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

[1]	Schneider D R, Saraiva A M, Azzoni A R, et al. Overexpression and purification of PWL2D, a mutant of the effector protein PWL2 from Magnaporthe grisea.[J]. Protein Expression & Purification, 2010,74(1):24-31. doi: 10.1016/j.pep.2010.04.020 URL pmid: 20438845
[2]	梁华兵, 王晓婉, 胡晓岚, 等. 水稻稻瘟病抗病基因Pi63顺式作用元件的载体构建[J]. 中国农学通报, 2015,31(30):220-224.
[3]	Couch B C, Kohn L M. A multilocus gene genealogy concordant with host preference indicates segregation of a new species, Magnaporthe oryzae, from M. grisea[J]. Mycologia, 2002,94(4):683-693. doi: 10.1080/15572536.2003.11833196 URL pmid: 21156541
[4]	He Y, Zhu M, Huang J, et al. Biocontrol potential of a Bacillus subtilis strain BJ-1 against the rice blast fungus Magnaporthe oryzae[J]. Canadian Journal of Plant Pathology, 2019,41(1):47-59. doi: 10.1080/07060661.2018.1564792 URL
[5]	Tang W, Jiang H, Zheng Q, et al. Isopropylmalate isomerase MoLeu1 orchestrates leucine biosynjournal, fungal development, and pathogenicity in Magnaporthe oryzae[J]. Applied Microbiology and Biotechnology, 2018,103(1):327-337. doi: 10.1007/s00253-018-9456-9 URL pmid: 30357439
[6]	Yuemin P, Rui P, Leyong T, et al. Pleiotropic roles of O-mannosyltransferase MoPmt4 in development and pathogenicity of Magnaporthe oryzae[J]. Current Genetics, 2018. doi: 10.1007/s00294-020-01073-z URL pmid: 32249353
[7]	Talbot N J. On the Trail of a Cereal Killer: Exploring the Biology of Magnaporthe grisea[J]. Annual Review of Microbiology, 2003,57(1):177-202. doi: 10.1146/annurev.micro.57.030502.090957 URL
[8]	Jones K, Khang C H. Vacuole Dynamics in Rice Cells Invaded by the Blast Fungus Magnaporthe oryzae[J]. Plant Vacuolar Trafficking, 2018,1789:195-203.
[9]	Shao Z, Li Z, Fu Y, et al. Induction of defense responses against Magnaporthe oryzae in rice seedling by a new potential biocontrol agent Streptomyces JD211.[J]. Journal of Basic Microbiology, 2018,58(8):686-697. doi: 10.1002/jobm.201800100 URL pmid: 29901825
[10]	Gladieux P, Condon A B, Ravel B S, et al. Gene Flow between Divergent Cereal- and Grass-Specific Lineages of the Rice Blast Fungus Magnaporthe oryzae[J]. mBio, 2018,9(1):e01219-17. doi: 10.1128/mBio.01219-17 URL pmid: 29487238
[11]	Liang Y, Zhao J, Wang C, et al. Infection with blast fungus (Magnaporthe orzyae), leads to increased expression of an arabinogalactan-protein epitope in both susceptible and resistant rice cultivars[J]. Physiological and Molecular Plant Pathology, 2018: S088557651730334X.
[12]	Allen R L, Bittner-Eddy P D, Grenville-Briggs L J, et al. Host-parasite coevolutionary conflict between Arabidopsis and downy mildew[J]. Science, 2004,306(5703):1957-1960. doi: 10.1126/science.1104022 URL pmid: 15591208
[13]	Zheng Y, Zheng W, Lin F, et al. AVR1-CO39 is a predominant locus governing the broad avirulence of Magnaporthe oryzae 2539 on cultivated rice (Oryza sativa L.)[J]. Mol Plant Microbe Interact, 2011,24(1):13-17. doi: 10.1094/MPMI-10-09-0240 URL pmid: 20879839
[14]	徐鑫, 郭旭梦, 刘松青, 等. 稻瘟病抗性基因Pi63启动子4个构建体的表达及抗性分析[J]. 中南民族大学学报:自然科学版, 2018,37(2):36-40.
[15]	田红刚. 黑龙江省稻瘟病菌致病性和品种抗性改良研究[D]. 沈阳:沈阳农业大学, 2016.
[16]	Liu J L, Wang X J, Mitchell T, et al. Recent progress and understanding of the molecular mechanisms of the rice-Magnaporthe oryzae interaction[J]. Molecular Plant Pathology, 2010,11(3):419-427. doi: 10.1111/j.1364-3703.2009.00607.x URL pmid: 20447289
[17]	姜华, 余欢, 王艳丽, 等. 稻瘟病菌无毒基因序列变异研究进展[J]. 浙江农业学报, 2015,27(3):512-520.
[18]	Orbach M J, Farrall L, Sweigard J A, et al. A Telomeric Avirulence Gene Determines Efficacy for the Rice Blast Resistance Gene Pi-ta[J]. The Plant Cell, 2000,12(11):2019-2032. doi: 10.1105/tpc.12.11.2019 URL pmid: 11090206
[19]	Heidi U Böhnert, Fudal I, Dioh W, et al. A Putative Polyketide Synthase/Peptide Synthetase from Magnaporthe grisea Signals Pathogen Attack to Resistant Rice[J]. The Plant Cell, 2004,16(9):2499-2513. doi: 10.1105/tpc.104.022715 URL pmid: 15319478
[20]	Li W, Wang B, Wu J, et al. The Magnaporthe oryzae avirulence gene AvrPiz-t encodes a predicted secreted protein that triggers the immunity in rice mediated by the blast resistance gene Piz-t[J]. Mol Plant Microbe Interact, 2009,22(4):411-420. doi: 10.1094/MPMI-22-4-0411 URL pmid: 19271956
[21]	Yoshida K, Saitoh H, Fujisawa S, et al. Association Genetics Reveals Three Novel Avirulence Genes from the Rice Blast Fungal Pathogen Magnaporthe oryzae[J]. The plant cell online, 2009,21(5):1573-1591. doi: 10.1105/tpc.109.066324 URL
[22]	龙子文, 周杰, 王春台, 等. 鄂西地区稻瘟病菌生理小种文库的构建及初步鉴定[J]. 安徽农业科学, 2012,40(24):12065-12067.
[23]	李泌, 王春台, 周杰, 等. 2011年鄂西地区水稻稻瘟病菌遗传多样性的分析[J]. 安徽农业科学, 2014,42(14):4249-4251.
[24]	田珂, 杨武, 李泌, 等. 鄂西南地区2012-2014年稻瘟病菌致病性变化分析[J]. 华中农业大学学报, 2017,36(05):10-14.
[25]	Xin X, Wu Y, Ke T, et al. Genetic diversity and pathogenicity dynamics of Magnaporthe oryzae in the Wuling Mountain area of China[J]. European Journal of Plant Pathology, 2019,153(3):731-742. doi: 10.1007/s10658-018-1587-4 URL
[26]	Song M Y, Kim C Y, Han M, et al. Differential requirement of Oryza sativa RAR1 in immune receptor-mediated resistance of rice to Magnaporthe oryzae[J]. Molecules and Cells, 2013,35(4):327-334. doi: 10.1007/s10059-013-2317-6 URL
[27]	Chang-Jie J, Xiao-Long L, Xin-Qiong L, et al. Stunted Growth Caused by Blast Disease in Rice Seedlings Is Associated with Changes in Phytohormone Signaling Pathways[J]. Frontiers in Plant Science, 2017,8:1558. doi: 10.3389/fpls.2017.01558 URL pmid: 28932234
[28]	Wan J, Wenli Z, Ron A, et al. A method for determining zygosity of transgenic zebrafish by TaqMan real-time PCR[J]. Analytical Biochemistry, 2005,344(2):240-246. doi: 10.1016/j.ab.2005.06.046 URL pmid: 16061191
[29]	Livak . Analysis of relative gene expression data using real-time quantitative PCR and the 2 (-Delta Delta C (T)) Method[J]. Methods, 2001,25(4):402-408. doi: 10.1006/meth.2001.1262 URL pmid: 11846609

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

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

图/表 7

参考文献 29

相关文章 15

编辑推荐

Metrics

本文评价

关于本刊

作者中心

审者中心

在线期刊

联系我们

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

[1]	张宇阳, 周雪, 刘灵艺, 许吴俊, 任旭琴, 王广龙, 熊爱生. 大蒜几丁质酶基因AsCHI1的鉴定及其对盐胁迫的响应[J]. 中国农学通报, 2022, 38(5): 23-29.
[2]	王盛昊, 于冰. 甜菜M14品系BvM14-UNG基因克隆及生物信息学分析[J]. 中国农学通报, 2022, 38(4): 16-22.
[3]	张琼, 王锦霞, 孟诗琪, 钟鑫爱, 刘大丽, 兴旺. 甜菜热激蛋白基因BvHSP18.2的克隆和生物信息学分析[J]. 中国农学通报, 2022, 38(27): 111-118.
[4]	韩旭晨, 董岩, 韩豪杰, 谭艳平, 刘学群, 刘新琼, 徐鑫, 李开, 王春台. 鄂西南地区稻瘟病菌AVR-Pia动态变化研究[J]. 中国农学通报, 2022, 38(21): 112-121.
[5]	王长丽, 廖巍, 叶广彬, 葛菁萍, 刘磊, 马毓坚, 黄霞, 宾晓芸. 编码酿酒酵母丙酮酸脱羧酶(Pdc6)基因克隆及其生物信息学分析[J]. 中国农学通报, 2021, 37(9): 103-108.
[6]	金龙飞, 尹欣幸, 冯美利, 周丽霞, 曹红星. 油棕硼转运通道蛋白基因NIP5的克隆及其在缺硼下的表达分析[J]. 中国农学通报, 2021, 37(9): 22-27.
[7]	黄剑强, 魏雯璐, 钟如卓, 张家炜, 杨创业, 王庆恒. 光裸星虫Sn-hsc70基因克隆及表达分析[J]. 中国农学通报, 2021, 37(9): 95-102.
[8]	刘霞, 张哲, 钱红洁, 张利杰, 杨艳丽. 致病疫霉木瓜蛋白酶亚家族基因C1A-PH-SCH1的克隆与表达分析[J]. 中国农学通报, 2021, 37(29): 13-19.
[9]	梁琪, 付歆崴, 李云鹏, 杨胜甫. 3种植物提取物对稻瘟病菌的抑菌活性及其抑菌机理研究[J]. 中国农学通报, 2021, 37(15): 120-127.
[10]	肖政, 苏家乐, 刘晓青, 孙晓波, 何丽斯, 陈尚平, 周惠民, 李畅. 羊踯躅八氢番茄红素脱氢酶基因的克隆及表达分析[J]. 中国农学通报, 2021, 37(15): 99-105.
[11]	贾惠旭, 李海英, 端木慧子. 甜菜M14品系BvM14-RIN4基因的克隆及应答盐胁迫分析[J]. 中国农学通报, 2021, 37(14): 27-33.
[12]	夏树立, 韩静, 王丽学, 杨春蕾, 刘长悦, 王海亮, 吴贶, 史慧玲. 一种高产L-赖氨酸枯草芽孢杆菌的构建研究[J]. 中国农学通报, 2020, 36(30): 98-105.
[13]	李晓雪, 孙继奇, 王天宇, 田丰收, 张程, 朱晔荣, 向蓓蓓, 王勇. 川西獐牙菜SmGES基因的克隆、生物信息学分析与原核表达[J]. 中国农学通报, 2020, 36(25): 19-25.
[14]	李珊, 杜春梅. 稻瘟病菌与水稻互作研究进展[J]. 中国农学通报, 2020, 36(24): 125-131.
[15]	孙伟, 包向男, 乌云高娃, 王建国, 李永胜, 李善铎, 张铁柱, 王飞飞, 李喜和. 体细胞克隆荷斯坦奶牛出生后死亡犊牛内脏器官的组织病理学观察[J]. 中国农学通报, 2020, 36(20): 114-118.