Cloning Storage Protein (SP3) Gene From Bombyx Mori And Researching SP3Regulate Melanization Reaction

Storage protein (SP) is storage protein in Bombyx mori.Usually synthesis in fat bodyof larvae and then released into the blood lymph.Taking silkworm as material, theStorage protein gene(sp3)of silkworm utilizing cDNA derived from fat body of silkwormas template was cloned and analyzed the characteristics of amino acid sequence. comparedwith other insectsamino acid sequence，tested the sp3genes on the distribution of the ageof4and5instars silkworm three days in the fat body, hemolymph,midgut, silkglands,testis,ovary,epidermis,tested the sp3genes on participating melanization reactioninduced by exogenous siRNA，the main results were as follows:1cloning and sequence analysis of sp3from Bombyx moriSp3was amplified by PCR utilizing cDNA derived from fat body of silkworm astemplate. The sequence analysis displayed that the open reading frame of Bmsp3gene witha length of2091bp coding696amino acids; with a predicted signal peptide consisting ofthe first27-74residues. The calculated molecular mass and isoelectric point of the matureprotein were82.10kDa and5.90, respectively.The nucleic acid and amino acid sequence of Bmsp3was aligned with otherpreviously identified insect storage protein,such as Manduca sexta, Helicoverpaarmigera,Papilio xuthus,the results showed that the nucleic acid sequence alignmenthomology is above75%,and Bombyx mori sp3mature protein revealed the highestsimilarity with amino acid sequence of them. As with beta-aryl storage protein subunitshomology was62.5%, aryl storage protein homology is above53.4%, the larvae serumprotein1homology of60.6%.2Bmsp3tissue specific expressionAnalysis Bmsp3transcription level of the age of4and5instars silkworm three daysin the fat body, hemolymph,midgut, silk glands,testis,ovary,epidermis,the results showedthat sp3in the silkworm larva stages organizations all have distribution, the most abundantin the blood and the fat body, and5instars of the content of sp3to4age more generally. 3sp3to participate in the silkworm melanization reaction induced by exogenoussiRNAwe measured PO activity in Bombyx mori larval hemolymph after siRNA fragmenttransfection of silkworm0h,12h,24h,48h，results show that the activity of PPO insiRNA fragments were increased after processing, in dealing with a24h after the highestactivity. After the segments of siRNA interference detection of silkworm melanizationreaction relatedgene(BmPPAE,Bmserpin-3,BmBGRP1,BmBGRPB2,BmPPO2,BmDDC)expression level inthe blood, the results showed BmPPAE, Bmserpin-3expression quantity on the rise, andBmBGRP1BmBGRPB2expression quantity declined, BmPPO2, BmDDC expressionquantity slightly decreased.