Function Study Of Serine Protease Ser2 And Osp In The Regulation Of Reproduction In Bombyx Mori

Insects are the most numerous group of animals on earth,and they have strong reproductive fecundity.Gonads,as important internal genitalia,secret variety of proteins and function at diversity ways.These proteins affect not only male reproductive ability,but also the physiological process and behavior of female reproduction.Lepidoptera is the second largest Order in the class of insects after Coleoptera,among which the silkworm,Bombyx mori is the lepidoptera model insect due to its clear research background and important economic significance.The study on secretory proteins of gonads of silkworm,which can reveal the roles of growth,development and reproduction,and help to understand the insect life history and evolutionary process.The relevant research results can not only be applied to the traits improvement of beneficial insects,but also can be extended to the biological control of pests.This study focused on the gonad transcriptome of silkworm and the function of its gonad specific-expression genes.The male sterility and female sterility mutant lines were constructed by using the transgenic CRISPR/Cas9 technology.The main results contain the following aspects: 1.Transcriptome analysis of silkworm gonadsIn silkworm,the gonads include testis(TE),Ovary(OV)and the accessory glands(AG).According to gender differences,accessory glands include male accessory gland(MAG)and female accessory gland(FAG).The gonads contain a variety of secreted proteins related to mating and reproduction.In this study,we selected TE,OV,MAG and FAG of silkworm virgin adult for transcriptome sequencing(RNA-seq).The analysis showed that there were 9385 genes expressed in the testis;4552 genes expressed in ovary;4054 genes expressed in male accessory gland;8205 genes expressed in female accessory gland;2923 genes expressed in the four tissues.Gene functions and metabolic pathways were diverse,and they accurately regulated reproductive process in different ways.2.Construction of Ser2 mutant and analysis of sperm regulation in silkwormThe protein encoded by the Serine protease 2(Ser2)gene is a member of serine protease family and an important component of seminal fluid proteins(SFPs),which is relatively conserved in evolution.We used CRISPR/Cas9 technology to knock out the Ser2 gene in silkworm.Disruption of Ser2 resulted in heritable male sterility.We stained sperm bundles and sperm of Ser2-mutant and wild-type individuals and the results showed that there was no significant morphological difference compared with control.Staining eggs that were laid after 5 hours of mating.In the control,sperm and egg nuclei were observed immediately after egg deposition,and cleavage nuclei were visible at 5 and 10 h post-mating.In contrast,when wild-type females were mated with mutant males,only nuclei from eggs were observed,and no cleavage nuclei were observed.This indicates that the deletion of Ser2 gene in silkworm,resulting in the failure of the sperm to enter the egg normally and ultimately causing male sterility.3.Construction of Osp mutant and analysis of oogenesis regulation in silkwormThe protein encoded by the Ovarian serine protease(Osp)gene is a member of the serine protease family.Similarly,we used CRISPR/Cas9 to knock out the Osp gene in silkworm.We found that the disruption of Osp gene resulted in heritable female sterility.We analyzed the ovaries of Osp-mutant and wild-type individuals at the wandering stage Mutations of Osp gene resulted in morphological changes in the ovaries and irregular oviducts.In the virgin female mutants,the oviducts were shrunken and were shorter than those of control.We observed abnormal eggs in the oviducts of the mutants,ultimately leading to female infertility.In summary,the study explored the reproductive regulatory role of gonadalsecreted proteins in silkworm.The reproductive function of Ser2 and Osp genes were analyzed by using the CRISPR/Cas9 technique.To explore the key genes that have no effect on competitiveness,attractiveness,growth and development but lead to male or female sterility.It can provide potential targets for the construction of novel sterile insects and developed new strategies for biological pest control.