A Study On The Female Infertility Of Black Dilute Mutant Of Silkworm,Bombyx Mori

Strong reproductive capacity is an important foundation for insects to survive,and oogenesis,as a key issue of insect reproduction,is a hotspot in developmental biology.Oogenesis provides an important basis for late embryonic development: in which oocytes meiosis to form a female pronucleus to provide gametes for fertilization;the yolk proteins were absorbed and stored by the oocyte in egg,which provides nutrients for the development of fertilization embryos;morphological construction of egg provides a stable place for embryonic development.It has been found that the oogenesis of insect is involved with complex molecular regulation networks.Studying on the molecular level of the oogenesis process can provide a basis for further understanding the genetic mechanism of reproduction and embryonic development,and also provide clues for exploring insect reproduction and evolution.Silkworm is an important agricultural economic insect and has important historical and cultural values.Since the beginning of the 20 th century,generations of collection and preservation,mutagenesis to create preserve,and has accumulated a wealth of mutants,which provide valuable resources for biological research.With the publication of silkworm genome data and long-term biological concern,it is used as a Lepidoptera model insect for the biological study of Lepidoptera insects.In this study,we used black dilute(bd)as the material,and the bd mutant larvae intugement are significant melanism and females are infertility.In the previous study,the mutant gene responsible for the mutant was identified by means of localized cloning and functional validation,which was a transcription factor encoding the BTB-ZF type,Bmbd.In this study,we conducted a series of phenotypic analysis of the bd mutant,and identified the key reason of female infertility is oogenesis anomaly,meanwhile,and analyzed the critical period of the oogenesis developmental defect;then we analyzed the Bmbd gene by molecular biology,bioinformatics and cell biology to explore how it affects the female infertility of silkworm bd mutant.Studies on this mutant can provide a reference for exploring the mechanism of female reproductive development and oogenesis in Lepidoptera insects,and also provide potential targets for the control of Lepidoptera pests.The main findings are as follows: 1.Analysis of the causes of bd sterile mutant in silkwormWe expore the key reason of female infertility by comparing and analyzing the biological processes of female reproductive development,oogenesis and mating fertilization of wild type and bd mutants of silkworm.The reproductive system development and oogenesis process were analyzed through observing the female reproductive system dissection and ovariole DAPI staining of the bd mutant,and results showes no significant difference compared with the wild type;combined with the results of scanning electron microscopy and fertilized egg DNA staining,we found that bd mutant micropyle channel abnormalities lead to sperm unable to enter the eggs;artificial parthenogenesis test showed that development potential of bd mutant egg was defects.In summary,the main factors resulting in bd mutant sterility was the micropyle channel defects and egg development potential defects.The formation of micropyle channel is related to the formation and migration of border cells of egg chamber,suggesting that abnormalities in the bd mutants may be involved with the formation or migration of border cells.2.A analysis of temporal and spatial expression patterns of Bmbd gene in the oogenesis process of silkwormBmbd gene was the responsible gene of silkworm bd mutant.To analyze the function time and location in the oogenesis process of silkworm,we draw materials of ovary and different developmental stages egg chamber of ovariole respectively,and the Bmbd gene was detected by fluorescence quantitative PCR and in situ hybridization in the process of ovarian development and oogenesis.The results of fluorescence quantitative PCR showed that the Bmbd gene had a highly relative expression level in the critical period of germ cell differentiation(before and after fourth instar larva)and the mature stage of egg chamer in the oogenesis process(early fifth instar larva and wandering stage to pupal stage)of silkworm;the further in situ hybridization experiments showed that the Bmbd gene was highly expressed in the early stages of oogenesis,and expression was mainly concentrated in follicular cells.3.Screening and analysis of upstream and downstream regulatory genes of Bmbd geneIn this part,to identify the genes that may interact with the Bmbd gene,we analyzed the transcription factor binding sites of the Bmbd gene transcription initiation region,and analysis the potential downstream gene by bd mutant transcriptome data screening.The binding sites of STAT and slow border cells,which closely related to the formation and migration of border cells,are found in the upstream of the Bmbd gene by genomatix.The Bmovo-1 gene associated with the oogenesis process and the BmHp1b-l gene associated with the chromatin formation were selected by screening the transcriptome data.The expression pattern of these two genes were analyzed in wild type,and then we analyzed their expression levels in key period during in the mutant.The results showed that the two genes had significant differences between the wild type and the bd mutants.Further analysis revealed that these two genes could be involved with the oogenesis processes of the bd mutants of silkworm,as the downstream target gene of Bmbd gene.It was further speculated that the functional defects of Bmbd gene in bd mutant could cause abnormal formation or migration of border cells,which then would cause damage of micropyle channel,and the Bmbd gene deficiency may affect the expression of Bmovo-1 and BmHp1b-l causing bd mutant reproductive abnormality.4.Verification of Bmbd gene function and identification of interaction proteinIn this part,we constructed the cell expression vector for two splicing variants of Bmbd gene.The results showed that Bmbd-1 protein mainly expressed in cytoplasm,while Bmbd-2 protein concentrated in the nucleus and periphery,the difference localization of the proteins encoded by the two splicing variants suggested that they have different modes of function.By constructing the overexpression vector of Bmbd gene in cells and synthesizing dsRNA,the Bmbd gene was overexpressed and interfered in the silkworm ovarian cell lines respectively.The results showed that the down-regulation of Bmbd gene could cause the migration of silkworm ovarian cells to be blocked,which further confirmed the Bmbd gene plays an important role in the migration of border cells.The Bmbd gene encodes a protein with a BTB domain,whereas proteins containing the BTB domain active in conjunction with other protein-forming protein complexes.Here we identified the binding of Actin and Ral-a protein,important proteins associated with border cell migration,by immunoprecipitation experiments.In this part,we confirmed that the Bmbd gene affects cell migration at the cellular level and may interact with Actin and Ral-a proteins to influence this process.