Transcriptome Sequencing Of Antheraea Pernyi Silk Gland,and Cloning And Expression Analysis Of A. Pernyi Cocoon Color-related Genes

The Antheraea pernyi silk,which is mainly composed of the fibroin and sericin proteins,is produced by silk gland.Generally,the outer layer of A.pernyi cocoon is naturally brown.The colourful silk for daily use comes from chemical stainin,which might cause environmental pollution.Nowadays,there is no way to produce naturally colourful silk.To dig out some genetic resources underling cocoon color from A.pernyi genome and lay a foundation for the improvement of cocoon color in the future,the full-length(FL)transcriptome of A.pernyi silk gland was first conducted to improve the structure annotations of genes expressed in the silk gland.And then,the structure of A.pernyi fibroin heavy chain gene(ApFibH)was analyzed,and its promoter was cloned.After that,the transgenic vector with the expression of green fluorescence protein gene(EGFP)drived by the promoter of ApFibH was constructed.In addition,the homologous genes of Bombyx mori carotenoid-binding gene(BmCBP),which controls the transport of carotenoids into B.mori silk gland,was identified and cloned from A.pernyi.Moreover,a transgenic vector controlling the specific expression of ApCBP in silk gland was also constructed,which built up a basis for stuying the formation mechanism and improvement method of the cocoon color in A.pernyi.The main results are as follows:1.Based on the FL transcriptome of A.pernyi silk gland,the structure annotations of genes expressed in silk gland were improved.A total of 12,572 FL transcripts were identified to be transcripted from 7,568 genes.Thereby,1,121 long chain noncoding RNAs(lnc RNAs)and 2,569 transcriptor factors(TFs)were discovered.These TFs were the members of 68 different TF families.There were 1,492 alternateive splicing events(ASs)and 3,068 alternative polyadenylation events(ASAs),which happened during the transcription and processing of the expressed genes in A.pernyi silk gland.These genetic resources lay a foundation for stuying the formation and improvement of the cocoon color in A.pernyi.2.After analysis,we found that ApFibH located on the chromosome 47,and had2 exons and 1 intron.Furthermore,its promoter was cloned,and three transgenic vectors were simultaneously constructed using the ApFibH promoter to drive the expression of EGFP gene,including pBac[3xp3EGFP-ApFibHp2-EGFP-hpA],pBac[3xp3EGFP-ApFibHp-EGFP-ser1 pA],and pBac[3xp3EGFP-ApFibHp-EGFP-hpA].These results have built up a basis for studying the driving potential of ApFibH promoter to the expression of exogenous gene and its application on the improvement of cocoon colour.3.After cloning the homologous genes of BmCBP in A.pernyi,ApCBP,we found that ApCBP located on chromosome 13,and contained 6 exons and 5 introns.The gene encoded a protein with the length of 297 amino acids.Although the protein possessed a START domain,which has the function of transporting carotenoids,ApCBP didn’t express in the A.pernyi silk gland.These might be the reason why the cocoon colour of A.pernyi was naturally brown.To study the role of ApCBP in the formation and improvement of A.pernyi cocoon colour in the future,we have constructed a transgenic vetor to control the specific expression of CBP in silk gland.4.We also tried our best to build the process of embryo microinjection in A.pernyi.In the end,the hatchability rate of eggs after microinjection has already reached 80%,which could meet the need of the performation of A.pernyi transgene,and were also the good foundation for choosing the positively transgenic individuals,studying the activity of ApFibH promoter,clarifying the transportation function of ApCBP to carotenoids,and improving the cocoon colour of A.pernyi in the future.In conclusion,the study has first established the map of FL transcriptome of A.pernyi silk gland and also improved the structure and functional annotation of genes expressed in silk gland.In addition,two genetic resources(ApFibH promoter and ApCBP),which might have role in the cocoon colour of A.pernyi,were analyzed and cloned,and their transgenic vectors were also constructed.Finally,a primary process of A.pernyi embryo microinjection was also built.These results have laid important foundations for stuying the formation mechanism and improvement of the A.pernyi cocoon color.