Cloning, Expression And Feeding RNAi Analysis Of Serpin Gene From Cotton Bollworm

Serine protease inhibitors(Serpins) are the largest and most broadly distributed superfamily of protease inhibitors which can centrallycontrol many important proteolytic cascades and affect protein metabolism by inhibiting serine protease activity, so it perform a variety of biological functions in the organism.In insects, the serpins participate in the innate immune response mainly by adjusting the melanization reaction and Toll pathway. In plants, the serpins are important defense proteins to resist the infection of pests and pathogens for it can impede the growth and development of insects. It has became a new kind of insecticidal protein and also became a target protein which researched and applied more and more widely in the plant gene engineering because of its broad insect-resistant spectrum and unique mechanism.Obtaining serine protease inhibitor genes is the foundation of researching their structure, function and mechanism. In this paper, we successfully cloned several new serine protease inhibitor genes from Helicoverpa armigera and preliminarily studied the basic characteristics, distribution and function of these genes bybioinformatics, Real-Time PCR and RNAi. The assay results are as follows:(1) Through aligned with the reported serpin gene of Bombyx mori. three serine protease inhibitor genes which had high similarity with the serpin of Bombyx mori were successfully cloned from Helicoverpa armigera by RT-PCR and respectively named Haspi-5、Haspi-6 and Haspi-10. Using Bioinformatics techniques, we analyzed the c DNA sequence and amino acid sequence of Haspi-5、Haspi-6 and Haspi-10 and constructed Phylogenetic tree of serpin from H. armigera and other insects.(2) Using gene recombination technology, prokaryotic expression vectors p ET17b-Haspi-5, p ET17b-Haspi-6 and p ET17b-Haspi-10 was constructed. Then inducing its expression in Escherichia coli BL21(DE3) with 0.8 mmol/L IPTG for 5 h at 30 ℃. Prokaryotic expression results showed that high efficient expression of Haspi-5 and Haspi-6 protein could be realized. SDS-PAGE analysis showed that the fused protein mainly existed as inclusion bodies and the molecular weigh of protein bands were consistent with prediction, however, the expression of p ET17b-Haspi-10 was not detected.(3)The transcription level of Haspi-5, Haspi-6 and Haspi-10 in various tissues of the 5th instar H. armigera larvae were detected by real-time quantitative PCR. The results revealed that the transcription levels of three genes were all observed in every tissues, but existed significant differences in different tissues. Haspi-5 gene had the highest expression levels in epidermis; Haspi-6 gene transcription level in head was much higher than other tissues; Haspi-10 gene had the highest expression levels inforegut and thetranscription level in midgut also higher than other tissues.(4)Interference vector p L4440-Haspi-5 and p L4440-Haspi-6 were successfully constructed. we carry out RNA interference to Haspi-5 and Haspi-6 gene from H. armigera larvae of the second instar by the way of live bacteria feeding. The results showed that the mortality rate of Helicoverpa armigera had increased after interference and real-time quantitative PCR revealed that the transcription level of Haspi-5 gene in Helicoverpa armigera decreased significantly, about 6.5 times, while the transcription level of Haspi-6 gene decreased slightly but not significant, about 1.2 times after RNA interference.