Guiyang Pythium class iturins prokaryotic expression of protease.docVIP

 PAGE \* MERGEFORMAT 13 Guiyang Pythium class iturins prokaryotic expression of protease [Abstract] Objective: To achieve Guiyang Pythium (Pythium guiyangense Su) class iturins protease (subtilisin-like protease, Pr1) activity in Escherichia coli expression of the gene sequence has been verified (YP1977) whether the subtilisin-like protease gene family members. Methods: (1) to extract total Pythium Guiyang RNA, in order to have obtained sequence (YP1977) as template primers were designed using the reverse transcription-polymerase chain reaction (RT-PCR) fragment was amplified by Pr1 gene ORF; (2) construct containing the Pr1 gene ORF sequence of green fluorescent protein gene ORF sequence of the recombinant plasmid pTWIN1-Pr1-EGFP, expressing transformed strain E.coli ER2566, IPTG-induced expression of Pr1 protease strain. Results: The successfully constructed, containing the Pr1 gene ORF sequence of green fluorescent protein ORF sequences of the recombinant plasmid pTWIN1-Pr1-EGFP, achieved the Pr1 gene expression in Escherichia coli activity. Conclusion: The evidence has been obtained DNA sequence is one of Guiyang Pythium Pr1 gene coding region. [Keywords:] mosquito control; subtilisin category; reverse transcription polymerase chain reaction; genes, fungi; Guiyang Pythium; class iturins protease; prokaryotic expression [Abstract] Objective: To verify if a DNA fragment we obtained from Pythium guiyangense Su before is the ORF of a subtilisin-like protease (Pr1) of P. guiyangense, a fungal pathogen of mosquitoes. Methods: A prokaryotic expression system was established: The total RNA of P. guiyangense was extracted at the time point of most abundant expression of Pr1, and cDNA was obtained with reverse transcription PCR. The subject sequence was cloned. A plasmid pTWIN1-Pr1-EGFP containing the sequence as well as an EGFP gene was constructed, and transformed into ER2566 cells. The gene expression was observed after IPTG induction. Results: Green fluorescen