| Bombyx mori is not only a kind of crucial economic insect,but also one model organism of lepidopteran insects.Besides,it plays an important role in physiological,genetic,pathological and immunological fields of insects.Beauveria bassiana,a pathogenic fungus,has serious harms to Bombyx mori.As a result,the infected Bombyx mori cannot cocoon or pupate anything,which causes great losses to production and economical effect of sericulture.In this study,by using digital gene expression profile constructed by B.bassiana to infect Bombyx mori larvae that is at the 3th star,filtrating stored protein gene----BmSP2?Bombyx mori sex-specific storage-protein 2,SP2?with specific expression,as well as cloning,expressing and studying its immunologic function of B.bassiana.The experimental results are as follows:1.Cloning Open Reading Frame?ORF?of BmSP2,expressing BmSP2 protein by prokaryotic system,and verifying inhibitory effect that it has against B.bassiana.Then,regarding BmSP2 as an object of research,and using Bombyx mori P50 as experimental material.By RT-PCR to clone and gain the gene's order of ORF,of which the order has2112 basic groups and encodes 703 amino acids,and among them,from the 1st to the 18th,the amino acids are signal peptides.Ligating BmSP2 gene into pET-28a expression vector to construct pET-28a-BmSP2 vector,and succeed in inducible expression of soluble target protein at concentration of 0.8 mM IPTG and at 16°C.Besides,measuring concentration of BmSP2 protein is 200?g/mL by using standard solution of BSA.After using solid bacteriostatic test,it is found that although the filter paper of BmSP2 solution is not on itself,and the amount of protein increases,the filter paper of 0.01%Tween-20 solution that in negative control group is overgrown with B.bassiana after 40-hours'culture.The range of inhibition also enlarges.In liquid bacteriostatic experiment,when cultured at 25°C for 8h,spores of B.bassiana in culture dish of 0.01%Tween-20 solution have basically germinated,and hyphae grow longer,no matter whether the BmSP2 solution in culture dish is B.bassiana or not.And speed of spore germination is slow.In a word,it indicates that BmSP2 protein has an inhibitory influence on B.bassiana.By real-time fluorescent quantitative technology----PCR,constructing temporal and spatial expression profiles of BmSP2 gene in Bombyx mori,and detecting change of expression of this gene of Bombyx mori after infecting by B.bassiana.Among expression profiles of BmSP2 at different developmental stages of Bombyx mori,it is the highest point at final instar of a larva,and the lowest one at stage of egg,pupa and adult.Each stage of larvae,the number of expression will reach a peak on the day after last day's lunch.In addition,among these tissue expression profiles,the largest number exists in fat body,and next are epidermis,ovary and testis,but the other tissues are barely expressed.Later,detecting expression of BmSP2's five stages at fat body and it is found that the highest level exists in the 4th day,and then begin to decline.Comparing with normal Bombyx mori,the expression level of BmSP2 of Bombyx mori that are infected by B.bassiana increases,especially after 16h of being infected.3.Constructing transgenic Bombyx mori to verify inhibitory function of BmSP2 that has against B.bassiana.Ligating specific promoter A3 and enhancer HR5 sequences into vector pBAC-HSKS,and building an overexpression vector of HR5-A3-pBAC-HSKS.After adding 8His tag in front of BmSP2 sequence,connect this fragment into vector,and take advantage of PCR,double digestion and sequencing to verify overexpression vector of HR5-A3-BmSP2-pBAC-HSKS that has been established.After extracting and purifying target plasmid,then inject it into eggs of Bombyx mori,and via red fluorescence to select transgenic Bombyx mori.Comparing survival time and phenotype of transgenic Bombyx mori and normal one that are both infected by B.bassiana,to test whether BmSP2 had inhibitory effect on B.bassiana... |
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