Cloning and expression analysis of heat shock cognate 70 gene promoter in tiger shrimp ( Penaeus monodon)

Abstract

Heat shock cognate 70 (HSC70) functions as a molecular chaperon and plays an important role in protein folding. HSC70 cDNA of tiger shrimp ( Penaeus monodon) was cloned and characterized in our previous study. After shrimps were treated with the 1-hr heat shock, the HSC70 mRNA level in hemocytes increased (∼ 8 fold) using real-time quantitative PCR. An hsc70 clone was obtained from genomic library screening. The gene contains 2 exons separated by a 1557-bp intron. The 5′-flanking region sequence (∼ 1 kb) ahead of the hsc70 gene contains a putative core promoter region and transcription elements including perfect heat shock element (HSE), imperfect HSE, CAAT elements, SP1, NF-κB and GC box. In insect Sf21 cells, the region could drive expression of the enhanced green fluorescent protein (EGFP) and luciferase gene to verify its promoter function. In the luciferase assay system, the effects of serial deletions on the hsc70 promoter were elucidated. Autographa californica multiple nuclear polyhedrosis virus infection (MOI = 0.1) on Sf21 cells significantly increased the hsc70 promoter activity. In addition, the effects of amino acid analogs and arsenic acid incubation with the cells on the hsc70 promoter activity were examined.