Gene Cloning And Function Analysis Of Peptidoglycan Recognition Proteins And Antimicrobial Peptides Of Cotesia Vestalis Teratocyte

Wasp parasitoids use a variety of methods to regulate host physiology and biochemistry to create a favorable environment for the parasitoid progeny’s development. Teratocytes, cells released at egg hatch from extra-embryonic serosal membranes of some wasp parasitioids, is one of the important parasitic factors. Manipulation of teratocytes on host mainly goes by serving as a nutrient source for the parasitioid lavae, arresting the growth and development of the host and triggering immunosuppression of the host. Antimicrobial peptides are important factors of the innate immunity in insects. When insects are infected by microbes, a large number of antimicrobial peptides with biological activity against bacteria, fungi, virus and even some cancer cells are synthesized and released rapidly by curtain tissue or organ of insects. In this thesis, we cloned six antimicrobial peptide genes based on the selected EST sequences of anti-microbial peptide genes, and we detected the RNA transcriptional levels when teratocytes were induced by different microbes with rt-qPCR. We also did a preliminary study on their function. The results were summarized as follows:1) Based on Nr annotation of teratocyte transcriptome,18EST sequences were matched to10known antimicrobial peptides families.15genes were verified by reverse transcript PCR, including defensin, lysozyme, hymenoptaecin, and peptidoglycan-recognition protein (PGRP).2) Six open reading frames (ORFs) encoding antimicrobial peptides were cloned by the methods of rapid application of cDNA end (RACE), including3defensin genes,2PGRP genes and1crustin gene. The ORFs of CvTdef-A, CvTdef-B, CvTdef-C, CvTpgrp-SA, CvTpgrp-LC and CvTcru were369bp,222bp,237bp,336bp,525bp and426bp long, which encoded putative proteins of123,74,79,112,175and142amino acids with calculated molecular weights of13.25kDa,7.97kDa,8.56kDa,12.69kDa,35.29kDa and16.39kDa. The analysis of sequences characteristics predicted that CvTdef-A, CvTdef-B and CvTdef-C contained signal peptides and six conserved sites of cysteine.3) We investigated the transcriptional patterns of CvTdef, CvTpgrp and CvTcru genes in microbe-challenged teratocytes. Escherichia coli, Bacillus subtilis and Monilia albican were used to challenge the teratocytes for6h,12h,24h and48h respectively. The real-time PCR analyses showed that six genes were all positively regulated and over-expressed after microbial induction. The transcriptional patterns of different genes were different, but the trend of transcriptional levels was similar, reaching the peak level and going down afterwards. The relationship between transcriptional patterns of these genes and microbes was different. The transcriptional levels of three defensin genes and CvTpgrp-SA induced by E. coli and B. subtilis were higher than that induced by M. albican. The transcriptional levels of CvTpgrp-LC induced by bacteria and fungus were similar. The up-regulated transcriptional level of CvTcru induced by B. subtilis was higher than induced by E. coli and M. albican. The up-regulated transcriptional level of CvTdef-C was highest among three defensin genes when induced by the same microbe for the same time. The up-regulated transcriptional level of CvTpgrp-LC was higher than CvTpgrp-SA.4) The inhibitory activity of the synthetic CvTdef-A was verified by detecting the growth curve of bacteria. The results revealed that the synthetic peptide of CvTdef-A had an antimicrobial activity both on E. coli and B. subtilis, and the inhibitory effect on E. coli was more powerful than on B. subtilis.In order to investigate if the teratocytes really have antimicrobial function in the later stage of host larvae, a microbial infection experiment was designed. We used the following microbes:E. coli, Pseudomonas aeruginosa (Gram-negative bacteria); Staphylococcus aureus, B. subtilis (Gram-positive bacteria); M. albican and Beauveria bassiana (fungus). Three different treatments were conducted for host larvae:non-parasitization, normal parasitization and pseudo-parasitization (the female parasitoids with radiation treatment could lay eggs into host larvae but the eggs were not able to hatch, resulting in no release of teratocytes). Then all the host larvae were infected by the microbes mentioned above. The results revealed that the mortality of pseudo-parasitized host larvae was significantly higher than the normal-parasitized ones, indicating that teratocytes might be involved in antimicrobial activity.