无毒基因在稻瘟病菌株007中的克隆鉴定及表达分析

doi:10.11924/j.issn.1000-6850.casb20190400047

中国农学通报 ›› 2020, Vol. 36 ›› Issue (18): 124-129.doi: 10.11924/j.issn.1000-6850.casb20190400047

所属专题：生物技术；水稻

无毒基因在稻瘟病菌株007中的克隆鉴定及表达分析

熊田田, 韩豪杰, 卢艳梅, 张宁, 王春台, 刘新琼, 程钢()

中南民族大学生命科学学院,武陵山特色资源植物种质保护与利用湖北省重点实验室,国家民委生物技术重点实验室,武汉 430074

收稿日期:2019-04-25 修回日期:2019-05-24 出版日期:2020-06-25 发布日期:2020-07-10
通讯作者: 程钢
作者简介:熊田田,女,1995年出生,安徽定远人,研究生,硕士,研究方向：分子生物学。通信地址：430074 中南民族大学生命科学学院8栋306A,Tel：027-67842689,E-mail: 13051382878@163.com。
基金资助:
国家自然科学基金委员会资助项目面上项目“水杨酸应答新基因OsAAA-ATPase-1在水稻广谱抗病反应中的分子机理研究”(31370306);湖北省自然科学重点项目“武陵山区稻瘟病菌小种流行演替与抗病育种研究”(2015CFA015);中央高校专项基金项目“恩施地区特色植物农药资源的筛选与鉴定及初步应用”(CZP17080);湖北重点实验室建设基金(2018BFC360)

Avirulence Genes in Magnaporthe oryzae 007: Cloning, Identification and Expression Analysis

Xiong Tiantian, Han Haojie, Lu Yanmei, Zhang Ning, Wang Chuntai, Liu Xingqiong, Cheng Gang()

Hubei Provincial Key Laboratory for Protection and Application of Special Plant Germplasm in Wuling Area of China, Key Lab for Biotechnology of State Ethnic Affairs Commission, College of Life Sciences, South-Central University for Nationalities, Wuhan 430074

Received:2019-04-25 Revised:2019-05-24 Online:2020-06-25 Published:2020-07-10
Contact: Cheng Gang

摘要/Abstract

摘要：

为了能明确稻瘟病国际标准菌株race 007中包含的无毒基因及其可能的主效无毒基因,本研究设计5个常见的无毒基因特异性引物进行克隆鉴定,并分别检测液体培养条件下和侵染水稻过程中race 007菌株中5个无毒基因的表达情况。本研究发现,稻瘟病菌株race 007中含有PWL2、AvrPiz-t、Avr-pik、Avr-pita、ACE1 5个无毒基因,克隆鉴定的结果表明这5个无毒基因的变异不大。相比较于液体培养条件下,在侵染水稻的过程中,PWL2表达上调,ACE1、AvrPiz-t、Avr-Pik表达基本不变,而Avr-Pita基因表达出现了下调。因此PWL2基因表达变化较为显著,本研究进一步对PWL2启动子区域进行分析,结果表明该PWL2基因启动子区域存在着一些顺式作用元件来影响PWL2基因的表达。本研究表明,稻瘟病菌race 007含有5种常见的无毒基因且序列变异不大,而且PWL2基因的表达是受到调控并可能发挥主要的致病作用。

关键词: 稻瘟病菌, 无毒基因, 克隆, 表达量分析

Abstract:

To clarify the avirulence genes contained in the international standard strain Magnaporthe oryzae race 007, and the possible mainly effective avirulence genes, we designed five common avirulence gene-specific primers for cloning and identification. And the expression of five avirulence genes in Magnaporthe oryzae race 007 strain were detected separately in the liquid culture condition and the process of infecting rice. The results showed that Magnaporthe oryzae race 007 strain contained five avirulence genes including PWL2, AvrPiz-t, Avr-pik, Avr-pita and ACE1. The result of cloning and identification indicated that the five avirulent genes did not change much. Compared with that under the liquid culture condition, in the process of infecting rice, the expression of PWL2 was up-regulated, and the expressions of ACE1, AvrPiz-t and Avr-Pik were basically unchanged, while the expression of Avr-Pita gene was down-regulated. Thus, the expression level of PWL2 gene was significantly changed. We further analyzed promoter region sequences of PWL2 gene, and the results showed that there were some cis-acting elements in the promoter region of PWL2 gene to affect the expression of PWL2 gene. This study show that Magnaporthe oryzae race 007 strain contains five common avirulent genes with little sequence variation and the expression of PWL2 gene is regulated and it may play a major pathogenic role in Magnaporthe oryzae race 007.

Key words: Magnaporthe oryzae, avirulence gene, cloning, expression analysis

中图分类号:

S432.1

熊田田, 韩豪杰, 卢艳梅, 张宁, 王春台, 刘新琼, 程钢. 无毒基因在稻瘟病菌株007中的克隆鉴定及表达分析[J]. 中国农学通报, 2020, 36(18): 124-129.

Xiong Tiantian, Han Haojie, Lu Yanmei, Zhang Ning, Wang Chuntai, Liu Xingqiong, Cheng Gang. Avirulence Genes in Magnaporthe oryzae 007: Cloning, Identification and Expression Analysis[J]. Chinese Agricultural Science Bulletin, 2020, 36(18): 124-129.

图/表 7

参考文献 29

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无毒基因在稻瘟病菌株007中的克隆鉴定及表达分析

Avirulence Genes in Magnaporthe oryzae 007: Cloning, Identification and Expression Analysis

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引物名称	引物序列(5’-3’)			Tm值/℃
PWL2	CATCCCTTACTCCGCCACTT			61
PWL2	CTCTTCTCGCTGTTCACGGT			61
AvrPiz-t	GCGACTTTCTTTCTTTGTCTCCT			55
AvrPiz-t	GACAGTGACCATGTTTTATACCG			55
Avr-pik	TCCTGCTGCTAACTCCATTC			60
Avr-pik	TCAACCAAGCGTAAACCTCG			60
Avr-Pita		CGACCCGTTTCCGCCTTTATT	56.5
Avr-Pita		TCCCTCCATTCCAACACTAACG	56.5
ACE1		CCCAGAGTTGGCGATGATGC	64
ACE1		ATGTGGCGGTGACAGAGGAC	64

引物名称	引物序列(5’-3’)	Tm值/℃
actin	ACTCCTGCTTCGAGATCCACATC	61
actin	TCGACGTCCGAAAGGATCTGT	61
PWL2	TCCTCCCTTTTGCTTTGGTC	57
PWL2	CGGATGTTGCCGTTGTTTT	57
AvrPiz-t	GACACTGGGGCACGATAAGG	62
AvrPiz-t	CGGAGGAGAGAACATCAGTGG	62
Avr-pik	GTCGAGAGCGAGACCCTAAC	62
Avr-pik	CAACCTGGAGGGAAGTCGC	62
Avr-Pita	CAAGGAATGTAACAGGGACGC	60
Avr-Pita	CCTGCTGTTTACGGGGTTGT	60
ACE1	TGCGTGACGAGATGTGGAAT	57.8
ACE1	TACGAGTGGCGAACATTGCT	57.8

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