CN114196680A - 来源于拟南芥的与抗灰霉病相关的转录因子及应用 - Google Patents
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Abstract
本发明属于植物基因工程技术领域，具体涉及一个来源于拟南芥的转录因子C3H23在抗灰霉病的应用。本发明所提供的C3H23基因来源于拟南芥，其CDS序列如表1所示。在C3H23过表达和突变体植株中，发现突变体感病更明显，而过表达明显促进拟南芥对灰霉病的抗性，证明该基因是一种正调控因子增强植物抗病性。本发明可为提高植物抗灰霉病提供资源，对于作物的遗传改良上具有重要的意义。
Description
技术领域
本发明属于植物基因工程技术领域，具体涉及一个来源于拟南芥的转录因子C3H23在抗灰霉病的应用。
背景技术
灰霉病是一种由灰葡萄孢菌(B.cinerea)引起的真菌性病害，导致200多种农作物发生病变，造成了重要的经济损失。灰葡萄孢菌在穿过宿主表皮后会杀死宿主组织，最终引起宿主组织腐烂。由于灰葡萄孢菌具有多种感染模式和宿主类型等特点，使用较多的施用化学杀菌剂的措施仍不是较为理想的防治措施，为解决这一问题可通过提高作物自身对灰葡萄菌的抗性来降低经济损失。目前，应用基因工程技术育种已成为培育优良作物的重要方法。近年来，随着研究的深入，发现越来越多的转录因子参与植物免疫反应。
CCCH锌指蛋白含有三个串联的半胱氨酸(Cys)和一个组氨酸(His)组成的保守结构域，在植物中广泛存在并在免疫响应中发挥了重要的作用。棉花的ZFP1基因能提高烟草对Rhizoctonia solan的抗性(Guo YH等，New Phytologist,183(1),62-75,2009)。水稻C3H12基因能够激活JA信号通路来提高对细菌Xanthomonas oryzae pv oryzae的抗性(Deng H等,Plant Physiology,158(2),876,2012)。拟南芥中AtC3H14主要依靠WRKY来调节植物对灰葡萄孢的基础防御(Wang D等,Plant Cell Environment,1–15,2020)。以上研究证明CCCH可能在转录水平和转录后水平同时调控了植物抗病，而更多的CCCH家族成员在植物抗病中的作用有待进一步研究。借助拟南芥模式体系，挖掘CCCH家族C3H23对灰霉病的抗性，对于作物的遗传改良上具有重要的意义。
发明内容
本发明的目的是提供一种来源于拟南芥的与抗灰霉病相关的CCCH转录因子的应用。
本发明所提供的与抗灰霉病相关的CCCH转录因子，来源于拟南芥属拟南芥(Arabidopsis thaliana)，名称为C3H23，其DNA序列如SEQ1所示。
本发明提供所述植物转录因子C3H23过表达载体。
本发明提供植物转录因子C3H23蛋白的应用。
本发明提供了一个来源于拟南芥的与抗灰霉病相关的转录因子C3H23的应用。实验证明，将本发明的基因转化拟南芥可以显著提高拟南芥自身对灰霉病的抗性，因而本发明的基因及其蛋白对植物抗灰霉病的研究具有重要的应用前景。
附图说明
图1 c3h23突变体和野生型植株在接种灰葡萄孢菌后表现的发病症状。
图2 C3H23过表达和野生型植株在接种灰葡萄孢菌后表现的发病症状。
具体实施方式
下述实施例中所用方法如无特别说明均为常规方法。
实施案例1拟南芥C3H23基因的克隆及过表达植株的获得
1)在tair网站上下载拟南芥C3H23的cDNA序列，分析其特异性后设计引物在拟南芥的总cDNA中扩增该片段；
2)回收纯化扩增的片段后将其构建在过表达载体上，测序成功后提取质粒转入农杆菌；
3)使用农杆菌介导拟南芥花序的侵染方法，并在抗性板上筛选获得转基因阳性植株，提取转基因植株RNA后反转录，使用qRT-PCR鉴定得到表达量较高的转基因植株；
实施案例2拟南芥c3h23突变体植株的获得
对购买的c3h23的突变体SALK-024606和SALK-112158长到四周后，提取植株DNA后，PCR扩增后使用琼脂糖凝胶电泳鉴定得到纯和突变体。
实施案例3C3H23基因在灰葡萄孢菌致病性方面的作用
1)取B.cinerea菌株培养在PDA培养基上，生长10天后收取成熟孢子。
2)用液体PDA悬起孢子后调整孢子浓度为5×105CFU/mL，在25℃摇床100rpm活化2h，选择4周生长良好的拟南芥野生型、突变体和过表达植株，将6μL孢子悬浊液滴到每株同一位置的三片叶片上，并在潮湿环境中进行培养。
3)2天后进行表型观察统计病斑面积。结果如图1和图2所示，相较于野生型，接病后c3h23突变体SALK-024606和SALK-112158都表现出更大的病斑面积，而过表达植株的病斑面积显著减少，表明基因C3H23具有增加灰霉病抗性的能力。
序列表
<110> 青岛农业大学
<120> 来源于拟南芥的与抗灰霉病相关的转录因子及应用
<160> 1
<170> SIPOSequenceListing 1.0
<210> 1
<211> 948
<212> DNA
<213> 拟南芥(Arabidopsis thaliana)
<400> 1
atgatgatcg gcgaaaataa aaaccggcca catccaacga tccatatccc tcaatgggat 60
caaatcaacg atccaacggc cacaatctct tcaccattct cttccgtcaa ccttaacagc 120
gttaacgact acccacactc tccgtcaccg tatctcgact ccttcgcttc tctcttccgt 180
tacctcccgt caaacgagtt aacaaacgat tcagactcat caagtggcga cgagtcatca 240
ccactcaccg actcattctc ctccgacgag tttcgcatct acgagttcaa aatccggcga 300
tgcgctcgag gtcgatctca tgattggacg gagtgtccgt tcgcacatcc cggagaaaaa 360
gctcgacgac gtgatccgag aaagtttcat tactccggca ccgcttgtcc tgagtttcgt 420
aaaggaagtt gtagaagagg tgattcgtgt gagttctctc atggagtttt cgagtgttgg 480
ctccatcctt ctcgttaccg tactcagccg tgtaaagacg gaactagctg ccggagaaga 540
atctgtttct tcgctcatac gacggagcag ttacgtgtat taccttgttc gttagatcca 600
gatcttggat tcttctcagg attagctact tctccgactt cgattcttgt ttctccttcg 660
ttttcaccac cgtcggaatc tccgccgctt tctccgagta ccggtgaact tattgcgtcg 720
atgaggaaaa tgcaattgaa cggaggtggt tgttcgtgga gttctccgat gagatctgca 780
gttaggttac ctttttcgtc gtctctgcgt ccgattcagg cggcaacgtg gccgaggata 840
agagagtttg agatcgaaga agctccggcg atggaatttg tggaatctgg gaaagagctg 900
agagcggaga tgtatgcaag actcagtaga gagaactcac tcggttga 948
Claims (2)
1.含有序列SEQ1所述核苷酸序列的基因过表达载体。
2.权利要求1所述的基因过表达载体的应用，其特征在于将权利要求1的载体转入拟南芥，转化该载体后提高拟南芥自身对灰霉病的抗性。
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Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130227746A1 (en) * | 2010-11-11 | 2013-08-29 | Purdue Research Foundation | Methods and compositions to regulate plant transformation susceptibility |
| CN103468712A (zh) * | 2013-08-20 | 2013-12-25 | 华中农业大学 | GhTZF1基因在增强植物抗旱性及延缓衰老中的应用 |
| US20200123562A1 (en) * | 2018-10-19 | 2020-04-23 | Pioneer Hi-Bred International, Inc. | Compositions and methods for improving yield in plants |
| CN112501181A (zh) * | 2020-12-04 | 2021-03-16 | 福建省亚热带植物研究所 | 水稻抗逆相关基因OsTZF7及其编码蛋白与应用 |
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Patent Citations (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US20130227746A1 (en) * | 2010-11-11 | 2013-08-29 | Purdue Research Foundation | Methods and compositions to regulate plant transformation susceptibility |
| CN103468712A (zh) * | 2013-08-20 | 2013-12-25 | 华中农业大学 | GhTZF1基因在增强植物抗旱性及延缓衰老中的应用 |
| US20200123562A1 (en) * | 2018-10-19 | 2020-04-23 | Pioneer Hi-Bred International, Inc. | Compositions and methods for improving yield in plants |
| CN112501181A (zh) * | 2020-12-04 | 2021-03-16 | 福建省亚热带植物研究所 | 水稻抗逆相关基因OsTZF7及其编码蛋白与应用 |
Non-Patent Citations (2)
| Title |
|---|
| DIAN WANG等: "The Arabidopsis CCCH protein C3H14 contributes to basal defense against Botrytis cinerea mainly through the WRKY33-dependent pathway", 《PLANT CELL ENVIRON》 * |
| LAM,B.等: "Arabidopsis thaliana putative CCCH-type zinc finger protein (At2g25900; F17H15.7) mRNA, complete cds, GenBank: AF370590.1", 《GENBANK数据库》 * |
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