CN107698672A - 一种水稻雄性不育相关蛋白及其编码基因与应用 - Google Patents

一种水稻雄性不育相关蛋白及其编码基因与应用 Download PDF

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CN107698672A
CN107698672A CN201710778224.9A CN201710778224A CN107698672A CN 107698672 A CN107698672 A CN 107698672A CN 201710778224 A CN201710778224 A CN 201710778224A CN 107698672 A CN107698672 A CN 107698672A
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唐益苗
赵昌平
徐磊
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Beijing Academy of Agriculture and Forestry Sciences
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Abstract

本发明涉及基因工程领域，具体地，本发明涉及一种水稻雄性不育相关蛋白及其编码基因与应用。所述基因的核苷酸序列如SEQ ID NO.2所示，在水稻中沉默该基因，显著降低了水稻花粉育性，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水稻，为免去雄杂交水稻做出贡献。

Description

一种水稻雄性不育相关蛋白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，本发明涉及一种水稻雄性不育相关蛋白及 其编码基因与应用。
背景技术
水稻雄性不育的发现与利用为增加水稻产量，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。
目前发现一些控制水稻花器官数目的基因、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。Ole e 1在不同植物 中功能多样性，参与众多的生命活动。Ole e 1花粉蛋白超家族Ole结构域具有较高 的多样性。目前，在植物中已克隆和预测了571个Ole e 1和Ole e 1类花粉蛋白家族 成员。
发明内容
本发明的目的是提供控制水稻性不育的相关蛋白。
本发明再一目的所提供的控制水稻雄性不育相关基因。
本发明的另一目的提供上述水稻雄性不育相关蛋白和基因的应用。
本发明所提供的控制水稻雄性不育相关蛋白，来源于水稻中花11，氨基酸为251个，其氨基酸序列如SEQ ID NO.1所示。
SEQ ID NO.1： MGTRLVPRETAALLGAFVALLAVSFGAVAAPAPLVVGSIKCLDCSPDDVKAEDAFRGLQVGIMCNSGAGEAYETKMLSGLDENGGFSIPLAADLLRDDGELDKDCFAQLHSAPETPCAGQTPPRIAKAGPGNDTIAAAAADAAPTYLAVSDDTLFSPVACKCGKYKKKFMFAPPPPPPPRPPAPEYKPPTPTLTPIPTPEPSYGPPAPKPPAPPVEDEPQPFFHKHPKLKFMHKKKPCPPLVDVDIPRPNN*
根据本发明的控制水稻雄性不育的相关基因可具有如SEQ ID NO.2所示核苷酸序列，OsTMS1开放阅读框(ORF)长度为756bp。
SEQ ID NO.2： ATGGGGACTCGCTTAGTTCCTCGGGAAACTGCAGCTCTTCTCGGTGCTTTCGTTGCGCTTCTGGCCGTCAGCTTCGGCGCCGTGGCGGCGCCGGCGCCGCTCGTGGTTGGCTCCATCAAGTGCTTGGATTGCTCTCCCGACGACGTCAAAGCTGAGGATGCGTTCAGAGGGCTTCAAGTAGGCATCATGTGCAACTCCGGCGCCGGCGAGGCCTACGAGACGAAGATGCTCAGCGGCCTCGACGAGAACGGCGGCTTCAGCATCCCGCTCGCCGCCGACCTCCTCCGCGACGACGGCGAGCTGGACAAGGACTGCTTCGCACAGCTCCACAGCGCGCCGGAGACGCCGTGCGCCGGACAGACCCCGCCCAGGATCGCCAAGGCCGGGCCTGGCAACGACACCATCGCCGCCGCCGCCGCTGACGCCGCGCCGACCTACCTCGCGGTCTCCGACGACACGTTATTCTCTCCCGTCGCGTGCAAGTGCGGCAAGTACAAGAAGAAGTTCATGTTCGCCCCGCCGCCGCCGCCGCCGCCCAGGCCACCGGCCCCGGAGTACAAGCCCCCGACACCGACACTGACTCCGATTCCGACGCCGGAGCCATCGTACGGGCCACCGGCGCCGAAGCCACCAGCTCCGCCGGTGGAGGACGAGCCGCAGCCGTTCTTCCACAAGCACCCGAAGCTCAAGTTCATGCACAAGAAGAAGCCGTGCCCGCCGCTCGTCGACGTGGACATTCCCC GGCCCAACAACTGA
本发明还提供了包含上述控制水稻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水稻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水稻的方法。本发明所提供的培育雄性不育水稻的方法，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 体导入水稻细胞中，得到雄性不育水稻。
根据本发明的具体实施方式，本发明以水稻中花11为实验材料，得到了水稻雄 性不育OsTMS1基因，在水稻中沉默OsTMS1，显著降低了水稻花粉育性，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水稻，为免去雄杂交水稻做出贡献。
附图说明
图1显示了OsTMS1-RNAi转基因植株与对照中花11的对比情况，其中，A： 开花30天后，OsTMS1-RNAi转基因植株与对照的生长情况；B：开花30天后， OsTMS1-RNAi转基因植株与对照的穗子结实情况，C.开花前，OsTMS1-RNAi转基 因植株与对照的花粉碘染情况；D.开花前，OsTMS1-RNAi转基因植株与对照的雄蕊 发育情况。
具体实施方式
实施例1、OsTMS1基因的克隆及序列基序分析
中花11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提取试剂盒完成植物叶片组织的总RNA提取，之后以提取的总RNA 为模板，以OsTMS1-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：
OsTMS1-F:ACTCGCTTAGTTCCTCGGGA
OsTMS1-R:GCAATGCCAGCACAAACAG
本发明得到一个基因OsTMS1，OsTMS1开放阅读框(ORF)长度756bp，编码 251个氨基酸，在NCBI上比对分析，发现该基因是一个编码Ole e 1花粉蛋白基因家 族，证明首次在水稻中克隆OsTMS1基因。
实施例2：OsTMS1-RNAi转基因水稻导致雄性不育
以水稻OsTMS1的cDNA序列为模板，避开其保守结构域，选取OsTMS1基因 301bp左右片段，在其3’端加上一段水稻内含子序列和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，转化水稻中花11 幼胚诱导的愈伤组织，获得TMS1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1所示，显示了OsTMS1-RNAi转基因植株比对照 中花11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，OsTMS1-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，OsTMS1-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，OsTMS1-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，OsTMS1-RNAi转基因植株与对照雄蕊发育情况， 转基因材料雄蕊变小，发现在水稻沉默TMS1，比对照花粉败育率提高在80-100％以 上，导致水稻中花11雄性不育，说明TMS1是花粉发育过程必要基因之一，可用于 培育雄性不育小麦，为免去雄杂交水稻做出贡献。
序列表
<110> 北京市农林科学院
<120> 一种水稻雄性不育相关蛋白及其编码基因与应用
<160> 2
<170> SIPOSequenceListing 1.0
<210> 1
<211> 251
<212> PRT
<213> 小麦(Triticum aestivuml.)
<400> 1
Met Gly Thr Arg Leu Val Pro Arg Glu Thr Ala Ala Leu Leu Gly Ala
1 5 10 15
Phe Val Ala Leu Leu Ala Val Ser Phe Gly Ala Val Ala Ala Pro Ala
20 25 30
Pro Leu Val Val Gly Ser Ile Lys Cys Leu Asp Cys Ser Pro Asp Asp
35 40 45
Val Lys Ala Glu Asp Ala Phe Arg Gly Leu Gln Val Gly Ile Met Cys
50 55 60
Asn Ser Gly Ala Gly Glu Ala Tyr Glu Thr Lys Met Leu Ser Gly Leu
65 70 75 80
Asp Glu Asn Gly Gly Phe Ser Ile Pro Leu Ala Ala Asp Leu Leu Arg
85 90 95
Asp Asp Gly Glu Leu Asp Lys Asp Cys Phe Ala Gln Leu His Ser Ala
100 105 110
Pro Glu Thr Pro Cys Ala Gly Gln Thr Pro Pro Arg Ile Ala Lys Ala
115 120 125
Gly Pro Gly Asn Asp Thr Ile Ala Ala Ala Ala Ala Asp Ala Ala Pro
130 135 140
Thr Tyr Leu Ala Val Ser Asp Asp Thr Leu Phe Ser Pro Val Ala Cys
145 150 155 160
Lys Cys Gly Lys Tyr Lys Lys Lys Phe Met Phe Ala Pro Pro Pro Pro
165 170 175
Pro Pro Pro Arg Pro Pro Ala Pro Glu Tyr Lys Pro Pro Thr Pro Thr
180 185 190
Leu Thr Pro Ile Pro Thr Pro Glu Pro Ser Tyr Gly Pro Pro Ala Pro
195 200 205
Lys Pro Pro Ala Pro Pro Val Glu Asp Glu Pro Gln Pro Phe Phe His
210 215 220
Lys His Pro Lys Leu Lys Phe Met His Lys Lys Lys Pro Cys Pro Pro
225 230 235 240
Leu Val Asp Val Asp Ile Pro Arg Pro Asn Asn
245 250
<210> 2
<211> 756
<212> DNA
<213> 小麦(Triticum aestivuml.)
<400> 2
atggggactc gcttagttcc tcgggaaact gcagctcttc tcggtgcttt cgttgcgctt 60
ctggccgtca gcttcggcgc cgtggcggcg ccggcgccgc tcgtggttgg ctccatcaag 120
tgcttggatt gctctcccga cgacgtcaaa gctgaggatg cgttcagagg gcttcaagta 180
ggcatcatgt gcaactccgg cgccggcgag gcctacgaga cgaagatgct cagcggcctc 240
gacgagaacg gcggcttcag catcccgctc gccgccgacc tcctccgcga cgacggcgag 300
ctggacaagg actgcttcgc acagctccac agcgcgccgg agacgccgtg cgccggacag 360
accccgccca ggatcgccaa ggccgggcct ggcaacgaca ccatcgccgc cgccgccgct 420
gacgccgcgc cgacctacct cgcggtctcc gacgacacgt tattctctcc cgtcgcgtgc 480
aagtgcggca agtacaagaa gaagttcatg ttcgccccgc cgccgccgcc gccgcccagg 540
ccaccggccc cggagtacaa gcccccgaca ccgacactga ctccgattcc gacgccggag 600
ccatcgtacg ggccaccggc gccgaagcca ccagctccgc cggtggagga cgagccgcag 660
ccgttcttcc acaagcaccc gaagctcaag ttcatgcaca agaagaagcc gtgcccgccg 720
ctcgtcgacg tggacattcc ccggcccaac aactga 756

Claims (8)

1.一种水稻雄性不育相关蛋白，其特征在于，所述蛋白的氨基酸序列如SEQ ID NO.1所示。
2.一种水稻雄性不育相关基因，其特征在于，编码权利要求1所述的水稻雄性不育相关蛋白。
3.根据权利要求2所述的水稻雄性不育相关基因，其特征在于，所述基因的核苷酸序列如SEQ ID NO.2所示。
4.包含权利要求2所述的水稻雄性不育相关基因的重组表达载体。
5.包含权利要求2所述的水稻雄性不育相关基因的重组细胞。
6.权利要求1所述的水稻雄性不育相关蛋白的应用。
7.权利要求2所述的水稻雄性不育相关基因的应用。
8.一种培育雄性不育水稻的方法去，其特征在于，所述方法包括将含有权利要求2所述的水稻雄性不育相关基因的沉默片段的重组表达载体导入水稻细胞中的步骤。
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CN108341860A (zh) * 2018-05-11 2018-07-31 北京市农林科学院 控制小麦雄性不育的BURP花粉蛋白TaBURP4B及其基因和应用
CN112521473A (zh) * 2020-12-09 2021-03-19 北京市农林科学院 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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