CN112522305A - 一种抗纹枯病玉米品系育种方法 - Google Patents
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Abstract
本发明公开了一种抗纹枯病玉米品系的育种方法。所公开的方法包括对玉米ZmDREB1A基因进行变异处理得到抗纹枯病玉米品系。发明人发现并鉴定了玉米ZmDREB1A(GRMZM2G124037)基因突变体提高了玉米植株的抗病性，表明ZmDREB1A基因对玉米抵抗R.Solani起着至关重要的作用。
Description
技术领域
本发明涉及ZmDREB1A基因突变体的育种应用，具体涉及通过玉米ZmDREB1A突变体提高玉米抗纹枯病的应用。
背景技术
CBF家族转录因子CBF1，CBF2和CBF3(分别为DREB1B，DREB1C和DREB1A)在调节植物抵抗冷冻胁迫中发挥重要作用，被低温诱导并调节下游抗冷相关基因的表达，以玉米GRMZM2G124037(ZmDREB1A)基因(序列如SEQ ID NO:1所示，其编码的蛋白序列如SEQ IDNO:2所示)为例，其在调节植物抵抗冷冻胁迫中有相关作用。
发明内容
本发明发现并鉴定了玉米GRMZM2G124037基因(ZmDREB1A)突变体可增强玉米抗纹枯病的能力。
基于此，本发明提供了一种抗纹枯病玉米品系的育种方法。所提供的方法包括对玉米ZmDREB1A基因进行变异处理得到抗纹枯病玉米品系。
可选的方案是，所述变异处理包括降低玉米ZmDREB1A基因的表达量。
可选的方案是，所述变异处理包括在玉米ZmDREB1A基因转录起始位点上游-33bp处插入Mutator 3转座子。
具体的，所述Mutator 3转座子插入序列SEQ ID NO:3所示。
具体的，所述的玉米ZmDREB1A基因序列如SEQ ID NO:1、4或5所示。
具体的，将W22自交系玉米ZmDREB1A基因进行变异获得抗纹枯病玉米品系。
附图说明
图1为实施例中玉米zmdreb1a突变体鉴定；(A)ZmDREB1A基因结构及Mutator插入位点，黑色框表示编码序列，白色框表示5’UTR和3’UTR；(B)PCR鉴定NS及zmdreb1a突变体，引物位置在图(A)标注；(C)RT-PCR鉴定ZmDREB1A的mRNA表达水平，ZmGAPDH为内参；(D)Western blot鉴定ZmDREB1A的蛋白表达水平，ZmGAPDH为内参；NS，zmdreb1a自交分离野生型；zmdreb1a，ZmDREB1A突变体；
图2zmdreb1a突变体接种R.Solani表型鉴定；(A)zmdreb1a纯合突变体和NS接种R.Solani 10天后第二片叶表型；(B)zmdreb1a突变体接种R.Solani 10天后第二片叶电导率测定，数据表示均值±标准误(3-10个生物学重复)，*表示NS与zmdreb1a之间差异显著(Student’s T-test，p<0.05)。
具体实施方式
除非另有说明，本文中的术语根据相关领域技术人员的常规认识理解。
以下是发明人提供的具体实施例，以对本发明的技术方案做进一步解释说明。
实施例：
该实施例中发明人获得并鉴定了一个转座子插入zmdreb1a突变体，该突变体抗纹枯病。
该实施例中所用试剂为市售产品；
所用引物序列如下，由生工生物工程(上海)股份有限公司合成；
MU25:CTTCGTCYATAATGRCAATTATCTC；
R2a:TCCACGAGCAACCCTTCC；
F1a:CTGCACAGCATAAATTCTCCAG；
R1a:GGTAGGTGCCGAGCCACAG；
UFMu-07584材料来自Maize Genetics Cooperation Stock Center(http:// www.maizegdb.org/uniformmu)；
zmdreb1a突变系为UFMu-07584自交分离产生的纯合突变株系。
野生型为UFMu-07584自交分离后产生；
R.Solani为玉米纹枯病病原菌立枯丝核菌AG1-IA，菌株为西北农林科技大学申请人实验室保存。
该实施例的方案与相关结果如下：
通过对UFMu-07584自交产生分离的纯合突变株系和自交分离野生型(NS)进行zmdreb1a突变体鉴定(图1)；
使用基因特异性引物和转座子特异性引物(MU25:CTTCGTCYATAATGRCAATTATCTC和R2a:TCCACGAGCAACCCTTCC；F1a:CTGCACAGCATAAATTCTCCAG和R1a:GGTAGGTGCCGAGCCACAG)对自交分离得到的纯合突变株系和野生型进行基因型鉴定；
使用MU25和R2a、F1a和MU25对zmdreb1a纯合突变株系进行PCR扩增并连接到T-载体进行测序，对转座子插入位点进行精细定位，发现转座子(序列如SEQ ID NO:3所示)插入位点位于ZmDREB1A转录起始位点上游-33bp处。
之后，使用基因特异性引物(MU25:CTTCGTCYATAATGRCAATTATCTC和R2a:TCCACGAGCAACCCTTCC；F1a:CTGCACAGCATAAATTCTCCAG和R1a:GGTAGGTGCCGAGCCACAG)对自交分离得到的纯合突变株系和野生型进行基因型鉴定；
其中纯合突变株系的鉴定序列(即zmdreb1a突变体MU25和R2a引物扩增产物)为SEQ ID NO:4所示；野生型的鉴定序列(F1a和R1a引物扩增产物)为SEQ ID NO:5所示序列。
进一步，通过RT-PCR使用基因特异性引物，对上述野生型(NS)和zmdreb1a突变体(zmdreb1a)的ZmDREB1A mRNA水平鉴定，鉴定结果显示zmdreb1a纯合突变体中ZmDREB1A基因mRNA表达水平明显低于野生型(NS)；
其中RT-PCR扩增程序为：95℃预变性5min；95℃变性30s，60℃退火30s，72℃延伸30s，28个循环；72℃终延伸8min；RT-PCR扩增引物：
RT-F:CTCCTCGTCTTCCACCTCCACCTCG；
RT-R:AACCAGTCGGGCTCAAACAC。
另外，通过Western blot对野生型(NS)和zmdreb1a纯合突变体的ZmDREB1A进行蛋白表达水平鉴定，鉴定结果显示zmdreb1a纯合突变体中ZmDREB1A基因蛋白表达水平明显低于野生型(NS)。
其中Western blot步骤参照文献：Gu,L.et al.ZmGOLS2,a target oftranscription factor ZmDREB2A,offers similar protection against abioticstress as ZmDREB2A.Plant Mol Biol 90,157-70(2016).中公开的相关方法，ZmDREB1A和ZmGAPDH一抗为课题组免疫家兔制备，ZmDREB1A使用稀释倍数为1：5000，ZmGAPDH使用稀释倍数为1：200000；二抗(羊抗兔)购于康为公司，使用稀释倍数为1：10000。
并且，发明人对上述zmdreb1a纯合突变体和野生型NS抗病性进行了鉴定：
将zmdreb1a纯合突变体及野生型NS种子在发芽纸上萌发3天，取长势一致发芽种子种植于同一盆中，25℃，16h/8h光周期，培养至两叶一心期后去除长势不一致的幼苗；
幼苗茎基部接种R.Solani，用黄色枪头取直径约4mm活化后的菌块，挑至幼苗茎基部，紧贴茎秆，接种完成后在盆上加塑料盖保湿，25℃，16h/8h光周期培养继续培养并观察，第二片叶出现枯黄坏死表型后拍照并测定电导率。
叶片电导率检测：取适量叶片浸泡于15mL去离子水中，样品抽真空30min，25℃、120rpm处理1h后测定电导，以去离子水为对照，叶片电导减去水电导记录为C1；叶片经沸水浴20min后，冷却至室温后测定电导，此时叶片电导减去水电导记录为C2；(C2-C1)×100％/C2即为叶片相对电导率；电导测定仪型号：雷磁DDS-307。
上述实施例使用的变异手段为在ZmDREB1A转录起始位点上游-33bp处插入序列如SEQ ID NO:3所示转座子，使得纯化突变系中的ZmDREB1A表达量下降，且可抗纹枯病，但适用于本发明的变异不限于该手段，本领域技术人员在本发明公开的基础上，结合公知常识可知，通过基因变异手段使得普通或现有玉米品系或为获得/增强抗纹枯病的玉米品系中的ZmDREB1A基因发生碱基对组成改变、排列顺序改变或表达量下降等现象均适用于本发明。
核苷酸序列表电子文件
<110>西北农林科技大学
<120>一种抗纹枯病玉米品系育种方法
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<220>ZmDREB1A基因核苷酸序列
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ATGGACACGGCCGGCCTCGTCCAGCACGCGACCTCCTCGTCTTCCACCTCCACCTCGGCGTCGTCGTCCTCGTCCTCGTCCGAGCAGCAGAGCAAGGCGGCGTGGCCGCCGTCGCCCGCTTCCTCCCCGCAGCAGCCGCCCAAGAAGCGCCCCGCGGGGCGCACGAAGTTCCGGGAGACGCGGCACCCGGTGTTCCGCGGCGTGCGGCGGCGGGGCGCCGCGGGCCGGTGGGTGTGCGAGGTGCGCGTCCCGGGGAGGCGCGGCGCGCGGCTGTGGCTCGGCACCTACCTCGCCGCCGAGGCGGCGGCGCGCGCGCACGACGCCGCGATGCTCGCCCTGCAGGGCCGCGGCGCGGGGCGCCTCAACTTCCCGGACTCCGCGCGGCTGCTCGCCGTGCCGCCCCCGTCCGCGCTCCCGGGCCTGGACGACGCCCGCCGGGCGGCGCTCGAGGCCGTCGCGGAGTTCCAGCGCCGCTCCGGGGCCGCCGACGAAGCGACCTCGGGCGCGTCTCCTCCCTCCTCGTCGCCGTCGCTGCCGGACGTTTCTGCTGCTGGCTCGCCGGCGGCGGCGCTTGAGCACGTGCCTGTGAAGGCCGACGAAGCAGTGGCGTTGGACTTGGACGGCGATGTGTTTGAGCCCGACTGGTTCGGGGACATGGACCTGGAGTTGGATGCGTACTACGCCAGCCTCGCGGAAGGGTTGCTCGTGGAGCCGCCGCCGCCAGCGGCCGCCTGGGATCATGAAGACTGCTGTGACTCCGGAGCCGCTGACGTCGCGCTCTGGAGCTACTACTAG
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<220>Mutator 3转座子
<400>3
GCTTGGCCATTAGTATCGTGGTCAGTTTTAGTTGTTCTCTTTGTGTCGACTTTGAGATTCGCTATTCTATACTGTTGCTCATTAACAATTTTGACCACAGCAAGAGTACTCAGAGCAGTGGGCCTTGGCCTGTGGGAGATAATTGCCATTATAGAAGAAGAGAGAAGGGGATTCGACGAAATGGAGGCGTTGGCGTTGGCTTCTCTAGTTTGGAGACGCAGACAACAGACAAACTCTAAAATGGATACGAGACAACACTTAGAGCTGCGTAAACAGATATCAGTGTCCTGTCACCGTTTACCGTTCCTGTGTGCAGACGGTGTCTGGCGTACTCTAGACCCGACGACTGGGACCTGGGCCAGCGACTCATGCTAGGCGGCTGCGACCCGCTGCCGCGCCGCCGCTGCCTGGCCCCGGCCTCCAAGCTCTTCCGCCGCCCGCTGCCCGTCAACGAGTCGCTCTGGACGCTGTCCGACGACGGCAACGTCCGGTGGAGCCGCTACCACTGCCGTGGCTACAGGTGCCTGTCCGCCAGGAACCAGCGCCGCGGCTACGACCGCTGCGTGGGGTGCTTCGACATGGACCGCGAGAGGCAGCGGTGGGCCAACCGCACCGCGTCGTCGTCCCTCGCCGACTTCCTCGTCGACGACGTGCTCGCGGCGAAACCAGGCGAGGTGCGCATCGGGCTGGACATGAGCGTGGGCACGGGCAGCTTTGCGGCGCGCATGCGGGAGCGCGGCGTGACCATCGTGTCGGCGGCCATGAACCTGGGGCGCGCCGTTCGCGGAGACAAACCCGCTGCGCGGGCTGGTGCCCCTGTACACGACCATGAGCCAGCGGCTGCCGCTGTTCGACAACACCATGGACCTGGTGCACACGGCGAGGCTCTTCGAGGGGTGGGTGGACCTGCACCTGCTGGACTTCGTGCTCTTCGACTGGGACCGCGTGCTCCCCCGGTGGGCTGCTGTGGGTGGACAAGTTCGCCTGCGCGCGCAAGGACCTGGACGACTACATGTACATGTTCCTGCAGTTCAGGTACAAGAAGCACCGCTGGGCCGTCTCCTTCAAGTCCAAGGACGAGGTCTACCTGCAATGGAGATTTGGATGTGTGTTCTCCCAAATCCAATTTTGTCCTCTTCAGTGAATGTTCCTGAATCAGTAGCCATTTATATATATCATGAAGTGGATTGGGAAGGAGCCGAACGAAGTTGAACTGTTGAATGTCAGTGAATCTGATGATAGAGCAGAAGTTGAACAGCTGGAGGACAACAAAAGAGCAGAGTTTGAGTGCAATTCCGGGGGCAAATCGCAATATCCTGGACTGGGATACTCGTGATCCAAGCTTGGGGGCTTTACATGCAAAAAAAAATCCAGTTCCGCAATATCCTGGACTGGGATACTCGTGAACAGCTGGAGGTCAAGAGATGTGCAGTCCAGATCGATCAGTATTTGCAGATTGGTGCGTCCACACGAGAGTTTACGGCGGCCCAAACCCCCCAAATCCAGTTACACCCCAAATCGAGTTCGAATTCCGCCCCGACGGCAAGCTCCGCTACGCTAACAACTCCAACTACAAGAACGACACCATGATCCGCAAGGAGGTCTTCGTCTCCCCCTCCGTCCTCTGCTAGGCCAGGAGGATCATCCAGGAGTCTGATGTATGTGGGGCGCACCTGTTTTTCTTCCTTGTTCATCAGCAGCAGCAGCGATTGGGTCCTCGGTCCGCGTATCAGCAGCGGCGAAGGAGAAGCGACGGAGACGAGAAGAGTACGCCAGACGGCGTCTGCGCACGGGAACGGTAAACGGTGACAGGACACTGATACCTGTTTACGCAGTCCTAAGTGTTGTCTCGTATCCATTTTAGAGTTTGTCTGTCGTCTGCGTCTCTAAATCAGAGAAGCCAACGCCAACGCCTCCATTTCGTCGAATCCCCTTGTCTCTTCTTCCATAATGGCAATTATCTCGGCCTGTGGAGAAATTCTTAGGGTCTTAACTCACTACAATAGGCCAATATTCAAAGTTGAGCGCCAACATACTGAAGCTGAATGTAGCAGCACATCTGATCAGGCAACATCTAGTGATTCCACAGATAAGAGATCTAATAATTCTCCAGGAAATGAATCTCTCCGCGTGTGAA
<210>4
<211>938
<212>DAN
<213>
<220>纯合突变株系的鉴定序列
<400>4
CTTCGTCYATAATGRCAATTATCTCCTCCGCGTGTGAACTCCAACGTCGCAGGGCATACCTATAAATACACCTCCCACAAAACCACACGCTCCACACAGCTACCACTCAGCTCAAGCTCGAGACAAGAAACCAGAACCAGCTCACTCCTCACTCCACTTCCACTCCCAACAGCAAGCTCAAGCAGTCAGTCACCGGCAGGGGTCAGGGTCACAGTCACAGCAGCAGCCATGGACACGGCCGGCCTCGTCCAGCACGCGACCTCCTCGTCTTCCACCTCCACCTCGGCGTCGTCGTCCTCGTCCTCGTCCGAGCAGCAGAGCAAGGCGGCGTGGCCGCCGTCGCCCGCTTCCTCCCCGCAGCAGCCGCCCAAGAAGCGCCCCGCGGGGCGCACGAAGTTCCGGGAGACGCGGCACCCGGTGTTCCGCGGCGTGCGGCGGCGGGGCGCCGCGGGCCGGTGGGTGTGCGAGGTGCGCGTCCCGGGGAGGCGCGGCGCGCGGCTGTGGCTCGGCACCTACCTCGCCGCCGAGGCGGCGGCGCGCGCGCACGACGCCGCGATGCTCGCCCTGCAGGGCCGCGGCGCGGGGCGCCTCAACTTCCCGGACTCCGCGCGGCTGCTCGCCGTGCCGCCCCCGTCCGCGCTCCCGGGCCTGGACGACGCCCGCCGGGCGGCGCTCGAGGCCGTCGCGGAGTTCCAGCGCCGCTCCGGGGCCGCCGACGAAGCGACCTCGGGCGCGTCTCCTCCCTCCTCGTCGCCGTCGCTGCCGGACGTTTCTGCTGCTGGCTCGCCGGCGGCGGCGCTTGAGCACGTGCCTGTGAAGGCCGACGAAGCAGTGGCGTTGGACTTGGACGGCGATGTGTTTGAGCCCGACTGGTTCGGGGACATGGACCTGGAGTTGGATGCGTACTACGCCAGCCTCGCGGAAGGGTTGCTCGTGGA
<210>5
<211>689
<212>DAN
<213>
<220>野生型的鉴定序列
<400>5
CTGCACAGCATAAATTCTCCAGCCGGCCAGACCCCACGCGGCCCCAGCATCAGATAAAAAAAGCGTCCCAGCAGCTGAAACATATTTTAAGTACCTGGGCTCCCAAAGAATCTACTGGCACCAGCTGTTTCCTTTGCCGCGGCCAGCCGCCCAACCGCCGGCCCGGCGCCTTGTTCCGTTGTTCGTCACCACGGCTTCTCCGCGTGTGAACTCCAACGTCGCAGGGCATACCTATAAATACACCTCCCACAAAACCACACGCTCCACACAGCTACCACTCAGCTCAAGCTCGAGACAAGAAACCAGAACCAGCTCACTCCTCACTCCACTTCCACTCCCAACAGCAAGCTCAAGCAGTCAGTCACCGGCAGGGGTCAGGGTCACAGTCACAGCAGCAGCCATGGACACGGCCGGCCTCGTCCAGCACGCGACCTCCTCGTCTTCCACCTCCACCTCGGCGTCGTCGTCCTCGTCCTCGTCCGAGCAGCAGAGCAAGGCGGCGTGGCCGCCGTCGCCCGCTTCCTCCCCGCAGCAGCCGCCCAAGAAGCGCCCCGCGGGGCGCACGAAGTTCCGGGAGACGCGGCACCCGGTGTTCCGCGGCGTGCGGCGGCGGGGCGCCGCGGGCCGGTGGGTGTGCGAGGTGCGCGTCCCGGGGAGGCGCGGCGCGCGGCTGTGGCTCGGCACCTACC
Claims (6)
1.一种抗纹枯病玉米品系的育种方法，其特征在于，对玉米ZmDREB1A基因进行变异处理得到抗纹枯病玉米品系。
2.如权利要求1所述的抗纹枯病玉米品系的育种方法，其特征在于，所述变异处理包括降低玉米ZmDREB1A基因的表达量。
3.如权利要求2所述的抗纹枯病玉米品系的育种方法，其特征在于，所述变异处理包括在玉米ZmDREB1A基因转录起始位点上游-33bp处插入Mutator 3转座子。
4.如权利要求3所述的抗纹枯病玉米品系的育种方法，其特征在于，所述Mutator 3转座子序列SEQ ID NO:3所示。
5.如权利要求1所述的抗纹枯病玉米品系的育种方法，其特征在于，所述的玉米ZmDREB1A基因序列如SEQ ID NO:1、4或5所示。
6.如权利要求1所述的抗纹枯病玉米品系育种方法，其特征在于，将W22自交系玉米ZmDREB1A基因进行变异处理获得抗纹枯病玉米品系。
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| CN115948366A (zh) * | 2022-11-16 | 2023-04-11 | 西北农林科技大学 | 玉米ZmAGA1基因用于提高植物抗旱性的应用 |
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| US20030140381A1 (en) * | 2001-12-20 | 2003-07-24 | Pioneer Hi-Bred International, Inc. | Genes and regulatory DNA sequences associated with stress-related gene expression in plants and methods of using the same |
| CN1472222A (zh) * | 2002-07-29 | 2004-02-04 | 清华大学 | 玉米dreb类转录因子及其编码基因与应用 |
| CN106232818A (zh) * | 2013-10-25 | 2016-12-14 | 浙江大学 | 改良植株 |
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| US20030140381A1 (en) * | 2001-12-20 | 2003-07-24 | Pioneer Hi-Bred International, Inc. | Genes and regulatory DNA sequences associated with stress-related gene expression in plants and methods of using the same |
| CN1472222A (zh) * | 2002-07-29 | 2004-02-04 | 清华大学 | 玉米dreb类转录因子及其编码基因与应用 |
| CN106232818A (zh) * | 2013-10-25 | 2016-12-14 | 浙江大学 | 改良植株 |
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| FENG QIN ET AL.: "Cloning and Functional Analysis of a Novel DREB1/CBF Transcription Factor Involved in Cold-Responsive Gene Expression in Zea mays L.", 《PLANT CELL PHYSIOL》 * |
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Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN115948366A (zh) * | 2022-11-16 | 2023-04-11 | 西北农林科技大学 | 玉米ZmAGA1基因用于提高植物抗旱性的应用 |
| CN115948366B (zh) * | 2022-11-16 | 2024-04-09 | 西北农林科技大学 | 玉米ZmAGA1基因用于提高植物抗旱性的应用 |
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