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

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.

Key words:Moesziomyces antarcticus;heat shock protein 70 family;promoter activity;genetic transformation;enhanced green fluorescent protein