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

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P_Hsp701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。

Abstract:

[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. P_Hsp701 showed the highest expression activity, serving as a strong promoter in M.antarcticus. P_Hsp704, P_Hsp705, and P_Hsp706 had lower activities than P_Hsp701 but could be used as alternative promoters for further research.