稻曲病菌致病力丧失突变菌株B-1015的T-DNA标记基因的克隆

doi:10.3864/j.issn.0578-1752.2014.13.007

摘要/Abstract

摘要： 【目的】研究稻曲病菌（Ustilaginoidea virens）致病力丧失突变菌株B-1015，克隆和分析T-DNA插入位点的侧翼序列，为稻曲病菌的致病机制研究提供参考。【方法】分析稻曲病菌T-DNA插入突变体B-1015菌株的生长速率、产孢量、孢子萌发率以及致病力等生物学表型。用PCR检测T-DNA在B-1015基因组中插入的稳定性，用Southern杂交检测T-DNA插入的拷贝数，用hiTail-PCR扩增T-DNA侧翼序列，用RACE PCR克隆T-DNA标记基因，半定量RT-PCR分析所克隆基因的表达情况。【结果】与野生菌株P1相比，突变菌株B-1015的生长速率和产孢量都有较明显的降低，孢子萌发率无明显差异，致病力丧失。分子检测结果表明T-DNA在B-1015基因组中为稳定的单拷贝插入。hiTail-PCR得到的T-DNA两端侧翼序列在野生型菌株基因组上不相连，RACE PCR在两边的侧翼序列上分别克隆得到Uvt-1015R和Uvt-1015L。预测分析表明Uvt-1015R包含一个长度为948 bp的开放阅读框（open reading frame finder，ORF），可编码295个氨基酸，5′端非编码区（5′-untranslated regions，5′-UTR）长度为 341 bp，3′端非编码区（3′-untranslated regions，3′-UTR）长度为271 bp。Uvt-1015L包含一个长度为351 bp的ORF，可编码116个氨基酸，5′-UTR为31 bp，3′-UTR长度为174 bp。在已知功能的蛋白中检索，没有发现与Uvt-1015R和Uvt-1015L基因同源的蛋白。序列分析发现T-DNA插入在两个基因序列中间，RT-PCR结果显示，在突变菌株B-1015中，Uvt-1015R表达量下降，Uvt-1015L不表达。【结论】稻曲病菌突变菌株B-1015致病力等重要生物学表型发生改变，可能是由于T-DNA的插入导致了B-1015基因组重排和Uvt-1015R、Uvt-1015L基因结构的破坏。

关键词: 稻曲病菌 , 致病力 , T-DNA , 基因克隆

Abstract: 【Objective】 The objective of this study is to analyze the biological phenotypes of the pathogenicity-defective mutant strain B-1015, to clone its T-DNA integration flanking sequences, and finally to reveal the functions of targeted genes in pathogenesis of Ustilaginoidea virens. The study will contribute to better understanding of the molecular pathogenesis of U. virens. 【Method】 Biological phenotypes of B-1015 were analyzed by testing growth rate, sporulation ability, spore germination rate and pathogenicity. The copy numbers of T-DNA inserted into B-1015 were identified by Southern blot. The flanking sequences of T-DNA were cloned by hiTail-PCR and the whole gene sequences of the T-DNA targeted genes were cloned by RACE PCR. The genes expression were detected by RT-PCR. 【Result】By comparing with wild type strain P1, the sporulation ability and growth rate of the B-1015 were significantly declined, but the spore germination rate had no significant difference. The inserted mutant lost its pathogenicity in rice. Molecular detection analysis confirmed that the B-1015 was a stable single T-DNA insertional mutant. The T-DNA flanking sequences were obtained by hiTail-PCR. Two genes, Uvt-1015R and Uvt-1015L, were cloned by RACE PCR. Gene sequence analysis showed that Uvt-1015R contained a 948 bp open reading frame, which could encode a 295 amino acid protein, a 341 bp 5′-UTR and a 271 bp 3′-UTR. Another gene, Uvt-1015L, comprised a 351 bp open reading frame coding a 116 amino acid protein, a 31 bp 5′-UTR and a 174 bp 3′-UTR. By searching in the known protein, the Uvt-1015R and Uvt-1015L genes showed no homology of any protein. Sequence alignment showed that the flanking sequences of T-DNA were non-adjacent in the wild type genome and the T-DNA disrupted the two genes sequence. RT-PCR analysis confirmed that compared to P1 strain, the transcripts of Uvt-1015R was significantly decreased, and the transcripts of Uvt-1015L was not detected any more. 【Conclusion】 In B-1015, both the T-DNA insertion and chromosomal rearrangement caused important biological phenotype mutant, thus leading to the loss of pathogenicity.

Key words: Ustilaginoidea virens , pathogenicity , T-DNA , gene clone

黄磊1, 2, 胡建坤2, 俞咪娜2, 于俊杰2, 王亚会2, 郑梦婷2, 郑睿2, 刘永锋1, 2. 稻曲病菌致病力丧失突变菌株B-1015的T-DNA标记基因的克隆[J]. 中国农业科学, 2014, 47(13): 2552-2562.

HUANG Lei-1, 2 , HU Jian-Kun-2, YU Mi-Na-2, YU Jun-Jie-2, WANG Ya-Hui-2, ZHENG Meng-Ting-2, ZHENG Rui-2, LIU Yong-Feng-1, 2 . Molecular Cloning of T-DNA Targeted Genes of Ustilaginoidea virens Pathogenicity-Defective Mutant Strain B-1015[J]. Scientia Agricultura Sinica, 2014, 47(13): 2552-2562.

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参考文献

[1]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)

[2]田鸿, 陶家凤. 水稻稻曲病菌厚垣孢子萌发特性及稻曲病菌毒素对水稻、玉米、小麦种子萌发的影响. 西南农业学报, 2000, 13(3): 113-116.

Tian H, Tao J F. The germinating characters of falsesmut chalmydospores and the influence of toxin produced by Ustilaginoidea virens on the growth of radicle and plumule of rice, tritici and madyis. Southwest China Journal of Agricultural Science, 2000, 13(3): 113-116. (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]De Groot M A, Bundock P, Hooykaas P J, Beijersbergen A G. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology, 1998, 16: 839-842.

[5]Jiang D, Zhu W, Wang Y, Sun C, Zhang K Q, Yang J K. Molecular tools for functional genomics in filamentous fungi: Recent advances and new strategies. Biotechnology Advances, 2013, 31(8): 1562-1574.

[6]Combier J P, Melayah D, Raffier C, Gilles G, Marmeisse R. Agrobacterium tumefaciens-mediated transformation as a tool for insertional mutagenesis in the symbiotic ectomycorrhizal fungus Hebeloma cylindrosporum. FEMS Microbiology Letters, 2003, 220(1): 141-148.

[7]Zwiers L H, De Waard M A. Efficient Agrobacterium tumefaciens- mediated gene disruption in the phytopathogen Mycosphaerella graminicola. Current Genetics, 2001, 39(5/6): 388-393.

[8]Sugui J A, Chang Y C, Kwon-Chung K J. Agrobacterium tumefaciens-mediated transformation of Aspergillus fumigatus: an efficient tool for insertional mutagenesis and targeted gene disruption. Applied and Environmental Microbiology, 2005, 71(4): 1798-1802.

[9]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.

[10]Nakamura M, Kuwahara H, Onoyama K, Iwai H. Agrobacterium tumefaciens-mediated transformation for investigating pathogenicity genes of the phytopathogenic fungus Colletorichum sansevieriae. Current Microbiology, 2012, 65: 176-182.

[11]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)

[12]张君成, 陈志谊, 张炳欣, 刘永锋, 陆凡. 稻曲病的接种技术研究. 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. Inoculation techniques used for inducing rice false smut efficiently. Acta Phytopathologica Sinica, 2004, 34(5): 463-467. (in Chinese)

[13]周永力, 樊金娟, 曾超珍, 刘小舟, 王疏, 赵开军. 稻曲病菌遗传多样性与群体结构的初步分析. 植物病理学报, 2004, 34(5): 442-448.

Zhou Y L, Fan J J, Zeng C Z, Liu X Z, Wang S, Zhao K J. Preliminary analysis of genetic diversity and population structure of Ustilaginoidea virens. Acta Phytopathologica Sinica, 2004, 35(5): 442-448. (in Chinese)

[14]陈莉, 许娟, 丁克坚, 李勋卓, 叶正和. 药剂对稻曲病菌的毒力测定及复配研究. 植物保护, 2012, 38(3): 159-161.

Chen L, Xu J, Ding K J, Li X Z, Ye Z H. Toxicity test of fungicides to Ustilaginoidea virens and mixed formulation. Plant Protection, 2012, 38(3): 159-161. (in Chinses)

[15]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)

[16]张震, 杜新法, 柴荣耀, 邱海萍, 王教瑜, 毛学琴, 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, Qiu H P, Wang J Y, Mao X Q, Sun G C. Cloning of a homologous gene of Magnaporthe grisea PMK1 type MAPK from Ustilaginoidea virens and functional identification by complement in Magnaporthe grisea corresponding mutant. Acta Microbiologica Sinica, 2008, 48(11): 1473-1478. (in Chinese)

[17]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. 中国农业科学, 2013, 46(9): 1790-1798.

Yu M N, Hu J K, Huang L, Yu J J, Yin X L, Nie Y F, Chen Z Y, Liu Y F. Molecular characterization of T-DNA integration of the Ustilaginoidea virens mutant 5062. Scientia Agricultura Sinica, 2013, 46(9): 1790-1798. (in Chinese)

[18]黄磊, 俞咪娜, 胡建坤, 于俊杰, 尹小乐, 聂亚峰, 陈志谊, 刘永锋. 稻曲病菌突变体B-726生物学性状分析及其T-DNA插入位点侧翼序列的克隆. 中国农业科学, 2013, 46(16): 3344-3353.

Huang L, Yu M N, Hu J K, Yu J J, Yin X Y, Nie Y F, Chen Z Y, Liu Y F. Analysis of biological phenotypes and molecular cloning of T-DNA integration flanking sequences of Ustilaginoidea virens mutant strain B-726. Scientia Agricultura Sinica, 2013, 46(16): 3344-3353. (in Chinese)

[19]罗婵, 汤刚彬, 谢体三, 石德顺. 感受态细胞制备与保存方法的比较研究. 生物技术, 2005, 15(1): 52-54.

Luo C, Tang G B, Xie T S, Shi D S. Comparative study on preparation and storage of competent Escherichia coli cells. Biotechnology, 2005, 15(1): 52-54. (in Chinese)

[20]Liu Y G, Chen Y L. High-efficiency thermal asymmetric interlaced PCR for amplification of unknown flanking sequences. BioTechniques, 2007, 43(5): 649-656.

[21]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 PCR. Chinese Journal of Rice Science, 2012, 26(5): 615-618. (in Chinese)

[22]Gupta A, Chattoo B B. A novel gene MGA1 is required for appressorium formation in Magnaporthe grisea. Fungal Genetics and Biology, 2007, 44(11): 1157-1169.

[23]Zhou Z, Li G, Lin C, He C. Conidiophore stalk-less1 encodes a putative zinc-finger protein involved in the early stage of conidiation and mycelial infection in Magnaporthe oryzae. Molecular Plant-Microbe Interactions, 2009, 22(4): 402-410.

[24]张震, 杜新法, 柴荣耀, 毛学琴, 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)

[25]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.

[26]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.