柱花草炭疽菌致病力丧失突变菌株1869的

doi:10.11686/cyxb2015094

草业学报 ›› 2015, Vol. 24 ›› Issue (8): 142-149.DOI: 10.11686/cyxb2015094

柱花草炭疽菌致病力丧失突变菌株1869的

许沛冬^1,2,郑肖兰²,赵艳¹,李秋洁²,吴伟怀²,贺春萍²,习金根²,梁艳琼²,郑金龙²,戚山江¹,张晓波^4,5*,易克贤^2,3*

1.海南大学农学院,海南海口 570228;
2.中国热带农业科学院环境与植物保护研究所,海南海口 571101;
3.中国热带农业科学院热带生物技术研究所,海南海口 571101;
4.热带作物种质资源保护与开发利用教育部重点实验室(海南大学),海南海口 570228;
5.海南大学旅游学院,海南海口 570228

出版日期:2015-08-20 发布日期:2015-08-20
通讯作者: E-mail:yikexian@126.com, angiaoo@126.com
作者简介:许沛冬(1990-),男,内蒙古集宁人,在读硕士。E-mail:xuridongshengxpd@163.com
基金资助:
国家自然科学基金(31072076,31101408),公益性行业科研专项(201303057)和中央级公益性科研院所基本科研业务专项(2015hzs1J002)资助

Molecular characterization of the flanking gene of T-DNA insertional, pathogenicity-defective Colletotrichum gloeosporioides mutant strain 1869

XU Pei-Dong^1,2, ZHENG Xiao-Lan², ZHAO Yan¹, LI Qiu-Jie², WU Wei-Huai², HE Chun-Ping², XI Jin-Gen², LIANG Yan-Qiong², ZHENG Jin-Long², QI Shan-Jiang¹, ZHANG Xiao-Bo^4,5*, YI Ke-Xian

1.College of Agriculture, Hainan University, Haikou 570228, China;
2.Environment and Plant Protection Institute, CATAS, Haikou 571101, China;
3.Institute of Tropical Bioscience and Biotechnology, CATAS, Haikou 571101, China;
4.Key Laboratory of Protection and Development Utilization of Tropical Crop Germplasm Resources(Hainan University), Ministry of Education, Haikou 570228, China;
5.College of Tourism, Hainan University, Haikou 570228, China

Online:2015-08-20 Published:2015-08-20

摘要/Abstract

摘要： 通过对实验室已构建的柱花草炭疽菌T-DNA突变体库中各转化子致病力的测定,获得致病力丧失突变菌株1869。对其进行PCR检测以验证T-DNA在1869基因组中插入情况,并测定观察其菌落直径、菌落形态及产孢量等生物学特性,利用TAIL-PCR克隆标记基因侧翼序列,采用生物信息学方法比对基因信息。结果表明,与柱花草炭疽菌野生型菌株CH008相比,突变菌株1869表现致病力丧失,菌落生长速率也显著低于野生型;然而,在分生孢子形态、产孢能力、孢子萌发率等方面与野生型并无明显差异。TAIL-PCR扩增得到T-DNA插入位点RB端序列467 bp,LB端侧翼序列388 bp,经两侧序列拼接比对,所得序列与Pac1同源性达到92%~96%,推测可能由于基因表达产物调节菌株对pH值的敏感性,从而影响了其致病过程。

Abstract: Colletotrichum gloeosporioides is a fungal pathogen of Stylosanthes guianensis causing anthracnose disease. Pathogenicity defective mutant strains of C. gloeosporioides were screened in the laboratory and strain 1869 selected for study. The mutant strain was compared with wild type strain CH008 for phenotypic characteristics, including colony diameter, colony morphology, sporulation ability and spore germination rate. Colony diameter of CH008 and strain 1869 differed, but the other traits did not. DNA of the mutant strain was extracted, and using thermal asymmetric interlaced PCR (TAIL-PCR) a T-DNA insertion mutation site believed responsible for loss of pathogenicity was identified. The RB and LB flanking sequences at the insertion site were cloned, also using TAIL-PCR. The RB flanking sequence was 467 bp in length, while the LB flanking sequence was 388 bp. The BLAST result showed that the sequence had 92%-96% homology to gene Pac1. By predicting the function of a protein coded by the flanking sequence,we inferred that the gene in question regulated the sensitivity to pH, and in this way affected the pathogenic potential of C. gloeosporioides.

T-DNA插入位点侧翼序列的克隆. 柱花草炭疽菌致病力丧失突变菌株1869的[J]. 草业学报, 2015, 24(8): 142-149.

XU Pei-Dong, ZHENG Xiao-Lan, ZHAO Yan, LI Qiu-Jie, WU Wei-Huai, HE Chun-Ping, XI Jin-Gen, LIANG Yan-Qiong, ZHENG Jin-Long, QI Shan-Jiang, ZHANG Xiao-Bo, YI Ke-Xian. Molecular characterization of the flanking gene of T-DNA insertional, pathogenicity-defective Colletotrichum gloeosporioides mutant strain 1869[J]. Acta Prataculturae Sinica, 2015, 24(8): 142-149.

参考文献

[1] Liu F M, Cui X D, Wu K R, et al . Biological characteristics of Colletotrichum gloeosporioides Penz. causing anthracnose of Stylosanthes guianensis . Guangdong Agricultural Science, 2012, 16: 65-68.
[2] Yi K X. Development and prospects and countermeasures on tropical forage industrial production in China. Chinese Journal of Tropical Agriculture, 2001, 4: 30-34.
[3] Jiang C S, Zhou D M, Zhang Z Y. Stylo anthracnose and the progress of its breeding for resistance to disease. Journal of Sichuan Grassland, 2002, (1): 22-27.
[4] Chakraborty S, Perrott R, Charchar M J D, et al . Biodiversity, epidemiology and virulence of Colletotrichum gloeosporioides .Ⅱ.Genetic and pathogenic diversity in isolates of Colletotrichum gloeosporioides from eight species of stylosanthes.Tropical Grasslands, 1997, 31: 393-401.
[5] Bian J, Chen T X, Chen X R. Pathogen identification and occurrence regularity of a novel disease—— Angelica anthracnose.Acta Prataculture Sinica, 2014, 23(6): 266-273.
[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] Liu P J, Wang Z Y, Wang Q H, et al . Agrobacterium tumefaciens mediated transformation of Magnaporthe grisea and identification of pathogenicity defective mutant. Chinese Journal of Rice Science, 2006, 20(3): 231-237.
[8] Yu M N, Hu J K, Huang L, et al . Molecular characterization of T-DNA integration of the Ustilaginoidea virens Mutant 5062. Scientia Agricultura Sinica, 2013, 46(9): 1790-1798.
[9] Bundock Paul, Dulk-Ras Amkeden, Beijersbergen Alice G M, et al . Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to saccharomyces cerevisiae. The EMBO Journal, 1995, 14(3): 3206-3214.
[10] Fang L, Liu H Q, Song F M, et al . Agrobacterium tumefaciens mediated transformation of Colletotrichum lagenarium the causal agent of cucumber anthracnose. Journal of Zhejiang University(Agriculture and Life Sciences), 2006, 32(4): 360-366.
[11] Zhen D X, Li M H, Jiang Z D. Agrobacterium tumefaciens -mediated transformation of Colletotrichum musae . Journal of Zhongkai Agrotechnical College, 2005, 18(4): 42-44.
[12] Li W, Zheng X L, He C P, et al . Establishment of Agrobacterium -mediated transformation systems for Colletotrichum gloeosporioides . Plant Protection, 2008, 34(1):76-81.
[13] Hu C P, Zheng J L, Gao J M, et al . Optimization of genetic transformation system of Stylo Anthracnose mediated by Agrobacterium tumefaciens .Chinese Journal of Tropical Crops, 2013, 34(6): 1007-1012.
[14] Stephenson S A, Hatfield J, Rusu A G, et al . CgDN 3: An essential pathogenieity gene of Colletotrichum gloeosporioides necessary to avert a hypersensitive-like response in the host Stylosanthes guianensis . Molecular Plant-Microbe Interactions, 2000, 13: 929-941.
[15] Thon M R, Nuekles E M, Takaeh J E, et al . CPR1: A gene encoding a putative signal peptidase that functions in pathogenicity of Colletotrichum graminicola to maize. Molecular Plant-Microbe Interactions, 2002, 15: 120-128.
[16] Yi K X, Huang J S, Liu G D. Genetic diversity analysis of Chinese Stylo anthracnose pathogens using random amplified polymorphic DNA.Acta Microbiologica Sinica, 2003, 43(3): 379-387.
[17] Gao J M, Hu C P, Zheng J L, et al . Construction and analysis of a T-DNA insertional mutant library for Colletotrichum gloeosporioides . Guangdong Agricultural Science, 2014, 19(10): 142-145.
[18] Mullins E D, Chen P X, Romaine P, et al . Agrobacterium -mediated transformation of Fusarium oxysporum: An efficient tool for inserlional mutagenesis and gene transfer. Phytopathology, 2001, 91: 173-180.
[19] Combier J P, Melayah D, Raffier C, et al . 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.
[20] Liu M, Zhang W, Zhou Y, et al . Research progress on grape anthracnose. China Plant Protection, 2014, 34(1): 29-33.
[21] Wharton P S, Julian A M. A cytological study of compatible and incompatible interactions between Sorghum bicolor and Colletotrichum sublineolum . New Phytologist, 1996, 134(1): 25-34.
[22] Kim Y K, Wang Y H, Liu Z M, et al . Identification of a hard surface contact-induced gene in Colletotrichum gloeosporioides conidia as a sterol glycosyl transferase, a novel fungal virulence factor. The Plant Journal, 2002, 30: 177-187.
[23] Thon M R, Nuckles E M, Takach J E, et al . CPRl: A gene encoding a putative signal peptidase that functions in pathogenicity of Colletotrichum graminicola to maize. Molecular Plant-Microbe Interactions, 2002, 15: 120-128.
[24] Tsuji G, Fujii S, Tsuge S, et al . The Colletotrichum lagenarium Ste12-Like gene CSTl is essential for appressorium penetration. Molecular Plant-Microbe Interactions, 2003, 16: 315-325.
[25] Sun B Y, Piao H L, Park S H, et al . Selection of optimal primers for TAIL-PCR in identifying Ds flanking sequences from Ac/Ds insertion rice lines. Chinese Journal of Biotechnology, 2004, 20(6): 821-826.
[26] Miyara I, Shafran H, Haimovich H K, et al . Multi-factor regulation of pectate lyase secretion by Colletotrichum gloeosporioides pathogenic on avocado fruits. Molecular Plant Pathology, 2008, 9(3): 281-291.
[27] Zhang C C, Liang Y C, Meng J R, et al . Identification and biological character research on anthracnose of Stylosanthes spp. Journal of Guangxi Agricultural University, 1998, 17(1): 87-91.
[28] Feng S F, Li F E, He C Z, et al . Bionomics and epidemiology of anthracnose on Stylosanthes spp. Chinese Journal of Tropical Crops, 1994, 15(1): 90-93.
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[2] 易克贤. 我国热带牧草产业化的发展现状、前景与对策. 热带农业科学, 2001, 4: 30-34.
[3] 蒋昌顺, 邹冬梅, 张正义. 柱花草炭疽病及其抗病育种研究进展. 四川草原, 2002, (1): 22-27.
[5] 卞静, 陈泰祥, 陈秀蓉. 当归新病害——炭疽病病原鉴定及发病规律研究. 草业学报, 2014, 23(6): 266-273.
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[8] 俞咪娜, 胡建坤, 黄磊, 等. 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.
[10] 方丽, 刘海青, 宋凤鸣, 等. 农杆菌介导的黄瓜炭疽菌遗传转化. 浙江大学学报(农业与生命科学版), 2006, 32(4): 360-366.
[11] 曾大兴, 李敏慧, 姜子德. 根癌农杆菌介导的香蕉炭疽菌转化. 仲恺农业技术学院学报, 2005, 18(4): 42-44.
[12] 李伟, 郑肖兰, 贺春萍, 等. 根癌农杆菌介导的胶孢炭疽菌遗传转化体系的建立. 植物保护, 2008, 34(1): 76-81.
[13] 胡彩平, 郑金龙, 高建明, 等. 根癌农杆菌介导柱花草炭疽菌遗传转化体系的优化. 热带作物学报, 2013, 34(6): 1007-1012.
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[17] 高建明, 胡彩平, 郑金龙, 等. 柱花草炭疽病菌T-DNA插入突变体库的构建与分析. 广东农业科学, 2014, 19(10): 142-145.
[20] 刘梅, 张玮, 周莹, 等. 葡萄炭疽病研究进展. 中国植保导刊, 2014, 34(1): 29-33.
[27] 张超冲, 梁英彩, 蒙绞荣, 等. 柱花草炭疽病菌的鉴定及生物学特性研究. 广西农业大学学报, 1998, 17(1): 87-91.
[28] 冯淑芬, 李凤娥, 何朝族, 等. 笔花豆炭疽病菌生物学特性和流行条件研究. 热带作物学报, 1994, 15(1): 90-93.

柱花草炭疽菌致病力丧失突变菌株1869的

Molecular characterization of the flanking gene of T-DNA insertional, pathogenicity-defective Colletotrichum gloeosporioides mutant strain 1869

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