ile Ala Ile Leu Thr Val Asn Arg Asp Thr Ile Cys Ser
705 710 715 720
Leu Ile Thr Glu Asn His Pro Pro Asn Ile Lys Ser Phe Ala Ser Lys
725 730 735
Asn Gln Lys Leu Glu Met Gly Glu Asn Leu Thr Pro Glu Ala Phe Ile
740 745 750
Thr Cys Pro Asp Gln Lys Lys Ile Thr Ala Val Glu Phe Ala Ser Phe
755 760 765
Gly Asp Pro Ser Gly Phe Cys Gly Asn Phe Thr Met Gly Lys Cys Asn
770 775 780
Ala Pro Ser Ser Lys Lys Ile Val Glu Gln Leu Cys Leu Gly Lys Ala
785 790 795 800
Thr Cys Ser Val Pro Met Val Lys Ala Thr Phe Thr Gly Gly Asn Asp
805 810 815
Gly Cys Pro Asp Val Thr Lys Thr Leu Ala Ile Gln Val Lys Cys Gly
820 825 830
<210> 5
<211> 473
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 5
caatggaatt acttaaatta attaatatgt ctttaactct caaacttctc tctattacct 60
tcctttccat catcagcatt gtttgtgctc aaaataccgc gcaaactatt acttatgatg 120
gtcgctcact ccttcttgat ggaaaacgag aacttttctt ctccggttcc atccattatc 180
cacgaagcac ccctgatatg tggccagata ttcttgataa agcaagacgt ggaggactaa 240
acgtaatcca aacatatgtg ttttggaatg gtcatgaacc tgagaaagac aaggtgaatt 300
tcgaaggccg gtatgatttg gtaaaattcc ttaagcttgt acaagagaaa ggaatgtatg 360
ttacccttag aattggccct ttcatccaag ctgaatggaa tcacggagga cttccatatt 420
ggctaagaga ggtccctgac atcatattcc gttctaacaa tgaacctttt aat 473
<210> 6
<211> 473
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
caatggaatt acttaaatta attaatatgt ctttaactct caaacttctc tctattacct 60
tcctttccat catcagcatt gtttgtgctc aaaataccgc gcaaactatt acttatgatg 120
gtcgctcact ccttcttgat ggaaaacgag aacttttctt ctccggttcc atccattatc 180
cacgaagcac ccctgatatg tggccagata ttcttgataa agcaagacgt ggaggactaa 240
acgtaatcca aacatatgtg ttttggaatg gtcatgaacc tgagaaagac aaggtgaatt 300
tcgaaggccg gtatgatttg gtaaaattcc ttaagcttgt acaagagaaa ggaatgtatg 360
ttacccttag aattggccct ttcatccaag ctgaatggaa tcacggagga cttccatatt 420
ggctaagaga ggtccctgac atcatattcc gttctaacaa tgaacctttt aat 473
<210> 6
<211> 473
<212> DNA
<213> Artificial Sequence (Artificial Sequence)
<400> 6
attaaaaggt tcattgttag aacggaatat gatgtcaggg acctctctta gccaatatgg 60
aagtcctccg tgattccatt cagcttggat gaaagggcca attctaaggg taacatacat 120
tcctttctct tgtacaagct taaggaattt taccaaatca taccggcctt cgaaattcac 180
cttgtctttc tcaggttcat gaccattcca aaacacatat gtttggatta cgtttagtcc 240
tccacgtctt gctttatcaa gaatatctgg ccacatatca ggggtgcttc gtggataatg 300
gatggaaccg gagaagaaaa gttctcgttt tccatcaaga aggagtgagc gaccatcata 360
agtaatagtt tgcgcggtat tttgagcaca aacaatgctg atgatggaaa ggaaggtaat 420
agagagaagt ttgagagtta aagacatatt aattaattta agtaattcca ttg 473
Claims (5)
1. A male sterility gene MsGAL is characterized in that the nucleotide sequence of the gene MsGAL is shown as SEQ ID NO. 1.
2. The application of a male sterile MsGAL gene is characterized in that: obtaining a medicago sativa male sterile line by inhibiting the expression of the MsGAL gene, and producing seeds by using the medicago sativa male sterile line.
3. A method for restoring the male sterility of MsGAL alfalfa, characterized in that the male sterile line of alfalfa according to claim 2 is transformed by genetic transformation means by constructing an overexpression vector, so that the line can be restored to fertility and a wild-type phenotype.
4. The method of claim 3, wherein the method comprises the steps of:
transferring agrobacterium subjected to MsGAL complementary construction into the alfalfa male sterile line, and culturing to obtain the alfalfa male sterile line; wherein the MsGAL complementary construction vector contains a nucleotide sequence shown as SEQ ID NO. 1.
5. The method for restoring the male sterility of Msgal alfalfa according to claim 4, comprising the steps of:
(a) providing agrobacterium LBA4404 carrying a complementary construction vector for expressing Msgal;
XbaI and SacI enzyme cutting sites are added at two ends of the full length of the MsGAL gene, a specific primer MsGAL-F-pBI/MsGAL-G-pBI is designed, and PCR amplification is carried out by using high-fidelity enzyme;
MsGAL-F-pBI: 5'-cgagctctccacacttcacttggattgca-3' and
MsGAL-R-pBI：5’-gctctagaatggaattacttaaattaattaatatgtctttaactct-3’
carrying out enzyme digestion on a pBI-121 expression vector for 3 hours at 37 ℃ by using the same double enzymes, connecting a target fragment with a linear long vector with GUS removed, transferring into an escherichia coli competent cell to culture on an LB/Kan solid culture medium, selecting a carrier primer M13-F/R for single cloning, carrying out PCR detection and sequencing;
M13-F: 5'-cgccagggttttcccagtcacgac-3' and
M13-R：5’-cacacaggaaacagctatgac-3’
expanding propagation of the bacterial liquid in LB/Kan liquid culture solution, successfully constructing a pBI121-MsGAL overexpression vector after correct sequencing, and pouring the obtained pBI121-MsGAL overexpression vector into agrobacterium;
(b) contacting the sterile line cell or tissue of the alfalfa with the agrobacterium-infected cell in the step (a), so that the nucleotide sequence shown as SEQ ID NO.1 is transferred into the sterile line cell of the alfalfa and is integrated on the chromosome of the sterile line cell 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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| CN103184218A (en) * | 2005-02-09 | 2013-07-03 | 巴斯福植物科学有限公司 | Expression cassettes for regulation of expression in monocotyledonous plants |
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| 紫花苜蓿雄性不育系的研究;于洪柱等;《黑龙江畜牧兽医》;20130910(第17期);全文 * |
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