Cloning Of The Signal Peptide Sequence And The Promoter In Allatostatin Gene From Gryllus Bimaculatus

Gryllus bimaculatus allatostatin is a kind of insect neuropeptide that inhibits Juvenile Hormone (JH) biosynthesis by the corpora allata(CA). Recently, more and more studies of AST have concentrated on its molecular biology. Grb-AST gene has a high GC content, and the cDNA encoding Grb-AST precursor encodes fifteen ASTs, which are characterized by a common carboxy terminus (Y/FXFGL/I/V). This paper studied the cloning signal peptide and promoter of Grb-AST gene.Based on the 5' end conservative sequence of Grb-AST precursor cDNA and the adaptor sequence, two gene specific primers(GSP) and an adaptor primer(AP) were synthesized respectively for the cloning of signal peptide. By using the method of genome walking, two fragments were cloned from Genome Walker library after the semi-nested PCR. The result of sequencing the bigger fragment showed that this 706bp sequence contained the ATG start codon. Among the 70 amino acids of AST N-end signal peptide prediction was carried out by using Signal? V2.0 program. The result demonstrated that there was a typical signal peptide with the front 30 amino acids (MAPRSACVAALLRASLLTVALLQAPPLA RA).Furthermore, Based on the cloned 706bp fragment, another GSP was designed and synthesized for a further cloning of promoter with AP. By the same method of genome walking again, a new 138bp fragment was cloned from Genome Walker library. It is suggested that 538bp sequence of AST gene 5' end had been cloned after the 138bp fragment was linked up with the 706bp fragment. The analysis of 538bp sequence with the software of promoter prediction indicated that there maybe exist four transcriptional initiator sites, one CAAT-box and two GC-boxes.In addition, The function of secreting protein by Grb-AST signal peptide was also studied in this paper. A GUS report gene, gained by the method of PCR, was inserted into the pET22b vector between the sites of HindIII and Xho I to construct a transitional vector named pET22G Then the signal peptide(pelB leader)in the pET22G vector was replaced by a containing Grb-AST signal peptide fragment to construct expression vector pET22ASG which had been transformed into expression host E.coli BL21 (DE3) pLysS. After the induction of IPTG, the extracellular proteins of pET22ASG/BL21 (DE3) pLysS showed blue when treated with GUS staining solution; But the extracellular proteins of CK:BL21 (DE3 )pLysS showed no color change with the same treat. These results verified the function of secreting protein by Grb-AST signal peptide.