磷酸甘露酶基因的阻断对刺糖多孢菌的形态及其多杀菌素合成的影响

doi:10.16409/j.cnki.2095-039x.2017.01.019

中国生物防治学报 ›› 2017, Vol. 33 ›› Issue (1): 134-141.DOI: 10.16409/j.cnki.2095-039x.2017.01.019

磷酸甘露酶基因的阻断对刺糖多孢菌的形态及其多杀菌素合成的影响

刘红雪, 蔡妹, 张由恒, 肖洁, 丁学知, 孙运军, 夏立秋

湖南师范大学生命科学学院/微生物分子生物学国家重点实验室培育基地, 长沙 410081

收稿日期:2016-04-09 出版日期:2017-02-08 发布日期:2017-01-24
通讯作者: 夏立秋,教授,E-mail:xialq@hunnu.edu.cn
作者简介:刘红雪,硕士研究生,E-mail:1018762199@qq.com
基金资助:
国家国际合作重点项目（2011DFA32610）；国家“973”计划（2011CB7221301）；国家“863”计划（2011AA10A203）；湖南省协同创新中心项目（20134486）

Impact on Strain Phenotypes and Spinosad Biosynthesis by Disruption of Phosphomannomutase Gene in Saccharopolyspora spinosa

LIU Hongxue, CAI Mei, ZHANG Youheng, XIAO Jie, DING Xuezhi, SUN Yunjun, XIA Liqiu

State Key Laboratory Breeding Base of Microbial Molecular Biology/College of Life Science, Hunan Normal University, Changsha 410081, China

Received:2016-04-09 Online:2017-02-08 Published:2017-01-24

摘要/Abstract

摘要： 磷酸甘露酶（PMM）是由manB基因编码的广泛存在于真核生物中的蛋白酶，在糖代谢过程中磷酸-6-甘露糖可在磷酸甘露酶的作用下转化为磷酸-1-甘露糖，与细胞的初级代谢过程及次级代谢产物的合成过程密切相关，可通过对刺糖多孢菌中与初级代谢途径相关的基因进行分析改造，促使多杀菌素的合成显著提高。本研究在刺糖多孢菌基因组中克隆了磷酸甘露酶编码基因manB的部分片段，将其与穿梭载体pOJ260连接，构建了阻断型载体pOJ260-manB。通过接合转移将其导入刺糖多孢菌野生菌中，获得刺糖多孢菌突变菌株S. spinosa-△manB。将突变菌株接种至不同培养基，观察突变菌株与野生菌株的孢子形成情况，发现突变菌株的孢子萌发有所提前，且孢子产生量比野生菌略多；在HPLC上检测多杀菌素的产量发现突变菌株的产量相对于野生菌株提高了180%，这说明manB基因对刺糖多孢菌的生长发育有一定的影响，并对调控多杀菌素的合成具有重要作用。

关键词: 刺糖多孢菌, manB基因, 阻断表达, 多杀菌素

Abstract: The phosphomannomutase encoded by manB gene widely exists in eukaryotes, phosphate-6-mannose can be converted into phosphate-1-mannose under the action of phosphomannomutase, which was closely related to primary metabolism and secondary metabolites synthesis process. Previous reports showed that genetic modification on genes related to primary metabolism in Saccharopolyspora spinosa could improve spinosad yields. The 1.0 kb partial segment of manB gene was amplified by PCR and cloned into the vector pOJ260, generating manB disruption vector pOJ260-manB. This vector was introduced into S. spinosa by conjugation, and transconjugant S. spinosa-△manB was obtained successfully. On BHI and TSB agar, the spore germination of the mutant strain S. spinosa-△manB was advanced and the spore yield was more than the wild type strain. HPLC analysis showed that spinosad yield of the mutant strain increased 180% than the wild strain. Our results revealed that disruption of manB affected the development of S. spinosa and the biosynthesis of spinosad.

Key words: Saccharopolyspora spinosa, phosphomannomutase gene, disruption, spinosad

中图分类号:

S476

刘红雪, 蔡妹, 张由恒, 肖洁, 丁学知, 孙运军, 夏立秋. 磷酸甘露酶基因的阻断对刺糖多孢菌的形态及其多杀菌素合成的影响[J]. 中国生物防治学报, 2017, 33(1): 134-141.

LIU Hongxue, CAI Mei, ZHANG Youheng, XIAO Jie, DING Xuezhi, SUN Yunjun, XIA Liqiu. Impact on Strain Phenotypes and Spinosad Biosynthesis by Disruption of Phosphomannomutase Gene in Saccharopolyspora spinosa[J]. journal1, 2017, 33(1): 134-141.

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磷酸甘露酶基因的阻断对刺糖多孢菌的形态及其多杀菌素合成的影响

Impact on Strain Phenotypes and Spinosad Biosynthesis by Disruption of Phosphomannomutase Gene in Saccharopolyspora spinosa

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