两启动子影响egfp在绿僵菌中的荧光表达

doi:10.3864/j.issn.0578-1752.2015.S.003

中国农业科学 ›› 2015, Vol. 48 ›› Issue (S): 23-31.doi: 10.3864/j.issn.0578-1752.2015.S.003

两启动子影响egfp在绿僵菌中的荧光表达

刘少芳¹，王苗苗¹，涂雄兵¹，张雄鹏²，农向群¹，曹广春¹，王广君¹，张泽华¹

¹中国农业科学院植物保护研究所植物病虫害生物学国家重点实验室，北京 100193
²乌兰察布市凉城县农牧业局，内蒙古乌兰察布 013750

收稿日期:2015-09-24 出版日期:2015-10-20 发布日期:2015-10-20
通讯作者: 农向群，Tel：010-82109569；E-mail：xqnong@sina.com
作者简介:刘少芳，E-mail：15652794633@163.com
基金资助:
农业部“948”计划（2011-G4）、国家农业科技成果转化资金（2013GB23260581）、现代农业牧草体系（CARS-35）

Two Promoters Cause Different Fluorescence Expression of egfp in Metarhizium anisopliae

LIU Shao-fang¹, WANG Miao-miao¹, TU Xiong-bing¹, ZHANG Xiong-peng², NONG Xiang-qun¹, CAO Guang-chun¹, WANG Guang-jun¹, ZHANG Ze-hua¹

¹State Key Laboratory for Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193
² The Agricultural and Animal Husbandry Bureau for Liangcheng County of Ulanqab City, Ulanqab 013750, Inner Mongolia

Received:2015-09-24 Online:2015-10-20 Published:2015-10-20

摘要/Abstract

摘要： 【目的】通过研究高效组成型三磷酸甘油醛脱氢酶基因启动子PgpdA和稻瘟病菌核糖体蛋白基因启动子RP27对绿色荧光蛋白基因egfp在绿僵菌转化子中表达的影响，为构建具有稳定报告基因的绿僵菌工程菌株提供高表达启动子，并为研究绿僵菌侵染寄主过程及宿存扩散提供依据。【方法】构建含潮霉素B抗性基因hph为标记基因、egfp为报告基因、分别以RP27和PgpdA为启动子的PAN-r-egfp和PAN-p-egfp表达载体。采用CaCl₂-PEG介导方法转化金龟子绿僵菌（Metarhizium anisopliae）菌株Ma9。以激光共聚焦显微镜观察转化子菌丝和分生孢子的荧光强度。【结果】在PAN-r-egfp和PAN-p-egfp转化的hph抗性转化子中，分别有40%和31%实现了egfp在绿僵菌的高强表达。以RP27启动的转化子菌丝和分生孢子均能表达强烈荧光，以PgpdA启动的转化子只有菌丝可观察到较强荧光，但分生孢子无荧光或荧光极弱。【结论】绿色荧光蛋白基因egfp的表达受启动子和绿僵菌生长发育的影响。RP27启动的表达不受金龟子绿僵菌Ma9生长阶段的影响，在菌丝和分生孢子阶段均可启动egfp表达。而PgpdA的启动作用在分生孢子阶段可能受到抑制，不能启动egfp的表达。

关键词: egfp, 启动子RP27, 启动子PgpdA, 金龟子绿僵菌, 遗传转化

Abstract: 【Objective】The study aimed to determine the effect of two promoters, PgpdA and RP27, on egfp gene expression. It would be important for construction of engineered Metarhizium anisopliae with a new and high-expression promoter in future. 【Method】Two expression vectors, PAN-r-egfp including promoter RP27 and PAN-p-egfp including promoter PgpdA, were constructed with a hph marker gene and a egfp reporter gene, respectively. CaCl₂-PEG meditated transformation was used to transfer the two vectors into M. anisopliae strainMa9. The transformants were observed fluorescence intensity of hypha and conidia by Laser Scanning Confocal Microscope.【Result】Among of the hph-resistance transformants from PAN-r-egfp and from PAN-p-egfp transformed, 40% and 31% achieved egfp expression with high fluorescence in M. anisopliae, respectively. Both hypha and conidia of transformants from PAN-r-egfp transformation shined strong fluorescence. But only hypha of transformants from PAN-p-egfp transformation was observed fluorescence, its conidia failed to produce fluorescence.【Conclusion】The egfp gene expression was influenced by different promoters and different development stages of M. anisopliae. The promoter RP27 could drive egfp gene expression either in hypha or in conidia of M. anisopliae Ma9. The initiating action of promoter PgpdA may be inhibited at conidiating stage of M. anisopliae Ma9, resulting in failure of egfp expression at the stage.

Key words: egfp, promoter RP27, promoter PgpdA, Metarhizium anisopliae, transformation

刘少芳，王苗苗，涂雄兵，张雄鹏，农向群，曹广春，王广君，张泽华. 两启动子影响egfp在绿僵菌中的荧光表达[J]. 中国农业科学, 2015, 48(S): 23-31.

LIU Shao-fang, WANG Miao-miao, TU Xiong-bing, ZHANG Xiong-peng, NONG Xiang-qun, CAO Guang-chun, WANG Guang-jun, ZHANG Ze-hua. Two Promoters Cause Different Fluorescence Expression of egfp in Metarhizium anisopliae[J]. Scientia Agricultura Sinica, 2015, 48(S): 23-31.

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两启动子影响egfp在绿僵菌中的荧光表达

Two Promoters Cause Different Fluorescence Expression of egfp in Metarhizium anisopliae

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