The Cloning, Prokaryotic Expression And Temporal Expression Pattern Of Serine Proteinase Inhibitor From Bemisia Tabaci

Bemisia tabaci has a wide range of host plants, and also has a strong resistance to chemical pesticides. These characteristics make it one of the most remarkable invasive pests in China. It has caused huge losses to agricultural production. Chemical pesticide abuse leads to pesticide residue which is a major threat to food safety and ecological balance. Research about pesticides that are environmental friendly and specific to target insects, is becoming an urgent task. Serine Proteinase Inhibitor(Serpins) is involved in a series of proteinase dependent reactions in organisms. Serpins mainly suppress excessive melanization, and play a role in innate immunity in insects. The purpose of this study is to clarify the structure and function based on the immune system of B. tabaci, and develop new pesticides that target on immunity pathways. RT-PCR and RACE technology was conducted to obtain the full-lenth c DNA sequence from unigene predicted as putative serine protease inhibitor 4 on the basis of transcriptome. QRT-PCR was employed to detect the stage-specific expression model of Serpin in B. tabaci. Prokaryotic expression was also conducted in Escherichia Coli. All these experiments provided theoretical foundation for the future study of immune system of B. tabaci.The main results are as follows:1. The unigene in B. tabaci transcriptome that is predicted as Serpin was cloned, and then its full-lenth c DNA sequence was obtained through RT-PCR and RACE technology.After that, the c DNA sequence was uploaded to Genbank, its accession number is No.KT326952. The length of Serpin c DNA is 1899 bp, and its open reading frame(ORF) is1206 bp, this ORF encodes 401 amino acids. Align the deduced amino acid sequences with the sequences of serpins from other insects, and then construct a phylogeny tree. The result indicted that the amino acid sequence of B. tabaci is highly homologous to those Hemiptera insects such as Diaphorina citri and Nilaparvata lugens.2. To elucidate the expression pattern of Serpin from B. tabaci at the level of nucleicacid, q RT-PCR was performed to detect the relative expression of serpin in eggs, 1-4thinstar nymph and adults. The result shows that serpin is expressed at all stages. The expression level of eggs and adults is significantly higher than other stages, while in 4thinstar nymph,the expression is much lower than other stages. In a word, Serpin is differential expressed in B.tabaci of distinct developmental stages.3. Expression vector pET-32a-Serpin was constructed and then transferred to E. coli BL21(DE3) strain. Large amount of protein expression was induced by IPTG. Results of SDS-PAGE and Western blot indict that pET-32a-Serpin expressed protein with relative molecular weight of about 45 k D. We used Ni-NTA affinity chromatography and gel extraction to purify serpin. It provided material for the research of structure and function of serpin. Animals was immunized with purified serpin to produce polyclonal antibody.Western blot was performed to reveal the stage-specific differential expression of serpin at protein level in B.tabaci, which verified the result of q RT-PCR.