[1]杜艳,刘永锋,常有宏,等.梨炭疽病菌原生质体遗传转化体系的建立及GFP标记菌株的获得[J].江苏农业学报,2017,(02):295-300.[doi:doi:10.3969/j.issn.1000-4440.2017.02.009]
　DU Yan,LIU Yong-feng,CHANG You-hong,et al.Establishment of genetic transformation system of Colletotrichum gloeosporioides protoplast and generation of GFP-tagged transformants[J].,2017,(02):295-300.[doi:doi:10.3969/j.issn.1000-4440.2017.02.009]
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梨炭疽病菌原生质体遗传转化体系的建立及GFP标记菌株的获得()
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江苏农业学报[ISSN:1006-6977/CN:61-1281/TN]

卷:
期数:
2017年02期
页码:
295-300
栏目:
植物保护
出版日期:
2017-04-30

文章信息/Info

Title:
Establishment of genetic transformation system of Colletotrichum gloeosporioides protoplast and generation of GFP-tagged transformants
作者:
杜艳1; 刘永锋1; 常有宏2; 蔺经2; 乔俊卿1; 刘邮洲1
（1. 江苏省农业科学院植物保护研究所，江苏南京210014；2. 江苏省农业科学院园艺研究所，江苏南京210014）
Author(s):
DU Yan1; LIU Yong-feng1; CHANG You-hong2; LIN Jing2; QIAO Jun-qing1; LIU You-zhou1
(1. Institute of Plant Protection, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China; 2. Institute of Vegetable Crops, Jiangsu Academy of Agricultural Sciences, Nanjing 210014, China)
关键词:
梨炭疽病菌; 遗传转化; 原生质体; GFP标记菌株
Keywords:
Colletotrichum gloeosporioides;  genetic transformation;  protoplast;  GFP-tagged transformants
分类号:
S661.2
DOI:
doi:10.3969/j.issn.1000-4440.2017.02.009
文献标志码:
A
摘要:
为建立梨炭疽病菌的原生质体遗传转化体系，以梨炭疽病菌菌株II-17为供试菌株，研究了菌龄、酶解液组合及酶解时间等对梨炭疽病菌原生质体制备的影响。结果表明，制备梨炭疽病菌原生质体的适宜条件为：CM液体培养基培养分生孢子24 h后，再转接到新的CM液体培养基中培养24 h，获得新鲜的菌丝；最适酶解液组合为9.0 mg/ml裂解酶+1.0 mg/ml崩溃酶+1.0 mg/ml蜗牛酶；酶解时间为2.5～3.0 h时酶解效率最高。对获得的转化子进行PCR鉴定和荧光观察,结果表明，GFP标记基因已成功整合到梨炭疽病菌的基因组中，转化子发出强烈的绿色荧光且遗传性稳定。致病性测定结果表明，转化子致病性与野生型菌株无明显差别，成功获得了梨炭疽病菌的GFP标记菌株。
Abstract:
To establish PEG-mediated genetic transformation of Colletotrichum gloeosporioides, single spore isolate II-17 of Colletotrichum gloeosporioides was used as a material of genetic transformation, culture time, enzyme mixtures and digestion time was studied in this study. The result showed that mycelia was cultured in CM liquid medium for 24 h, and then transferred to new CM liquid medium for 24 h, which was suitable for protoplast generation. The enzyme mixture was 9.0 mg/ml lysing enzyme, 1.0 mg/ml driselase and 1.0 mg/ml snailase. In addition, when the enzymolysis time ranged from 2.5 h to 3.0 h, the efficiency was the highest. PCR and fluorescence observation results showed that the fluorescence gene GFP had been successfully integrated into the genome of the II-17 strain, and the heredity of transformants was steady. The pathogenicity test indicated that there was no significant difference between GFP transformants and the wild type strain, and the GFP-tagged transformants of Colletotrichum gloeosporioides were successfully generated.

参考文献/References:

［1］吴良庆, 朱立武, 衡伟, 等. 砀山梨炭疽病病原鉴定及其抑菌药剂筛选[J].中国农业科学, 2010, 43(18): 3750-3758.
［2］沈静霆. 砀山酥梨炭疽病菌生物学特性及有效药剂筛选[D].合肥：安徽农业大学, 2012.
［3］SHEAR C L, WOOD A K. Ascogenous forms of Gloeosporium and Colletotrichum [J].Botanical Gazette, 1907, 43(4): 259-266.
［4］高正辉, 张金云, 伊兴凯, 等. 砀山酥梨炭疽病发生特征与防治技术[J].安徽农业科学, 2010, 38(19): 10445-10446.
［5］张磊, 常有宏, 刘邮洲, 等. 梨轮纹病和炭疽病病原菌PCR检测[J].江苏农业学报, 2012, 28(2): 415-420.
［6］IVIC D, VONCINA D, SEVER Z，et al. Identification of Colletotrichum species causing bitter rot of apple and pear in croatia[J].Journal of Phytopathology, 2013, 161(4): 284-286.
［7］刘邮洲, 陈志谊, 钱国良, 等. 梨胶胞炭疽病菌的分离、鉴定及其生物学特性[J].江苏农业学报, 2013, 29(1): 60-64.
［8］刘邮洲, 陈志谊, 刘永锋, 等. 枯草芽胞杆菌sf628对梨炭疽病的控制作用[J].植物保护学报, 2012, 39(6): 492-496.
［9］付余波, 钱国良, 胡白石, 等. 21种杀菌剂对梨炭疽病菌、轮纹病菌、黑斑病菌的室内毒力测定[J].江苏农业科学, 2011, 39(2): 178-180.
［10］RODRIGUEZ R J, YODER O C. Selectable genes for transformation of the fungal plant pathogen Glomerella cingulata f. sp. phaseoli (Colletotrichum lindemuthianum) [J]. Gene, 1987, 54 (1): 73-81．
［11］李思蒙, 王永林, 黄冬辉, 等. 杨树炭疽病菌原生质体遗传转化的建立及绿色荧光蛋白的表达[J]. 林业科学, 2013, 49(5): 121-127.
［12］林春花, 刘先宝, 蔡吉苗, 等. 橡胶树尖孢炭疽菌绿色荧光蛋白(GFP)标记转化株的获得[J].热带作物学报, 2009, 30(10): 1495-1500.
［13］LIN C H,CAI Z Y,SHI T,et al. The use of T-DNA tagging to isolate mutants of Colletotrichum gloeosporioides and Colletotrichum acutatum with reduced virulence against Hevea brasiliensis[J].Forest Pathology, 2013, 43(4): 289-296.
［14］ARMESTO C,MAIA F G M,ABREU M S D, et al. Genetic transformation with the gfp gene of Colletotrichum gloeosporioides isolates from coffee with blister spot[J].Brazilian Journal of Microbiology, 2012, 43(3): 1222-1229.
［15］张俊,吕延超,刘晓妹,等.芒果尖孢炭疽病病原菌的绿色荧光蛋白基因标记[J].热带作物学报, 2011, 32(9): 1708-1710.
［16］刘朋娟,王政逸,王秋华,等.农杆菌介导的稻瘟病菌转化及致病缺陷突变体筛选[J].中国水稻科学, 2006, 20(3): 231-237.
［17］王海艳,李保华,张清明,等.农杆菌介导苹果炭疽病菌的遗传转化及转化子鉴定[J].中国农业科学, 2013, 46(9): 1799-1807.
［18］ROBINSON M,SHARON A. Transformation of the bioherbicide Colletotrichum gloeosporioides f. sp. aeschynomene by electroporation of germinated conidia [J]. Current Genetics, 1999, 36(1/2): 98-104.
［19］李伟.芒果胶孢炭疽病菌遗传转化体系的建立[D]. 儋州：华南热带农业大学, 2004.
［20］YELTON M M, HAMER J E, TIMBERLAKE W E. Transformation of Aspergillus nidulans by using a trpC plasmid [J]. Proceedings of the National Academy of Sciences, 1984, 81(5): 1470-1474.
［21］张岳平,罗绍春,黄燕萍,等.芦笋茎枯病菌原生质体的制备及再生[J].植物保护学报,2014，41(2): 182-186.
［22］周礼红,李国琴,王正祥,等.红曲霉原生质体的制备、再生及其遗传转化系统[J].遗传,2005,27(3): 423-428.
［23］姚婷婷,王正祥.黑曲霉原生质体的制备、再生及转化条件[J].食品与生物技术学报, 2006，25(4)：116-120.
［24］徐莹,刘太国,何月秋,等.绿色荧光蛋白及其在丝状真菌研究中的应用[J].植物保护,2008,34(6): 1-6.

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备注/Memo

备注/Memo:
收稿日期：2016-04-27 基金项目：江苏省农业科技自主创新基金项目[CX(15)1023] 作者简介：杜艳 (1984-), 女, 安徽阜阳人, 博士, 助理研究员, 主要从事植物真菌病害研究。(Tel) 13951738556; (E-mail) dy411246508@126.com。通讯作者：刘邮洲, (Tel) 025-84390228; (E-mail) shitouren88888@163.com
更新日期/Last Update: 2017-05-02