Studies On Inner Eggshell Proteins And A Lethal Egg Mutant (L-e~m) Of The Silkworm,Bombyx Mori

Oogenesis is a critical process for reproduction and population keeping in insect. In insect, the development of oogenesis is a successive process consisting of previtellogenesis, vitellogenesis, choriogenesis, oviposition and fertilization.Proteins are the major content of eggshell in insect. Eggshell is a complex specialized extracellular architecture which surrounds the surface of oocyte, and it is secreted by follicular epithelium cells (FCs) in ovarian tissue during the choriogenesis stage. The eggshell in insects has two major layers:the external chorion layer and the inner layer (vitelline membrane layer, VM layer). Eggshell functions in preventing embryo from physical injury, pathogen invasion, preventing loss of water, keeping gas change and determining the embryonic pattern. The structure and function of eggshell is a hotspot of biological research in insect.The studies of vitelline membrane proteins (VMPs) is limited in dipteran insects due to the divergence of them, this situation is unbalance to functional importance of VM layer. Besides it, the assembly process of a functional VM network, the component of VMPs, the expression profiles of VMP coding genes and the genomic location of VMP coding genes are sitll unknown biological issues.Here, we used silkworm, Bombyx mori, which is a model species for Lepidoptera as research material. After construction the profiles of silkworm ovary and egg proteins, comparative proteomics was then carried out to analyze the change pattern of the proteins. Bioinformatics analyses were used to screen the lepidopteran VMPs and VMP coding genes, and then compared the conservatism and diversity of VMPs in insects. Combining a lethal eggshell mutant (l-em), the function of a lepidopteran VMP-BmEP80was explored in depth. The main results are as follows:1. Proteomics analysis of egg and ovarian tissue in silkworm, Bombyx moriFollicle is the functional developing unit for oogenesis process in insect. In B. mori, the major developmental processes of follicles are completed during pupal stage, and the follicles display as an asynchronous developmental pattern within the ovarioles. Each follicle represents a unique developmental stage that differs from its immediate neighbors by approximately2-2.5h in the developmental program.The24h and48h fertilized eggs,24h and48h unfertilized eggs, diapause-destined and nondiapause-destined eggs, eggshell and egg content, and ovarian tissues from Day-7pupae from Dazao and small egg mutant (emi, sm, sm-2) strains were dissected. The extract proteins were displayed through two-dimensional gel electrophoresis (2-DE) and the resultant maps were made. Spots with high abundance in2-DE maps of ovarian tissues and24h fertilized eggs were selected for MALDI-TOF-MS analyses, and then33and42proteins were identified, respectively. Finally, the protein profiles of above tissues were constructed.Comparative proteomics analysis was carried out to compare the2-DE maps of the24h and48h fertilized/unfertilized egg proteins and five development-related spots were displayed. Two of them were identified by MALDI-TOF-MS for the first time in this study, and named as BmEP80and Peritrophin-like27. BmEP80was synthesized by ovarian tissues during the pupal stage and deposited within the eggs, and disappeared after fertilization. We then presumed BmEP80as an inner eggshell structure protein according to above features. Peritrophin-like27has three chitin-binding type2domain and appeared in48h fertilized eggs. This phenomenon suggested that Peritrophin-like27might function as a structure protein participating in the formation of organ or tissue during the early embryogenesis.Comparative proteomics analyses also showed the different protein spots between diapause-destined and nondiapause-destined eggs, and between Dazao and small egg mutant strains’ovarian tissues. The above information useful for understanding the formation of diapause and the genetic mechanism of small egg mutants.2. The research on expression profiles and function of the BmEP80Comparative proteomics analyses of egg proteins revealed BmEP80may act as an inner eggshell protein in B. mori. According to the genome information, BmEP80was predicted as a high molecular weight protein coding by an intronless gene. In this section, the expression pattern of BmEP80and its functions were investigated.Expression profiles analyses showed BmEP80specifically expressed in ovarian tissue during middle/late pupal stage and mainly at the late vitellogenesis/early choriogenesis stage (from-5to+10). In situ hybridization (ISH) indicated BmEP80was expressed in FCs, not in the oocyte of silkworm follicles. The expression profiles of BmEP80were coincided with the expression pattern of inner eggshell protein coding genes in insect.Western blot detection showed the expression of BmEP80began in～-10stage follicles.2-DE maps of eggshell and egg content extract proteins showed BmEP80present in eggshell extract, not in the egg content. Immunohischemistry analysis showed BmEP80located within a thin layer between the trabecular layer and the VM layer. All of above results indicated BmEP80is a high molecular weight inner eggshell protein coding gene in B. mori.The function of BmEP80was evaluated by RNA interference (RNAi). After injection of siRNAs, the mRNA level of BmEP80was decreased and the amount of BmEP80protein in eggshell was reduced～20%. The moths were normally copulated and laid eggs after RNAi treatment. But the eggs were obviously collapsed and appeared a triangle on the surface. The results indicated BmEP80was necessary to structural integrity of eggshell, and it might have a function in preventing loss of water.3. Prediction, expression profiles and functional domain analyses of the VMP coding genes in lepidoptern insectsThe studies of VMPs are limited in dipteran insects and only one of non-dipteran VMP (BmVMP30) has been reported. Sequence cluster analysis indicated BmVMP30show no similarity with dipteran VMPs.In Drosophila melanogaster, VMPs coding genes (VM26Ab, VM26Ac and VM26Aa) were clustered near to each other on26A position on the left arm of chromosome2. According to this characteristic, other three putative VMP coding genes were identified on the side of BmEP80, which indicated that a similar VMPs coding region present in B. mori. Putative VMP coding genes within this region were named as BmVMP25(-),BmVMP23(+), BmEP80(+) and BmVMP30.1(+).Expression profiles analyses showed BmVMP25, BmVMP23and BmVMP30.1 specifically expressed in ovarian tissue during pupal stage and expressed prior to early choriogenesis stage. The expression profiles of them were coincided with the expression pattern of inner eggshell protein coding genes in insect.Recently, the genome sequencing of three lepidopteran insects(Manduca sexta, Danaus plexippus, Heliconius melpomene) were completed. tBlastn analyses revealed the VMPs coding region in these lepidopteran insects. Four, three and four putative VMP coding genes were identified in VMPs coding region of M. sexta, D. plexippus and H. melpomene, respectively. In addition, the order of the putative VMP coding genes was preserved among the four lepidopteran insects. The present study revealed that the linear arrangements of VMP coding genes were conserved between the Diptera and Lepidoptera.Lepidopteran VMPs were classified into three groups (Group Ⅰ～Ⅲ) according to the sequence divergence. Alignment of the VMPs in Group I has enabled us to find a hydrophobic conserved region in their C terminus (35aa) and named as VM domain. The VM domain in lepidopterans possessed the first/second conserved cysteine residue (CX7C), and non-classical redox active site motifs (CXXS and SXXC) were identified in the cysteine residue. The presence of VM domain in non-dipteran insects was identified for the first time, which indicated that assembly of inner eggshell through intermolecular disulfide bonds is coincident among insects. Comparing the VM domain between the VMPs in Lepidoptera and Diptera, we found that the second cysteine residue was conserved in all VMPs and the first cysteine residue was absent in two lepidopteran VMPs. Interestingly, the third cysteine residue which is conserved in dipteran VMPs was totally lost in their lepidopteran counterparts. Above results suggested that the function of VM domain depend on the hydrophobic motif, CX7C and the non-classical redox active site motifs, but not the conservatism of amino residue sequence.4. The study of a lethal egg mutant (l-em) in silkworm, Bombyx moril-em is a recessive lethal egg mutant inherited as a pseudo-maternal trait. Its homozygous female and male individuals (l-em/l-em) are able to develop and mate normally. However, the eggs laid by the l-em/l-em females collapse within1hour due to the loss of water. The phenotype suggested l-em may be as an inner eggshell protein coding gene mutant. Its candidate gene might function in water retention for eggshell.To explore the difference between eggshell proteins from the virgin l-em/l-em female and those from+/l-em females, we extracted eggshell proteins from l-em/l-em and+/l-em eggs and separated them on2-DE. Compared to the+/l-em2-DE map, we found a-90kDa spot (suspect as BmEP80) was absent on the l-em/l-em2-DE map. To further confirm the loss of this egg-shell protein in the l-em/l-em eggs, BmEP80specific antibody was used to conduct a Western blot analysis, which showed that BmEP80protein was indeed absent from the l-em/l-em eggs. Moreover, no transcript of BmEP80was detected in the ovary tissue dissected from l-em/l-em female. We therefore hypothesized that BmEP80was the culprit behind1-em mutation.Genomic PCRs, iPCRs, Genome DNA qPCR and Southern blot were carried out to examine the BmEP80locus. The data led us to a conclusion that intra-chromosomal segmental duplication occurred at BmEP80locus in l-em/l-em. During this event, a small fragment (-2kb) between BmVMP23and BmEP80in wild-type strain was replaced by a～96.3kb segment in l-em/l-em.This～96.3kb segment contains D-region1(～3.9kb, forward duplicated), iD-region2(～86.7kb, reverse duplicated), and a TE(Qian,～5.7kb). Eight genes BmVMP23, BmEP80, Gene1, Gene2, Gene3, Gene4, Gene5and cJHBP were involved in this event. Among them, the Genes1-5were completely duplicated, and a part sequence of the exon5in cJHBP was also duplicated. However, none of them showed alterations in mRNA levels between l-em/l-em and+/l-em, suggesting the duplication of those six genes could not account for the l-em/l-em phenotype. Gene and chromosomal segment duplications have been thought as the primary source of material for the origins of new gene functions. Duplication of Genes1-5might endow them with new potentialities.BmVMP23and BmEP80were identified to be VMPs in B. mori. In l-em/l-em,the3’ downstream sequence of BmVMP23was replaced by D-region1and resulted in deletion of polyadenylation signal sequence in3’untranslated region (3’UTR). Deficiency of the polyadenylation signal sequence for BmVMP23may have caused degradation of mRNA so that it was hard to detect BmVMP23mRNA in ovary tissues dissected from l-em/l-em individual. However, no signal peptide was predicted in BmVMP23. It might not be secreted by FCs to extracellular egg-shell and hence disruption of BmVMP23gene could not have negative effects on the structure of eggshell. On the other hand, l-em mutant has two truncated BmEP80genes. Both of them lack the5’ upstream sequence and the first160bp of the ORF, with no detectable transcript and protein in l-em/l-em individual. BmEP80is an abundant egg-shell protein, the absent of BmEP80in the eggs laid by l-em/l-em female disrupted the formation of the electron-lucent homogeneous layer, a counterpart of the wax/crystalline chorionic layers in D. melanogaster and M. sexta, which function in holding of water. Taken together, the loss of BmEP80protein is likely responsible for the severe egg-shell defects.Besides it, a LTR retrotransposon was identified on BmEP80locus in l-em genome and named as Qian. Based on the structure of BmEP80locus in the l-em genome, a model for the Qian-mediated chromosomal segmental duplication was proposed and described as "gap repair through illegitimate pairing". This study demonstrated that LTR retrotransposons could mediate large segmental duplication that may lead to immediate phenotypic variation and potential long term impact in evolution in animals.