CN113046377B - 一种雄性不育基因MsGAL及其应用 - Google Patents

一种雄性不育基因MsGAL及其应用 Download PDF

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CN113046377B
CN113046377B CN202110465414.1A CN202110465414A CN113046377B CN 113046377 B CN113046377 B CN 113046377B CN 202110465414 A CN202110465414 A CN 202110465414A CN 113046377 B CN113046377 B CN 113046377B
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徐博
王英哲
闫冬
孟悦
徐安凯
张南翼
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Abstract

本发明公开了一种雄性不育基因MsGAL的应用及恢复紫花苜蓿雄性不育的方法，属于生物工程技术领域。本发明提供了一种MsGAL基因的应用，所述MsGAL基因如SEQ IDNO.1所示的核苷酸序列，所述的应用是：通过抑制MsGAL基因的表达从而获得紫花苜蓿雄性不育株系并用其生产种子；本发明还提供一种恢复MsGAL基因缺失导致紫花苜蓿雄性不育的方法，通过构建过表达载体，利用遗传转化手段转化上述育成的紫花苜蓿雄性不育株系，使其恢复育性及野生型表型；本发明创制的紫花苜蓿雄性不育株系以及提供的恢复该雄性不育株系育性的方法，在杂交紫花苜蓿构建和农业生产上具有十分重要的应用。

Description

一种雄性不育基因MsGAL及其应用
技术领域
本发明涉及一种生物工程技术领域的紫花苜蓿株系创制方法，尤其涉及一种雄性不育基因MsGAL及其应用。
背景技术
紫花苜蓿(Medicago sativa L.)是发展草畜产业首选的牧草资源，在世界范围内广泛种植。作为典型的雌雄同花异花授粉植物，具有自交不亲和性，需采用杂交的方式进行制种。因有着明显的杂种优势，在实际应用中利用这种优势制种主要分为人工去雄杂交制种和利用核质互作雄性不育系杂交制种两条途径，后者不仅可以减少去雄用工，降低制种成本且被证明能有效培育优质苜蓿杂交品种。随着各国对优质牧草需求量的与日俱增，早日全面揭示紫花苜蓿雄性不育的机理，最大发挥其杂种优势效应是重要任务。
雄性不育对植物本身是不利于繁衍的性状，但却是在自然界中进化出的生存方式之一，利用这一性状进行重要遗传改良是目前关于紫花苜蓿育种工作的研究热点。在高等植物中，花粉发育是一个复杂的过程,其间涉及到大量基因的表达和调控。花粉作为雄配子体，包含与雌配子体结合完成受精过程的全部遗传信息,在开花植物的有性生殖过程中发挥着重要的作用，花粉发育期间的任一过程受阻都可能导致植物花粉败育，表现雄性不育性。紫花苜蓿雄性不育的发生有时空特异性，发掘和阐明其雄性不育相关基因的复杂调控网络通路，为生产实践中能更好地利用紫花苜蓿雄性不育系和杂种优势培育优质杂交种扩大紫花苜蓿产业提供理论依据。
紫花苜蓿在育种方面的研究虽一直在进行，但杂交种选育过程中优质雄性不育系与配套保持系材料筛选困难，同时作为四倍体植物，基因组相对复杂、生长周期较长等因素，也限制了其杂交、杂种优势利用在育性遗传基础和遗传模式等相关研究的进度。
发明内容
本发明的目的是针对目前技术上的缺陷和不足，提供一种MsGAL基因的应用及恢复MsGAL基因缺失导致紫花苜蓿雄性不育的方法，利用MsGAL基因及其蛋白参与调控紫花苜蓿花粉败育的特点，以及利用转基因技术控制紫花苜蓿雄性生殖发育，通过抑制该蛋白产生新的紫花苜蓿雄性不育株系，通过过表达该蛋白恢复紫花苜蓿的雄性不育性。
本发明是通过以下技术方案实现的：
一方面，本发明提供了一种雄性不育基因MsGAL，所述基因MsGAL核苷酸序列如SEQIDNO.1所示。
另一方面，本发明还提供了一种雄性不育MsGAL基因的应用，其特征在于，所述的应用是：通过抑制MsGAL基因的表达从而获得紫花苜蓿雄性不育株系，并用所述紫花苜蓿雄性不育株系来生产种子。
最后，本发明提供了一种恢复MsGAL基因缺失导致紫花苜蓿雄性不育的方法，通过构建含有MsGAL基因的过表达载体，利用遗传转化手段转化上述育成的紫花苜蓿雄性不育株系，能够使其恢复育性及野生型表型；
优选地，所述方法包括如下步骤：
将MsGAL互补构建的农杆菌转入所述紫花苜蓿雄性不育系，培育，即得；其中MsGAL互补构建载体中含有编码如SEQ IDNO.1所示的核苷酸序列；
优选地，具体包括以下步骤：
(a)提供携带表达MsGAL互补构建载体的农杆菌LBA4404；
在MsGAL基因全长两端添加XbaI和SacI酶切位点，设计特异性引物MsGAL-F-pBI/MsGAL-G-pBI，用高保真酶进行PCR扩增；
MsGAL-F-pBI：5’-cgagctctccacacttcacttggattgca-3’和
MsGAL-R-pBI：5’-gctctagaatggaattacttaaattaattaatatgtctttaactct-3’
对pBI-121表达载体使用同样的双酶进行37℃酶切3h后，将上述目的片段与去除GUS后的线性长载体相连接，转入大肠杆菌感受态细胞于LB/Kan固体培养基上培养，挑取单克隆用载体引物M13-F/R进行PCR检测并测序；
M13-F：5’-cgccagggttttcccagtcacgac-3’和
M13-R：5’-cacacaggaaacagctatgac-3’
将菌液在LB/Kan液体培养液中扩繁，测序正确后，成功构建pBI121-MsGAL过表达载体，将所得pBI121-MsGAL过表达载体倒入农杆菌；
(b)将紫花苜蓿不育系细胞或组织与步骤(a)中农杆菌感受态细胞接触，从而使编码如SEQ IDNO.1所示的核苷酸序列转入到紫花苜蓿不育系细胞，并且整合到紫花苜蓿不育系细胞的染色体上；
(c)选择转入所述核苷酸的紫花苜蓿细胞或组织，再生，获得紫花苜蓿植株。
本发明的有益效果：
本发明首次从豆科植物的花药中克隆全新基因，即紫花苜蓿β-半乳糖苷酶基因(MsGAL)，通过控制紫花苜蓿β-半乳糖苷酶MsGAL基因及其编码蛋白获得紫花苜蓿雄性生殖发育的变异株，实现控制紫花苜蓿花粉育性；本发明创制的紫花苜蓿雄性不育株系在营养期与来源亲本的产量及品质性状无明显差异，进入生殖生长阶段后雄性生殖发育异常，得到花粉败育植株，同时发明恢复该雄性不育株系育性的操作方式，在杂交紫花苜蓿构建和农业生产上具有十分重要的应用。
附图说明
图1为本发明提供的实施例1中pRNAi-MsGAL干扰表达载体构建示意图；
图2为本发明提供的实施例2中pBI121-MsGAL过表达载体构建示意图；
图3为本发明提供的不育系形态学观察示意图；
图4为本发明提供的恢复育性植株表征镜像图。
具体实施方式
下面结合实施例，进一步阐述本发明。这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验步骤，通常按照常规条件。
实施例1、紫花苜蓿雄性不育株系创制方法
1.1紫花苜蓿育性控制的MsGAL基因克隆
利用野生型紫花苜蓿公农1号材料，根据MsGAL基因的全长序列设计特异性引物MsGAL-F/R，依次进行提取花药总RNA,合成cDNA及PCR扩增MsGAL基因的cDNA全长。
MsGAL-F：5’-atggaattacttaaattaattaatatgtct-3’和
MsGAL-R：5’-tcatccacacttcacttggattg-3’
经测序鉴定cDNA序列全长2499bp，包括832个氨基酸，如SEQ IDNO.1所示的核苷酸序列；该基因编码的分泌型蛋白包含典型的糖苷水解酶35家族结构域，且C端存在一个半乳糖凝集素结构域。
1.2通过RNAi手段降低紫花苜蓿中MsGAL基因的表达水平
为抑制MsGAL基因表达，构建pRNAi-MsGAL干扰表达载体并转化野生型紫花苜蓿，以期调控育性，获得紫花苜蓿雄性不育株系，重组载体构建图谱如图1所示，该载体包含如SEQ IDNO.3(正向片段)和SEQ IDNO.4(反向片段)所示的核苷酸序列。
基于同源重组技术，在序列中选取长537bp的保守片段，设计特异性引物MsGALi-F/R，采用高保真酶进行PCR扩增。
MsGALi-F：5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTCAatggaattacttaaattaatt-3’和
MsGALi-R：5’-GGGGACCACTTTGTACAAGAAAGCTGGGTAttaaaaggttcattgttaga-3’
经BP重组及LR反应构建RNAi表达载体，于LB/Spe抗性平板上挑取单菌落，过夜摇菌后用引物RNAi-F/R进行PCR鉴定及测序验证。
RNAi-F：5’-actgacgtaagggatgacgcac-3’和
RNAi-R：5’-gatttgtagagagagactggt-3’
测序正确后将pRNAi-MsGAL重组载体质粒转化农杆菌感受态细胞LBA4404，重悬菌体匀涂布到YEP/Spe固体培养基(含20mg/Lrif)上，28℃倒置培养48h。挑取单菌落至对应的液体培养基中扩繁菌液，次日吸取100μL菌液，以1/50的比例取菌液转移到YEP液体培养基(不含任何抗生素)中扩繁活化，4000xg离心10min收集菌体，用MS液体培养基(4.43g/L MS+30g/L蔗糖)重悬菌体，调节菌液OD600值达0.6-0.8，此时即可用于紫花苜蓿的遗传转化。
选取长势良好的野生型紫花苜蓿无菌苗，将叶片剪成四边均有伤口的1cm2小块作外植体，于农杆菌重悬液中振荡侵染15min，无残留菌液后按照叶片上表面朝下的方式9片/皿排布于MS固体培养基(MS519 4.43g/L+蔗糖30g/L+琼脂粉6.8g/L)上，28℃暗处共培养3d。
共培养后的外植体用无菌水(含500mg/LCef)冲洗后，转移至MS1培养基(MS+2,4-D2mg/L+KT 0.25mg/L+Kan 25mg/L+AHC 2000mg/L)诱导长出愈伤组织，待其长至1cm及以上时剪下，接种到MS2培养基(MS+KT 1.5mg/L+Kan25mg/L+AHC 2000mg/L)上进一步分化培养，约30d后转至MS3培养基(1/2MS+Kan 25mg/L+AHC 2000mg/L)中诱导生根，长出大量不定根后进行炼苗、移栽至试验田培养。
对移栽成活的再生苗进行分子检测，提取叶片的基因组DNA进行PCR阳性鉴定，RT-PCR分析阳性苗中MsGAL基因的表达水平，综合选取有效RNA干扰植株。
1.3MsGAL基因缺失导致紫花苜蓿花粉发育异常
如图3所示，雄性不育系表型与野生型紫花苜蓿表型相比，抑制MsGAL基因表达后的阳性植株的花粉粒呈干瘪状，数量少、体型小且有缺失(B，C)；采用碘化钾染色测定法后进行镜检，镜检结果表明花粉在四分体时期的绒毡层细胞异常导致小孢子发育缺陷，进而表现释放花粉粒数量少或不能正常释放花粉粒(A)，导致紫花苜蓿花粉败育，创制新的紫花苜蓿雄性不育株系。
1.4上述创制的雄性不育系在紫花苜蓿制种中的用途
将MsGAL不育系作为父本与三系或两系杂交组合中的不育亲本杂交，得到Fl代。在F2代中筛选同时具有雄性不育及不育特征的植株，将该植株与原不育亲本对应的保持系杂交。再次在F2代中筛选同时具有雄性不育及不育特征的植株与保持系杂交，经多代杂交筛选后获得新的雄性不育不育系，适宜作为杂交组合中的母本。
实施例2、恢复MsGAL基因缺失导致紫花苜蓿雄性不育的方法
2.1通过过表达MsGAL基因恢复新雄性不育系的育性
为恢复新雄性不育系的育性，构建pBI121-MsGAL过表达载体并转入上述创制的新紫花苜蓿雄性不育系，以期恢复育性，重组载体构建图谱如图2所示，该载体包含如SEQIDNO.1所示的核苷酸序列。
在MsGAL基因全长两端添加XbaI和SacI酶切位点，设计特异性引物MsGAL-F-pBI/MsGAL-G-pBI，用高保真酶进行PCR扩增。
MsGAL-F-pBI：5’-cgagctctccacacttcacttggattgca-3’和
MsGAL-R-pBI：5’-gctctagaatggaattacttaaattaattaatatgtctttaactct-3’
对pBI-121表达载体使用同样的双酶进行37℃酶切3h后，将上述目的片段与去除GUS后的线性长载体相连接，转入大肠杆菌感受态细胞于LB/Kan固体培养基上培养，挑取单克隆用载体引物M13-F/R进行PCR检测并测序。
M13-F：5’-cgccagggttttcccagtcacgac-3’和
M13-R：5’-cacacaggaaacagctatgac-3’
将菌液在LB/Kan液体培养液中扩繁，测序正确后，利用农杆菌介导的紫花苜蓿遗传转化体系将成功构建的pBI121-MsGAL过表达载体导入上述创制的新雄性不育系植株，通过共培养、筛选、分化及诱导生根等培养过程，进行阳性鉴定及花粉育性观察，如图4所示，由雄性不育系转化得到的恢复植株镜像图内小孢子数远多于所述不育系，且在花期采用I2-KI溶液染色进行育性测定，紫花苜蓿新雄性不育株系的花粉由之前的黄褐色变为蓝色，即恢复了新雄性不育系的育性，同时与野生型紫花苜蓿表型基本无异。
序列表
<110> 吉林农业大学、吉林省农业科学院
<120> 一种雄性不育基因MsGAL及其应用
<141> 2021-04-28
<160> 5
<170> SIPOSequenceListing 1.0
<210> 5
<211> 2499
<212> DNA
<213> 人工序列(Artificial Sequence)
<400> 5
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
<210> 5
<211> 832
<212> PRT
<213> Alfalfa mosaic virus
<400> 5
Met Glu Leu Leu Lys Leu Ile Asn Met Ser Leu Thr Leu Lys Leu Leu
1 5 10 15
Ser Ile Thr Phe Leu Ser Ile Ile Ser Ile Val Cys Ala Gln Asn Thr
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Ala Gln Thr Ile Thr Tyr Asp Gly Arg Ser Leu Leu Leu Asp Gly Lys
35 40 45
Arg Glu Leu Phe Phe Ser Gly Ser Ile His Tyr Pro Arg Ser Thr Pro
50 55 60
Asp Met Trp Pro Asp Ile Leu Asp Lys Ala Arg Arg Gly Gly Leu Asn
65 70 75 80
Val Ile Gln Thr Tyr Val Phe Trp Asn Gly His Glu Pro Glu Lys Asp
85 90 95
Lys Val Asn Phe Glu Gly Arg Tyr Asp Leu Val Lys Phe Leu Lys Leu
100 105 110
Val Gln Glu Lys Gly Met Tyr Val Thr Leu Arg Ile Gly Pro Phe Ile
115 120 125
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
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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
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Lys Lys Thr Asp Val Ala Pro Val Leu Arg Ile Leu Ser Leu Gly His
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Ser Leu Gln Ala Phe Val Asn Gly Glu Tyr Ile Gly Ser Lys His Gly
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Ser His Glu Glu Lys Gly Phe Glu Phe Gln Lys Pro Val Ser Phe Lys
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545 550 555 560
Asp Ser Gly Ala Tyr Met Glu His Arg Tyr Ala Gly Pro Lys Thr Ile
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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
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Ala Pro Ser Ser Lys Lys Ile Val Glu Gln Leu Cys Leu Gly Lys Ala
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Thr Cys Ser Val Pro Met Val Lys Ala Thr Phe Thr Gly Gly Asn Asp
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<210> 5
<211> 473
<212> DNA
<213> 人工序列(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> 人工序列(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> 人工序列(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)

1.一种雄性不育基因MsGAL，其特征在于，所述基因MsGAL核苷酸序列如SEQ ID NO.1所示。
2.一种雄性不育MsGAL基因的应用，其特征在于，所述的应用是：通过抑制MsGAL基因的表达从而获得紫花苜蓿雄性不育株系，并用所述紫花苜蓿雄性不育株系来生产种子。
3.一种恢复MsGAL紫花苜蓿雄性不育的方法，其特征在于，通过构建过表达载体，利用遗传转化手段转化权利要求2中的紫花苜蓿雄性不育株系，能够使其恢复育性及野生型表型。
4.根据权利要求3所述的一种恢复MsGAL紫花苜蓿雄性不育的方法，其特征在于，所述方法包括如下步骤：
将MsGAL互补构建的农杆菌转入所述紫花苜蓿雄性不育系，培育，即得；其中MsGAL互补构建载体中含有编码如SEQ ID NO.1所示的核苷酸序列。
5.根据权利要求4所述的一种恢复MsGAL紫花苜蓿雄性不育的方法，具体包括以下步骤：
(a)提供携带表达MsGAL互补构建载体的农杆菌LBA4404；
在MsGAL基因全长两端添加XbaI和SacI酶切位点，设计特异性引物MsGAL-F-pBI/MsGAL-G-pBI，用高保真酶进行PCR扩增；
MsGAL-F-pBI：5’-cgagctctccacacttcacttggattgca-3’和
MsGAL-R-pBI：5’-gctctagaatggaattacttaaattaattaatatgtctttaactct-3’
对pBI-121表达载体使用同样的双酶进行37℃酶切3h后，将目的片段与去除GUS后的线性长载体相连接，转入大肠杆菌感受态细胞于LB/Kan固体培养基上培养，挑取单克隆用载体引物M13-F/R进行PCR检测并测序；
M13-F：5’-cgccagggttttcccagtcacgac-3’和
M13-R：5’-cacacaggaaacagctatgac-3’
将菌液在LB/Kan液体培养液中扩繁，测序正确后，成功构建pBI121-MsGAL过表达载体，将所得pBI121-MsGAL过表达载体倒入农杆菌；
(b)将紫花苜蓿不育系细胞或组织与步骤(a)中农杆菌感受态细胞接触，从而使编码如SEQ ID NO.1所示的核苷酸序列转入到紫花苜蓿不育系细胞，并且整合到紫花苜蓿不育系细胞的染色体上；
(c)选择转入所述核苷酸的紫花苜蓿细胞或组织，再生，获得紫花苜蓿植株。
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