Studies On The Mechanism Of Bombyx Mori Vps13d In Affecting The Silk Yield

Vacuolar protein sorting-associated proteins(VPS)is a proteins family that can specifically recognize,sort and transport cargo in cells.Intra-cellular transport of lipid and protein cargoes,mainly in and out of the plasma membrane,is precisely regulated to maintain the integrity and dynamics of the cell and its organelles.VPS proteins play an important role in cargo sorting and degradation in cells,and are of great significance for growth and development of cells.It has been reported that Vps13 d protein has conserved developmental regulatory functions in multiple species.Although Vps13 d protein has been reported in other species,its functional analysis has not been reported in Bombyx mori.In this study,the whole genome sequencing results of 137 representative silkworm strains with different cocoon characters previously available in our research group were used to find out Bombyx mori Vacuolar protein sorting associated protein 13d(BmVps13d)gene that might be related to the silk yield of B.mori.The biological functions in B.mori were studied by using CRISPR/Cas9 genome editing technique for the first time.The main research results are as follows:1.In this study,to preliminarily explore the function of BmVps13 d in B.mori,bioinformatics analysis and coding region sequencing of this gene was conducted.Homology comparison results of Vps13 d proteins showed that the Vps13 d proteins had the same functional domains in B.mori,Drosophila melanogaster and Homo sapiens,and BmVps13 d protein shared 28.70% and 28.18% identity with that of in D.melanogaster and H.sapiens,respectively.The results suggested that the three proteins might have similar functions.BmVps13 d sequences of JS(high silk quantity)and L10(low silk quantity)strains were compared,and the results showed that there was no difference between them,indicating that the coding region of this gene had no effect on the silk yield of JS and L10.2.To further explore the functions of BmVps13 d in B.mori,real-time fluorescence quantitative PCR(RT-q PCR)and promoter sequencing were used to detect the relative expression level and the difference of putative promoter sequence of this gene in JS and L10,respectively,and the difference of promoter activity was verified using double luciferase reporting assay.The result of RT-q PCR showed that BmVps13 d was significantly differentially expressed in the silk gland and midgut of JS and L10 strains at the third day of the fifth instar.The promoter region of BmVps13 d was predicted.The sequencing result showed that compared to JS strain,an insertion of 9 bp nucleotides and two deficiencies of adenine ribonucleotides in the putative promoter region of BmVps13 d in L10 strain.Therefore,we speculated that the difference in the predicted promoter region sequence of this gene might affect the promoter activity.Double luciferase reporting assay was conducted.The predicted promoter regions of BmVps13d(2.0 KB)from JS and L10 strain were respectively cloned and transfected into BmN cells.The results of double luciferase assay showed that the predicted promoter activity of BmVps13 d in JS strain was significantly higher than that of L10 strain in BmN cells.3.The transgenic plasmid p XL[IE1-DSred-U6-BmVps13d-sg RNA] was constructed by single-fragment homologous recombination,and △BmVps13 d mutant strain was screened out successfully.Targeted mutation of BmVps13 d gene in B.mori was induced by CRISPR/Cas9 gene editing technique.The sequencing result showed that the mutation of BmVps13 d gene mainly occurred near the adjacent motif(PAM)region in the prototypic spacer region,and the deletion of different number of bases appeared in this region.The investigation results of the ΔBmVps13 d mutant phenotypes showed after BmVps13 d mutation,the body weight of △BmVps13 d mutant decreased by 24.7%,the cocoon weight of △BmVps13 d mutant decreased by 27.1%(female)and 11.8%(male),and the cocoon layer ratio of △BmVps13 d mutant decreased by 2.4%(female)and 2.9%(male).The results of RTq PCR showed that BmVps13 d decreased the expression of sericin gene(ser-1)in △BmVps13 d mutants,suggesting that BmVps13 d might be involved in silk protein synthesis and played a significant role in the growth and development of silkworm.This study provided a clue to explore the molecular mechanism affecting the silk yield,and was of great reference value for the selection and breeding of silkworm with high quality.