菟丝子生防菌“鲁保一号”生物学特性及T-DNA插入突变体库的构建

doi:10.11686/cyxb2016238

草业学报 ›› 2017, Vol. 26 ›› Issue (1): 142-148.DOI: 10.11686/cyxb2016238

菟丝子生防菌“鲁保一号”生物学特性及T-DNA插入突变体库的构建

李健, 李美^*, 高兴祥, 房锋, 董连红

山东省农业科学院植物保护研究所,山东济南250100

收稿日期:2016-06-07 出版日期:2017-01-20 发布日期:2017-01-20
通讯作者: E-mail: limei9909@163.com
作者简介:李健(1986-),男,山东临沂人,助理研究员,博士。E-mail: lijian910@163.com
基金资助:
山东省自然科学基金(ZR2015CQ014)资助

Biological characteristics of Lubao No.1 biological control agent (Colletotrichum gloeosporioides) and construction of a T-DNA insertional mutant library

LI Jian, LI Mei^*, GAO Xing-Xiang, FANG Feng, DONG Lian-Hong

Institute of Plant Protection, Shandong Academy of Agricultural Science, Jinan 250100, China

Received:2016-06-07 Online:2017-01-20 Published:2017-01-20

摘要/Abstract

摘要： “鲁保一号”菌剂对杂草菟丝子生防效果良好,但是受制于菌种退化问题其进一步应用受到限制。本研究通过对“鲁保一号”菌的生物学性状进行分析,明确其菌种培养特性:最佳培养基为PDA培养基,最佳培养温度为25~28 ℃,最佳培养pH值为5~8。同时,利用农杆菌介导的遗传转化方法进行“鲁保一号”菌株转化,并初步构建了约含2300个阳性转化子的T-DNA插入突变体库。转化子经连续继代6次培养后潮霉素B抗性稳定,随机挑取的20个转化子均能PCR克隆出潮霉素基因,转化子是由T-DNA插入引起的,且筛选抗性能够稳定遗传。以上研究为后期筛选获得致病力稳定的遗传改良菌株,并进一步应用奠定了基础。

Abstract: The application of Lubao No.1 microbial agent has been limited because of its degradation, although the strain shows high bio-herbicidal activity against Cuscuta chinensis. The results of an investigation of the biological characteristics of Lubao No.1 showed that the optimal mycelium growth medium was PDA medium. The optimal culture temperature is 25-28 ℃, and optimal initial pH of culture media was 5-8. Construction of a T-DNA insertional mutant library of Lubao No.1 using an Agrobacterium mediated transformation method, produced about 2300 positive transformants. Randomly selected transformants were all resistant to hygromycin B. A hygromycin gene fragment was amplified by PCR from all 20 tested mutants, indicating that the resistance of the transformants to hygromycin B was stable and was caused by the inserted T-DNA. This study laid the foundation for obtaining further applications of a genetically modified Colletotrichum strains with stable pathogenicity.

李健, 李美, 高兴祥, 房锋, 董连红. 菟丝子生防菌“鲁保一号”生物学特性及T-DNA插入突变体库的构建[J]. 草业学报, 2017, 26(1): 142-148.

LI Jian, LI Mei, GAO Xing-Xiang, FANG Feng, DONG Lian-Hong. Biological characteristics of Lubao No.1 biological control agent (Colletotrichum gloeosporioides) and construction of a T-DNA insertional mutant library[J]. Acta Prataculturae Sinica, 2017, 26(1): 142-148.

参考文献

[1]　Li Y H. Identification, Control and Quarantine of Weed[M]. Nanjing: Jiangsu Science Press, 1981: 302-303.
李扬汉. 田园杂草的识别、防除与检疫[M]. 南京: 江苏科学出版社, 1981: 302-303.
[2]　Shan L H. The damage characteristics and control methods of Cuscuta chinensis. The Chinese Livestock and Poultry Breeding, 2013, (3): 17.
单丽华. 菟丝子的危害特点及防治方法. 中国畜禽种业, 2013, (3): 17.
[3]　Shi X F. The measures for control of dodder. Shaanxi Forest Science and Technology, 2014, (2): 101-103.
石晓峰. 菟丝子的发生及防治方法. 陕西林业科技, 2014, (2): 101-103.
[4]　Gao Z Y, Gan J E, Zhang Y J. The prevention and control of soybean dodder. China Rural Science and Technology, 1997, 4: 17-18.
高昭远, 干静娥, 张玉洁. 大豆菟丝子的防治. 中国农村科技, 1997, 4: 17-18.
[5]　Liu Z H, Zhu Q R. Lubao No.1 Strain[M]. Jinan: Shandong Science and Technology Press, 1980.
刘志海, 朱全让. 鲁保一号菌[M]. 济南: 山东科技出版社, 1980.
[6]　Yang C X. Study of Phyllosticta F-3 Strain to Control Dayflower Leaf Blight[D]. Shenyang: Shenyang Agricultural University, 2006.
杨春喜. 叶点霉F-3菌株对鸭跖草防除潜力的研究[D]. 沈阳: 沈阳农业大学, 2006.
[7]　Zhang T Y. Colletotrichum gloeosporioides (Penz.) Sacc.f.sp.cuseutae. Acta Fungus Sinica, 1985, 4(4): 234-239.
张天宇. 胶孢炭疽菌菟丝子专化型. 真菌学报, 1985, 4(4): 234-239.
[8]　Lin B Q, Nie Z, Guo H C. Bacterial degradation of the Lubao No.1 strain. Microbiology China, 1979, 6(2): 1-4.
林伯荃, 聂征, 郭恒聪. 鲁保一号菌的菌种退化. 微生物学通报, 1979, 6(2): 1-4.
[9]　Li G T. Agrobacterium tumefaciens-mediated Transformation of Trichoderma viride and Correlations between Mycoparasitism and Biological Control[D]. Zibo: Shandong University of Technology, 2007.
李国田. 土壤杆菌介导的绿色木霉插入突变及重复寄生与生物防治能力的关系[D]. 淄博: 山东理工大学, 2007.
[10]　Yao R, Shi M L, Pan S Y, et al. Progress on Agobacterium tumefaciens-mediated plant transformation. Current Biotechnology, 2011, (4): 260-265.
姚冉, 石美丽, 潘沈元, 等. 农杆菌介导的植物遗传转化研究进展. 生物技术进展, 2011, (4): 260-265.
[11]　Bundock P, den Dulk-Ras A, Beijersbergen A, et al. Trans-kingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae. The EMBO Journal, 1995, 14(13): 3206-3214.
[12]　De Groot M J, Bundock P, Hooykaas P J, et al. Agrobacterium tumefaciens-mediated transformation of filamentous fungi. Nature Biotechnology, 1998, 16: 839-842.
[13]　Abuodeh R O, Orbach M J, Mandel M A, et al. Genetic transformation of Coccidioides immitis facilitated by Agrobacterium tumefaciens. Journal of Infectious Diseases, 2000, 181(6): 2106-2110.
[14]　Mullins E, Chen X, Romaine P, et al. Agrobacterium-mediated transformation of Fusarium oxysporum: an efficient tool for insertional mutagenesis and gene transfer. Phytopathology, 2001, 91(2): 173-180.
[15]　Li H Y. T-DNA Insertional Mutagenesis of Magnaporthe grisea[D]. Fuzhou: Fujian Agriculture and Forestry University, 2003.
李宏宇. 稻瘟病菌T-DNA插入突变研究[D]. 福州: 福建农林大学, 2003.
[16]　Bundock P, Mroczek K, Winkler A, et al. T-DNA from Agrobacterium tumefaciens as an efficient tool for gene targeting in Kluyveromyces lactis. Molecular and General Genetics, 1999, 261(1): 115-121.
[17]　Michielse C, Arentshorst M, Ram A, et al. Agrobacterium-mediated transformation leads to improved gene replacement efficiency in Aspergillus awamori. Fungal Genetics and Biology, 2005, 42(1): 9-19.
[18]　Tsuji G, Fujii S, Fujihara N, et al. Agrobacterium tumefaciens-mediated transformation for random insertional mutagenesis in Colletotrichum lagenarium. Journal of General Plant Pathology, 2003, 69(4): 230-239.
[19]　Liu B W. The good effect of bacteria liquid of Lubao No.1 to control soybean dodder. Hebei Agriculture, 2003, (5): 20.
刘炳文. 用“鲁保一号”菌液防治大豆菟丝子效果好. 河北农业, 2003, (5): 20.
[20]　Li Y F, Li K C, Huang W G, et al. The first large-scale occurrence of Cuscuta chinensis in Ningxia plain. Plant Protection, 2008, (3): 151-153.
李寅菲, 李克昌, 黄文广, 等. 宁夏草原首次大面积发生菟丝子危害. 植物保护, 2008, (3): 151-153.
[21]　Li J, Li Y, Gao X X, et al. The biological characteristics of Digitaria sanguinalis pathogen Fusarium chlamydosporum strain ZC201301. Acta Prataculturae Sinica, 2016, 25(3): 234-239.
李健, 李岩, 高兴祥, 等. 马唐生防菌厚垣孢镰刀菌ZC201301的生物学特性研究. 草业学报, 2016, 25(3): 234-239.
[22]　Fang Z D. Plant Pathology Research Method[M]. Beijing: China Agriculture Press, 2001.
方中达. 植病研究法[M]. 北京: 中国农业出版社, 2001.
[23]　Huang G X, Xue Y C, Lu X. Construction of a T-DNA insertional mutant library of Colletotrichum gloeosporioides of mango and its molecular analysis. Chinese Journal of Tropical Crops, 2008, 29(1): 27-32.
黄贵修, 薛迎春, 卢昕. 胶孢炭疽菌T-DNA插入突变体库的构建及其分子分析. 热带作物学报, 2008, 29(1): 27-32.
[24]　Wang W J. Construction of a T-DNA Insertional Library of Colletotrichum gloeosporioides ES026[D]. Wuhan: Huazhong Agricultural University, 2014.
王文娟. 胶胞炭疽菌ES026 T-DNA插入突变体库的构建[D]. 武汉: 华中农业大学, 2014.
[25]　Zhang Y H, Wei D S, Xing L J, et al. A modified method for isolating DNA from fungus. Microbiology China, 2008, 35(3): 466-469.
张颖慧, 魏东盛, 邢来君, 等. 一种改进的丝状真菌DNA提取方法. 微生物学通报, 2008, 35(3): 466-469.
[26]　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.
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.
[27]　Cui A J. Study on the Investigate and Chemistry Controlling of Malignant Weed in Seed Product Field of Agropyron mongolicum Keng[D]. Huhhot: Inner Mongolia Agricultural University, 2009.
崔爱娇. 蒙农1号蒙古冰草种子田杂草调查与化学防除研究[D]. 呼和浩特: 内蒙古农业大学, 2009.
[28]　Zhang C X, Ni H W, Wei S H, et al. Current advances in research on herbicide resistance. Scientia Agricultura Sinica, 2009, 42(4): 1274-1289.
张朝贤, 倪汉文, 魏守辉, 等. 杂草抗药性研究进展. 中国农业科学, 2009, 42(4): 1274-1289.
[29]　Qiang S, Chen S G. Current status of bioherbicide research and development and its opportunities and challenges. Weed Science, 2011, 29(1): 1-6.
强胜, 陈世国. 生物除草剂研发现状及其面临的机遇与挑战. 杂草科学, 2011, 29(1): 1-6.
[30]　Zhao H, Zhou Y J, Liu X C, et al. Advances in bioherbicide formulation. Plant Protection, 2005, 31(5): 5-8.
赵航, 周勇军, 刘小川, 等. 生物除草剂剂型研究进展. 植物保护, 2005, 31(5): 5-8.

菟丝子生防菌“鲁保一号”生物学特性及T-DNA插入突变体库的构建

Biological characteristics of Lubao No.1 biological control agent (Colletotrichum gloeosporioides) and construction of a T-DNA insertional mutant library

RichHTML

PDF (PC)

可视化

摘要/Abstract

引用本文

使用本文

参考文献

相关文章 0

编辑推荐

Metrics