Genetic Manipulation Of Autophagy Related Genes In Bombyx And Its Effect On Lipid Metabolism

MacroAutophagy(hereafter referred to as autophagy)is a ubiquitous physiological phenomenon of self-eating in eukaryotic cells.Studies have shown that autophagy is highly conserved in evolution,orthologs of to autophagy related genes can be found from yeast,Drosophila to mammals.A series of autophagy related(ATG)proteins are involved in autophagy occurance.In mammals,a close connection is reported between some ATGs and lipid metabolism,and studies have shown that lipophagy is induced by upstream signal or genetic manipulation of some Atg genes.At present,there is no report on the relationship between autophagy and lipid metabolism in insects,and our preious study showed that BmAtg1 RNA interference(RNAi)blocked the lipid degradation in Bombyx fat body.However,the detailed mechanism underlying ATG protein in regulating lipid degradation have not been elucidated clearly and their interactions is also not well documented in insects.This thesis conducted RNAi and microinjection of sgNRA plus Cas9 mRNA to reveal the function of ATG proteins in lipid degradation in Bombyx.Moreover,stable Atg gene transgenic silkworm was preliminarily estabolish here for further deep studies.The main results are as follows:1.Bioinformatic analysis of Bombyx ATG proteinsBioinformatics of ATG proteins were analyzed and the results were as below:BmATG1 is an unstable protein and it harbors a protein kinase domain but not transmembrane region.BmATG5 and BmATG6 both are unstable protein without transmembrane region even other relative functional domain.BmATG8 has a transmembrane region between 75-100 amino acids.It is predicted as a transmembrane protein and whole sequence is an intact ubiquitin domain.BmATG13 is a stable protein with a transmembrane region between 120-140 amino acids as well as a HORMA domain.2.RNAi of Atg gene caused reduction of autophagy,lipid droplet accumulation in fat body and abnormal development of silkworm.Twelve hours before the initiation of wandering,the larvae were injected with the dsRNA of key Bombyx Atg genes including 1,5,6,8,13 in vivo.After the normal feeding for 24 h,the fat body was collected and detected for autophagy by Lyso-tracker Red,TEM etc..Results showed that Atg gene RNAi decreased autophagy and lysosome acidification significantly compared to the control,as well as blockage of lipid degradation,thus accumulation of lipid droplets in fat body,which lead to decrease of lipid content in the hemolymph,as well as increased lipid content in fat body.Atg gene RNAi led to abnormal development of silkworm at different developmental stages,with the highest arrest of development during larval-pupal metamorphosis.3.CRISPR/Cas9 knockout of Atg gene reduces autophagy in fat body and leads to accumulation of lipid dropletsKnockout of BmAtg1,5,6,8,13 genes was performed by injection of their sgRNA together with Cas9 mRNA into silkworm eggs.The hatched larvae were raised to early wandering stage,and collected for fat body to detect autophagy by Lyso-tracker Red,TEM detection and neutral lipid by BODIPY staining..Results showed that autophagy was significantly reduced,in contrast lipid droplet accumulated dramatically in fat body,thus proving that knockut of Atg gene reduced the lipid degradation in Bombyx.Moreover,the less weight lose was also detected in male or female Atg gene-knockout pupa,indicated that blockage of autophagy inhibited lipid degradation,resulting in heavier weight.4.Preliminary Establishment of stable transgenic silkworm strainsBy constructing UAS/GAL4 and CRISPR/Cas9 system,combined with silkworm egg microinjection,we established a stable transgenic silkworm strain.We have constructed the vectors as follow:In the UAS/GAL4 system:pBac[3×P3EGFP,Hsp70promoter-GAL4]?pBac[3×P3EGFP,LP3promoter-GAL4]pBac[3×P3DsRed,UAS-BmAtg1]?pBac[3×P3DsRed,UAS-BmAtg5]?pBac[3×P3DsRed,UAS-BmAtg6]?pBac[3×P3DsRed,UAS-BmAtg8]?pBac[3×P3DsRed,UAS-BmAtg13].In the CRISPR/Cas9 system:pBac[3×p3DsRed,Hsp70-Cas9]?pBac[3×p3DsRed,LP3-Cas9]?pBac[3×p3EGFP,BmAtg1sgRNA]?pBac[3×p3EGFP,BmAtg1sgRNA]?pBac[3×p3EGFP,BmAtg5sgRNA]?pBac[3×p3EGFP,BmAtg6sgRNA]?pBac[3×p3EGFP,BmAtg8sgRNA]?pBac[3×p3EGFP,BmAtg13sgRNA].The recombinant vector plasmid and helper plasmid were mixed for microinjection,and the hatched larvae were raised in normal condition,identified as G0 generation,and their offsprings are termed as G1 generation,which were conducted for fluorescent screening,and 5 positive transgenic lines were obtained,including CRISPR/Cas9 system:pBac[3×p3DsRed,LP3-Cas9];UAS/GAL4 system:pBac[3×p3DsRed,UAS-BmAtg5]?pBac[3×p3DsRed,UAS-BmAtg6]pBac[3×p3DsRed,UAS-BmAtg8]?pBac[3×p3DsRed,UAS-BmAtg13].