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CN112521473B - 一种小麦雄性不育相关蛋白TaMYB97及其编码基因与应用 - Google Patents

一种小麦雄性不育相关蛋白TaMYB97及其编码基因与应用 Download PDF

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CN112521473B
CN112521473B CN202011447765.1A CN202011447765A CN112521473B CN 112521473 B CN112521473 B CN 112521473B CN 202011447765 A CN202011447765 A CN 202011447765A CN 112521473 B CN112521473 B CN 112521473B
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王伟伟
唐益苗
赵昌平
马锦绣
汪德州
郝小聪
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Abstract

本发明涉及基因工程领域，具体地，本发明涉及一种小麦雄性不育相关蛋白TaMYB97及其编码基因与应用。所述蛋白的氨基酸序列如SEQ ID NO.1所示，在小麦中沉默该基因，显著降低了小麦花粉育性，可用于作物杂交育种以及提高产量、加速杂种优势分子育种进程，可用于培育雄性不育小麦，为免去雄杂交小麦做出贡献。

Description

一种小麦雄性不育相关蛋白TaMYB97及其编码基因与应用
技术领域
本发明涉及基因工程领域，具体地，本发明涉及一种小麦雄性不育相关蛋白及TaMYB97其编码基因与应用。
背景技术
小麦雄性不育的发现与利用为增加产量，改进品质，增加抗性和适应性提供优异种源，因而在植物育种上具有重要的应用价值。
目前发现一些控制小麦花器官数目的基因、控制花粉囊细胞的分开和分化的基因、控制雄性减数分裂基因、促进花粉粒发育的关键基因等。研究发现，MYB基因家族在植物基因组中分布较为广泛，通过调控花药花粉发育影响植物育性，利用该类转录因子创制雄性不育系可应用到杂种优势中，因此，研究该类转录因子具有重要的应用价值。拟南芥中的AtMYB21、AtMYB24、AtMYB57通过与茉莉酸甲酯等激素相互作用或调节苯丙烷代谢途径控制木质素的合成进而调控花药开裂。DYT1、TDF1、AMS、MS1以及R2R3-MYB转录因子是拟南芥中一系列调控花粉壁和绒毡层发育的转录因子，敲除这些基因产生的突变体特异性表现出绒毡层或花粉壁发育畸变，DYT1直接调控TDF1的表达从而影响绒毡层的发育和花粉壁的形成。水稻中的OsTDF1与拟南芥中的AtTDF1有着相似的作用，即突变体中间层的降解受到阻碍。ZmMYBC1是第一个在高等植物中被发现的MYB类基因，该基因参与了野生型玉米糊粉中色素的合成。MYB转录因子通过调控绒毡层的形态及其细胞程序性死亡(PCD)、花药开裂和光合产物的运输等影响开花时间、花型花色及花药的发育。小麦中的GAMYB转录因子通过控制GA信号转导影响开花诱导、花粉形成、花粉管伸长等过程。小麦中的TaMYB80是一个花药发育相关转录因子，小麦绒毡层程序性死亡(PCD)发生在四分体后期/小孢子初期，TaMYB80在四分体/小孢子阶段的花药中表达量达到最高，之后小麦绒毡层程序性死亡(PCD)的启动也随之开始。
发明内容
本发明的目的是提供控制小麦雄性不育的相关蛋白。
本发明再一目的所提供的控制小麦雄性不育相关基因。
本发明的另一目的提供上述小麦雄性不育相关蛋白和基因的应用。
本发明所提供的控制小麦雄性不育相关蛋白，来源于小麦中京冬18，氨基酸为400个，其氨基酸序列如SEQ ID NO:1所示。
SEQ ID NO:1：
MATAMDTTMPRSGVGEGGGGGGLKKGPWTQAEDQVLLDHVRRHGEGNWNAVRRETGLQRCGKSCRLRWANHLRPNLRKGPFSPEEERLILRLHGLIGNKWARISTHLPGRTDNEVKNFWNTRLKRRQRAGQSLYPPDVEREIALMRAQNINPFADADGNTVASPFLGPFALPPRPPSSTKQASHSHSHSSPLINQHYQLLNQMQGMQMRHHAVQHHHQQPAFHYHGGIRSPGLPPLPTRPRELPSNQIEMASCSGGGDGLLEALLLGVDEHQLPRPNHAVCRAGSMPDLMYGGVASGSDSDVTSQFPPAPGGQDPHHGGKWDFLVDDMKPTMRRATSAAENETSGMFGVAHGSISGEWFGTGVGSPGPSSVVTTDDEFGLEMQQLMSSLPLSADELNWNA*
根据本发明的控制小麦雄性不育的相关基因可具有如SEQ ID NO:2所示核苷酸序列，TaMYB97开放阅读框(ORF)长度为1203bp。
SEQ ID NO:2：
ATGGCGACGGCGATGGACACGACGATGCCGAGGAGCGGGGTAGGGGAAGGCGGCGGCGGCGGCGGGCTGAAGAAGGGGCCGTGGACGCAGGCGGAGGACCAGGTGCTGCTCGACCACGTGCGGCGGCACGGCGAGGGCAACTGGAACGCCGTGCGCCGGGAGACGGGGCTGCAGCGCTGCGGCAAGAGCTGCCGGCTCCGGTGGGCCAACCATCTCCGCCCCAACCTCCGCAAGGGCCCCTTCTCTCCCGAGGAGGAGCGCCTCATCCTCCGCCTTCACGGCCTCATCGGCAACAAGTGGGCGCGCATCTCAACACACCTGCCCGGGAGGACGGACAACGAGGTCAAGAACTTCTGGAACACGCGCCTCAAGCGCCGGCAGCGCGCCGGCCAGTCGCTCTACCCGCCGGACGTCGAGCGGGAGATCGCCCTCATGCGCGCCCAGAACATCAACCCGTTCGCCGACGCGGACGGCAACACCGTGGCGTCGCCGTTCTTGGGCCCGTTCGCGCTGCCTCCCAGGCCGCCGTCGTCCACCAAACAGGCCTCTCACTCTCATTCTCACTCCTCGCCGCTGATCAACCAGCACTACCAGCTCCTCAACCAGATGCAGGGGATGCAGATGCGCCACCACGCCGTCCAGCACCACCACCAGCAGCCGGCGTTCCACTACCACGGTGGCATCAGGTCGCCGGGGCTCCCGCCGTTGCCGACCAGGCCGCGGGAGCTCCCTTCGAACCAAATCGAGATGGCGAGCTGCAGCGGCGGCGGCGACGGCCTGCTGGAGGCTCTGCTGCTCGGCGTCGACGAACATCAACTCCCCCGTCCAAACCATGCCGTGTGCAGGGCGGGCTCCATGCCCGATCTCATGTACGGCGGCGTCGCGAGCGGGAGCGACAGCGACGTCACCTCGCAGTTCCCTCCCGCTCCCGGAGGCCAGGACCCGCACCATGGCGGGAAATGGGACTTCCTCGTCGATGACATGAAGCCAACAATGAGGAGGGCGACGAGCGCGGCGGAGAACGAGACTTCCGGTATGTTTGGTGTGGCCCATGGGTCAATATCCGGGGAGTGGTTTGGCACCGGTGTTGGCTCACCAGGGCCGTCGTCCGTCGTCACCACGGATGATGAGTTCGGCCTTGAGATGCAGCAGCTCATGTCGTCGTTGCCACTGTCGGCCGACGAGCTTAACTGGAACGCTTAA
本发明还提供了包含上述控制小麦雄性不育相关基因的重组载体。
本发明还提供了包含上述控制小麦雄性不育相关基因的重组细胞。
本发明的另一个目的是提供一种培育雄性不育小麦的方法。本发明所提供的培育雄性不育小麦的方法，是将含有上述雄性不育相关基因的沉默片段的重组表达载体导入小麦细胞中，得到雄性不育小麦。
根据本发明的具体实施方式，本发明以小麦中京冬18为实验材料，得到了小麦雄性不育TaMYB97基因，在小麦中沉默TaMYB97，显著降低了小麦花粉育性，本发明的温敏雄性不育基因相关蛋白及其编码基因可用于作物杂交育种以及提高产量、加速杂种优势分子育种进程，可用于培育雄性不育小麦，为免去雄杂交小麦做出贡献。
附图说明
图1显示了TaMYB97基因在雄蕊中特异表达；
图2显示了TaMYB97-RNAi转基因植株的结实率和花药的育性，其中，A.开花30天后，TaMYB97-RNAi转基因植株与对照生长情况，转基因材料结实率明显下降；B.开花30天后，TaMYB97-RNAi转基因植株与对照穗子，转基因材料结实率明显下降，C.开花前，TaMYB97-RNAi转基因植株与对照花粉碘染情况，转基因材料花粉育性明显下降；D.开花前，TaMYB97-RNAi转基因植株与对照花粉育性和结实率情况，转基因材料败育。
具体实施方式
实施例1：TaMYB97基因的克隆及序列基序分析
京冬18(J18)种植于北京田间，待挑旗以后，分别在花粉母细胞时期(PMC)、二分体(Dyad)、四分体时期(Tetrad)和成熟花粉期(MP)取样，迅速置于液氮冷冻，-80℃保存备用。根据RNA提取试剂盒完成花药组织的总RNA提取，之后以提取的总RNA为模板，以TaMYB97-F/R序列为引物，用反转录酶(M-MLV)进行反转录，得到用于后续实验的cDNA模板。设计一对引物：
TaMYB97-F:ATGGCGACGGCGATGGACACGA
TaMYB97-R:TTAAGCGTTCCAGTTAAGCT
本发明得到一个基因TaMYB97，TaMYB97开放阅读框(ORF)长度1203bp，编码400个氨基酸，在NCBI上比对分析，发现该基因是一个编码MYB转录因子家族，证明首次在小麦中克隆TaMYB97基因。
使用保存的太原806材料cDNA材料进行RT-qPCR，如图1所示，结果表明：TaMYB97基因在雄蕊中高度表达，其他组织中表达量极低。
实施例2TaMYB97-RNAi转基因小麦导致雄性不育
以小麦TaMYB97的cDNA序列为模板，避开其保守结构域，选取TaMYB97基因301bp左右片段，在其3’端加上一段小麦内含子序列和301bp序列的反向互补序列，进行全基因合成。分析该序列酶切位点后分别在其两端加上酶切位点BamHI和SacI，然后对pTCK303比载体和合成片段进行双酶切，再做一次连接将合成的反向互补序列构建到pTCK303载体上。将构建好的载体转化至农杆菌EHA105中，转化小麦J18幼胚诱导的愈伤组织，获得TaMYB97沉默转基因植株。获得T2代转基因株系，经过碘染转基因植株或未转基因植株，图2所示，显示了TaMYB97-RNAi转基因植株比对照J18降低了结实率和花药的育性，但基本不影响植株的形态建成和发育周期，其中，A.开花30天后，TaMYB97-RNAi转基因植株与对照生长情况，转基因材料结实率明显下降；B.开花30天后，TaMYB97-RNAi转基因植株与对照穗子，转基因材料结实率明显下降，C.开花前，TaMYB97-RNAi转基因植株与对照花粉碘染情况，转基因材料花粉育性明显下降；D.开花前，TaMYB97-RNAi转基因植株与对照雄蕊发育情况，转基因材料雄蕊变小，发现在小麦沉默TaMYB97，比对照花粉败育率提高在80-100％以上，导致小麦J18雄性不育，说明TaMYB97是花粉发育过程必要基因之一，可用于培育雄性不育小麦，为免去雄杂交小麦做出贡献。
序列表
<110> 北京市农林科学院
<120> 一种小麦雄性不育相关蛋白TaMYB97及其编码基因与应用
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<213> 小麦(Triticum aestivuml.)
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cgggagctcc cttcgaacca aatcgagatg gcgagctgca gcggcggcgg cgacggcctg 780
ctggaggctc tgctgctcgg cgtcgacgaa catcaactcc cccgtccaaa ccatgccgtg 840
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tgggacttcc tcgtcgatga catgaagcca acaatgagga gggcgacgag cgcggcggag 1020
aacgagactt ccggtatgtt tggtgtggcc catgggtcaa tatccgggga gtggtttggc 1080
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gagatgcagc agctcatgtc gtcgttgcca ctgtcggccg acgagcttaa ctggaacgct 1200
taa 1203

Claims (1)

1.一种培育雄性不育小麦的方法，其特征在于，所述方法包括将含有小麦雄性不育相关基因的沉默片段的重组表达载体导入小麦细胞中的步骤，其中，所述小麦雄性不育相关基因的核苷酸序列如SEQ ID NO：2所示。
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