南极莫氏黑粉菌热激蛋白70家族启动子鉴定及活性比较  

Identification and activity comparison of promoters of the heat shock protein 70 family of Moesziomyces antarcticus

作　　者：张聪 郭利桃 董国云 李毅 李新文 张政兵 林宇丰 侯春生 李智敏[1] ZHANG Cong;GUO Litao;DONG Guoyun;LI Yi;LI Xinwen;ZHANG Zhengbing;LIN Yufeng;HOU Chunsheng;LI Zhimin(Institute of Bast Fiber Crops,Chinese Academy of Agricultural Sciences,Changsha 410205,Hunan,China;Zhangjiajie Agricultural Science and Technology Research Institute,Zhangjiajie 427000,Hunan,China;Plant Protection and Plant Inspection Station,Department of Agriculture and Rural Affairs of Hunan Province,Changsha 410005,Hunan,China)

机构地区：[1]中国农业科学院麻类研究所,湖南长沙410205 [2]张家界市农业科学技术研究所,湖南张家界427000 [3]湖南省农业农村厅植保植检站,湖南长沙410005

出　　处：《微生物学通报》2024年第8期2934-2946,共13页Microbiology China

基　　金：湖南省植保植检站植物防疫防控科研项目(HNZB202104);中国农业科学院科技创新工程项目(ASTIP-IBFC)。

摘　　要：【背景】南极莫氏黑粉菌(Moesziomyces antarcticus)由于能够产生优良的生物表面活性剂和脂酶而被深入研究,但目前仍缺乏遗传操作相关的基因表达元件。【目的】检测南极莫氏黑粉菌热激蛋白70超家族全部7个基因的启动子表达活性,明确该菌中不同热激蛋白70家族成员的启动子活性差异,并筛选出可用于调控南极莫氏黑粉菌基因表达的强启动子。【方法】通过基因组数据库获得南极莫氏黑粉菌JCM10317菌株中7个热激蛋白70家族成员的基因信息,利用生物信息学方法进行热激蛋白70家族进化分析并预测热激蛋白70启动子(P_(Hsp70))序列中的关键顺式作用元件,然后构建7个热激蛋白70启动子连接增强型绿色荧光蛋白基因的重组表达载体,通过测定阳性转化子荧光值和荧光显微观察比较不同热激蛋白70家族成员启动子的活性差异。【结果】蛋白进化分析表明7个热激蛋白70家族成员分别属于不同亚家族,并且各成员启动子中的顺式作用元件种类和数量也具有明显差异。此外,以P_(Hsp701)、P_(Hsp702)、P_(Hsp703)、P_(Hsp704)、P_(Hsp705)、P_(Hsp706)和P_(Hsp707)构建的重组质粒转化菌株BDH3-1所获得的阳性转化子平均荧光值分别为对照的12.8、1.6、2.9、5.8、4.6、5.0和1.5倍,与荧光显微观察结果一致。【结论】根据生物信息学分析及绿色荧光蛋白表达结果发现,南极莫氏黑粉菌热激蛋白70家族不同成员间的启动子活性差异显著,其中PHsp701的活性最高,是南极莫氏黑粉菌中的强启动子,P_(Hsp704)、P_(Hsp705)、P_(Hsp706)启动子活性次之,可作为备选启动子用于后续研究。[Background]Moesziomyces antarcticus has been extensively studied for its ability to produce excellent biosurfactants and lipases,while little is known about its expression elements for genetic manipulation.[Objective]To measure and compare the promoter activities of all the seven genes of the heat shock protein 70 superfamily in M.antarcticus,and screen out the strong promoters that can be used for regulating gene expression in M.antarcticus.[Methods]The gene information on seven members of the heat shock protein 70 family was obtained from the genome database of M.antarcticus JCM10317.Bioinformatics tools were used for evolutionary analysis of the heat shock protein 70 family,and key cis-acting elements in the heat shock protein 70 promoter(P_(Hsp70))sequences were predicted.The recombinant expression vectors were constructed by fusing the promoters of the seven heat shock protein 70 genes with the gene encoding the enhanced green fluorescent protein.The positive transformants were measured for the fluorescence intensity and observed under a fluorescence microscope,on the basis of which the promoter activities of different members of the heat shock protein 70 family were compared.[Results]The seven heat shock protein 70 family members belonged to different subfamilies,and their promoters had different categories and number of cis-acting elements.Moreover,the average fluorescence intensities of transformants with P_(Hsp701)、P_(Hsp702)、P_(Hsp703)、P_(Hsp704)、P_(Hsp705)、P_(Hsp706) and P_(Hsp707) recombinant plasmids respectively were 12.8,1.6,2.9,5.8,4.6,5.0,and 1.5 times of that in the control.The results are consistent with the fluorescence observation results under the microscope.[Conclusion]The results of bioinformatics analysis and enhanced green fluorescent protein expression revealed significant differences in the promoter activity among the heat shock protein 70 family members in M.antarcticus.PHsp701 showed the highest expression activity,serving as a strong promoter in M.antarcticus.P_(Hsp704)�

关 键 词：南极莫氏黑粉菌 热激蛋白70家族 启动子活性 遗传转化 增强型绿色荧光蛋白 

分 类 号：S432.44[农业科学—植物病理学]