稻曲病菌T-DNA插入突变体5062的插入位点分析

doi:10.3864/j.issn.0578-1752.2013.09.006

摘要/Abstract

摘要： 【目的】研究稻曲病菌致病力增强的ATMT突变菌株5062，为稻曲病菌致病机制解析、致病相关基因克隆提供方法基础。【方法】测定和观察5062的菌落直径、菌落形态、产孢能力等生物学特性，进一步利用TAIL-PCR和RACE技术克隆5062基因组的T-DNA侧翼基因，并比对分析基因的信息，最后利用RT-PCR检测突变基因的表达量。【结果】与野生型菌株相比，突变菌株5062田间接种表现为致病力增强，在MM和水琼脂培养基上生长速率减慢，产生大量分生孢子，而在PSA和TB3培养基上，其菌落形态、色素等方面与野生型无明显差异。TAIL-PCR扩增得到的紧邻T-DNA两侧的侧翼序列在野生型中不相邻。TAIL-PCR结合RACE技术克隆了5062基因组的T-DNA侧翼基因，RT-PCR表明T-DNA插入位点右侧1个基因在突变体中上调表达。【结论】突变菌株5062的致病力增强，在营养贫乏条件下产孢能力增强，其表型的变化可能是染色体重排和T-DNA插入共同影响的结果。

关键词: 稻曲病菌 , 致病力 , T-DNA , 突变 , 染色体重排

Abstract: 【Objective】 The objective of the experiment is to establish a method for cloning genes of the Ustilaginoidea virens mutant, and to shed light on the pathogenic mechanism of the U. virens, depending on a new mutant 5062, which has increased virulence. 【Method】With the wild type strain 70-22 as a control, phenotypic analysis, such as growth rate, colonial morphologies and sporulation, were analyzed. TAIL-PCR combined with RACE were used to identify the T-DNA integration site and the genes of flanking right site of the T-DNA. The expression levels of the genes were analyzed by RT-PCR as well. 【Result】Colonial morphologies and pigment on PSA and TB3 medium were no different between 70-22 and 5062. But on MM and water-agar medium, 5062 grew slower and produced more conidiophores. The flanking sequences of T-DNA were non-adjacent in the wild type. The expression of the gene flanking right site of the T-DNA was upregulated.【Conclusion】In 5062, both the T-DNA insertion and inversion of chromosomal fragment caused a mutation of the increased virulence.

Key words: Ustilaginoidea virens , virulence , T-DNA , mutant , chromosomal rearrangement

俞咪娜, 胡建坤, 黄磊, 于俊杰, 尹小乐, 聂亚锋, 陈志谊, 刘永锋. 稻曲病菌T-DNA插入突变体5062的插入位点分析[J]. 中国农业科学, 2013, 46(9): 1790-1798.

YU Mi-Na, HU Jian-Kun, HUANG Lei, YU Jun-Jie, YIN Xiao-Le, NIE Ya-Feng, CHEN Zhi-Yi, LIU Yong-Feng. Molecular Characterization of T-DNA Integration of the Ustilaginoidea virens Mutant 5062[J]. Scientia Agricultura Sinica, 2013, 46(9): 1790-1798.

0
/ / 推荐

导出引用管理器 EndNote|Reference Manager|ProCite|BibTeX|RefWorks

链接本文: https://www.chinaagrisci.com/CN/10.3864/j.issn.0578-1752.2013.09.006

https://www.chinaagrisci.com/CN/Y2013/V46/I9/1790

参考文献

[1]杨健源, 曾列先, 陈深, 李传瑛, 韦靖鸾, 谭新球, 朱小源. 我国稻曲病研究进展. 广东农业科学, 2011(2): 77-79.

Yang J Y, Zeng X L, Chen S, Li C Y, Wei J L, Tan X Q, Zhu X Y. Research advance on rice false smut (Ustilaginoidea virens) in China. Guangdong Agricultural Sciences, 2011(2): 77-79.（in Chinese）

[2]李小娟, 刘二明, 肖启明, 刘年喜, 王金辉, 谭小平, 郑和斌. 水稻对稻曲病抗性的分级及相应级别的产量损失. 湖南农业大学学报: 自然科学版, 2011, 37(3): 275-279.

Li X J, Liu E M, Xiao Q M, Liu N X, Wang J H, Tan X P, Zheng H B. Rating of rice resistance against Ustilaginoidea virens and relationship between resistance and yield loss. Journal of Hunan Agricultural University: Natural Sciences, 2011, 37(3): 275-279. (in Chinese)

[3]尹小乐, 陈志谊, 刘永锋, 于俊杰, 李燕, 俞咪娜. 稻曲毒素A的相对含量分析及其与致病力的相关性. 中国农业科学, 2012, 45(22): 4720-4727.

Yin X L, Chen Z Y, Liu Y F, Yu J J, Li Y, Yu M N. Detection of the relative content of ustiloxin A in rice false smut balls and correlation analysis between pathogenicity and ustiloxin A production of Ustilaginoidea virens. Scientia Agricultura Sinica, 2012, 45(22): 4720-4727. (in Chinese)

[4]Li P X, Evans C D, Wu Y Z, Cao B, Hamel E, Joullié M M. Evolution of the total syntheses of ustiloxin natural products and their analogues. Journal of American Chemical Society, 2008, 130(7): 2351-2364.

[5]刘朋娟, 王政逸, 王秋华, 李德葆. 农杆菌介导的稻瘟病菌转化及致病缺陷突变体筛选. 中国水稻科学, 2006, 20(3): 231-237.

Liu P J, Wang Z Y, Wang Q H, Li D B. Agrobacterium tumefaciens- mediated transformation of Magnaporthe grisea and identification of pathogenicity defective mutant. Chinese Journal of Rice Science, 2006, 20(3): 231-237. (in Chinese)

[6]Chen X L, Yang J, Peng Y L. Large-scale insertional mutagenesis in Magnaporthe oryzae by Agrobacterium tumefaciens-mediated transformation. Methods in Molecular Biology, 2011, 722: 213-224.

[7]Maruthachalam K, Klosterman S J, Kang S, Hays R J, Subbarao K V. Identification of pathogenicity-related genes in the vascular wilt fungus Verticillium dahliae by Agrobacterium tumefaciens-mediated T-DNA insertional mutagenesis. Molecular Biotechnology, 2011, 49(3): 209-221.

[8]Chi M H, Park S Y, Kim S, Lee Y H. A novel pathogenicity gene is required in the rice blast fungus to suppress the basal defenses of the host. PLoS Pathogens, 2009, 5(4): e1000401.

[9]Li Y, Yan X, Wang H, Liang S, Ma W B, Fang M Y, Talbot N J, Wang A Y. MoRic8 is a novel component of G-protein signaling during plant infection by the rice blast fungus Magnaporthe oryzae. Molecular Plant Microbe Interactions, 2010, 23(3): 317-331.

[10]Goh J, Jeon J, Kim K S, Park J, Park S Y, Lee Y H. The PEX7-mediated peroxisomal import system is required for fungal development and pathogenicity in Magnaporthe oryzae. PLoS One, 2011, 6(12): e28220.

[11]Mullins E D, Chen X, Romaine P, Raina R, Geiser D M, Kang S. Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 2001, 91(2): 173-180.

[12]张震, 杜新法, 柴荣耀, 毛雪琴, 邱海萍, 王艳丽, 王教瑜, 孙国昌. 根癌农杆菌介导遗传转化稻曲病菌. 中国水稻科学, 2006, 20(4): 440-442.

Zhang Z, Du X F, Chai R Y, Mao X Q, Qiu H P, Wang Y L, Wang J Y, Sun G C. Agrobacterium tumefaciens-mediated transformation of the pathogen of Ustilaginoidea virens. Chinese Journal of Rice Science, 2006, 20(4): 440-442. (in Chinese)

[13]张君成, 陈志谊, 张炳欣, 刘永锋, 陆凡. 稻曲病菌接种技术研究. 植物病理学报, 2004, 34(5): 463-467.

Zhang J C, Chen Z Y, Zhang B X, Liu Y F, Lu F. Study on biology of conidia of Ustilaginoidea virens. Acta Phytopathologica Sinica, 2004, 34(5): 463-467. (in Chinese)

[14]张君成, 张炳欣, 陈志谊, 刘永锋, 陆凡. 稻曲病菌分生孢子的生物学研究. 植物病理学报, 2003, 33(1): 44-47.

Zhang J C, Zhang B X, Chen Z Y, Liu Y F, Lu F. Study on biology of conidia of Ustilaginoidea virens. Acta Phytopathologica Sinica, 2003, 33(1): 44-47. (in Chinese)

[15]顾志敏, 丁正中, 陈析丰, 郭龙彪, 曾大力, 钱前, 马伯军. 实时荧光定量PCR筛选稻曲病菌内参基因. 中国水稻科学, 2012, 26(5): 615-618.

Gu Z M, Ding Z Z, Chen X F, Guo L B, Zeng D L, Qian Q, Ma B J. Reference genes selection of Ustilaginoidea virens by real-time quantitative polymerase chain reaction (PCR). Chinese Journal of Rice Science, 2012, 26(5): 615-618. (in Chinese)

[16]胡东维, 王疏. 稻曲病菌侵染机制研究现状与展望. 中国农业科学, 2012, 45(22): 4604-4611.

Hu D W, Wang S. Progress and perspectives in infection mechanism of Ustilaginoidea virens. Scientia Agricultura Sinica, 2012, 45(22): 4604-4611. (in Chinese)

[17]刘见平, 唐涛, 张松柏, 郑和斌. 稻曲病初侵染源及病菌侵染适期初步研究. 杂交水稻, 2009, 24(1): 74-77.

Liu J P, Tang T, Zhang S B, Zheng H B. Preliminary studies on initial infection sources and pathogen-infecting favorable stage for rice false smut. Hybrid Rice, 2009, 24(1): 74-77. (in Chinese)

[18]Tang Y X, Jin J, Hu D W, Yong M L, Xu Y, He L P. Elucidation of the infection process of Ustilaginoidea virens (teleomorph: Villosiclava virens) in rice spikelets. Plant Pathology, 2013, 62: 1-8.

[19]李燕, 于俊杰, 刘永锋, 尹小乐, 张荣胜, 俞咪娜, 陈志谊. 稻曲病菌产孢能力及致病力测定. 中国农业科学, 2012, 45(20): 4166-4177.

Li Y, Yu J J, Liu Y F, Yin X L, Zhang R S, Yu M N, Chen Z Y. Determination of sporulation and pathogenicity of Ustilaginoidea virens. Scientia Agricultura Sinica, 2012, 45(20): 4166-4177. (in Chinese)

[20]Guo M, Chen Y, Du Y, Dong Y H, Guo W, Zhai S, Zhang H F, Dong S M, Zhang Z G, Wang Y C, Wang P, Zheng X B. The bZIP transcription factor MoAP1 mediates the oxidative stress response and is critical for pathogenicity of the rice blast fungus Magnaporthe oryzae. PLoS Pathogens, 2011, 7(2): e1001302.

[21]刘连盟, 王玲, 黄雯雯, 刘恩勇, 黄世文. 水稻稻曲病菌G蛋白β亚基基因的克隆、表达与序列分析. 中国水稻科学, 2010, 24(4): 353-359.

Liu L M, Wang L, Huang W W, Liu E Y, Huang S W. Cloning, expression and sequence analysis of G protein β subunit gene of rice false smut pathogen Ustilaginoidea virens. Chinese Journal of Rice Science, 2010, 24(4): 353-359. (in Chinese)

[22]张震, 杜新法, 柴荣耀, 王教瑜, 邱海萍, 毛雪琴, 孙国昌. 稻曲病菌PMK1类同源基因克隆及在稻瘟病菌遗传互补中的功能验证. 微生物学报, 2008, 48(11): 1473-1478.

Zhang Z, Du X F, Chai R Y, Wang J Y, Qiu H P, Mao X Q, Sun G C. Cloning of a homologous gene of Magnaporthe grisea PMK1 Ttype MAPK from Ustilaginoidea virens and functional identification by complement in Magnaporthe grisea corresponding mutant. Acta Microbiologica Sinica, 2008, 48(11): 1473-1478. (in Chinese)

[23]Tanaka E, Kumagawa T, Tanaka C, Koga H. Simple transformation of the rice false smut fungus Villosiclava virens by electroporation of intact conidia. Mycoscience, 2011, 52: 344-348.

[24]Zhu Q H, Ramm K, Eamens A L, Dennis E S, Upadhyaya N M. Transgene structures suggest that multiple mechanisms are involved in T-DNA integration in plants. Plant Science, 2006, 171: 308-322.

[25]Müller A E, Atkinson R G, Sandoval R B, Jorgensen R A. Microhomologies between T-DNA ends and target sites often occur in inverted orientation and may be responsible for the high frequency of T-DNA-associated inversions. Plant Cell Reports, 2007, 26: 617-630.

[26]Zhu C F, Wu J H, He C Z. Induction of chromosomal inversion by integration of T-DNA in the rice genome. Journal of Genetics and Genomics, 2010, 37: 189-196.

[27]Li G H, Zhou Z Z, Liu G F, Zheng F C, He C Z. Characterization of T-DNA insertion patterns in the genome of rice blast fungus Magnaporthe oryzae. Current Genetics, 2007, 51: 233-243.