Identification,Function And Expression Regulation Of Chorion Genes In Silkworm,Bombyx Mori

Insects,which have the character of wide variety and tremendous number,are an important contributor to biodiversity.During the long-term process of co-evolution,a complex and close relationship was established between insects and humans.On the one hand,insects provide a large number of living goods for humans,for example,a bright silk culture was depended on lepidoptera insects silkworms.On the other hand,insects also caused significant loss in economy,especially orthoptera locust,which is written in a human agriculture society's history of blood and tears.Prolific fecundity results in large number and wide variety of insects,most of which lay eggs in the hundreds.Therefore,the study of insect eggs will establish theoretical basis for pest control and improvement of fertility in economic insects.Chorion is a complexly protective structure on the surface of insect oocyte,whose main components are chorion proteins codon by chorion genes.Present study,silkworm,the representative of Lepidoptera was used to study the function and expression regulation of chorion genes.Firstly,we identified the chorion genes and constructed their expression pattern maps.Proteins component of chorion was analyzed using LC-MS/MS method.Secondly,for function study,the possible mutation mechanism of Grk mutant was analyzed at the level of proteomics.Finally,for expression regulation,the role of miRNAs in the regulation of chorion genes expression was analyzed.The mainly results in present study are as follows: 1.Identification and evolution analysis of chorion genes in silkwormThe silkworm chorion genes belong to a superfamily family and all members are located on a genome region of chromosome 2(chorion locus).Nearly one hundred chorion genes have been identified in silkworm in the past 40 years research.However,high repeat sequence makes obstacle for the chorion locus to be sequenced completely,which seriously hinders the comprehensive and systematic research in chorion.In this study,BAC sequencing method was used to obtain the complete sequences of chorion locus.Firstly,positions of more than 200 BAC clones in the chorion locus were analyzed,and seven BAC clones which covering complete chorion locus were selected.Then,871,711 bp covering chorion locus was obtained by sequencing of 7 BACs,chorion genes were predicted by fgenesh software,and 127 chorion genes and 5 non-chorion genes were annotated,of which chorion genes were labeled BmCho-1~BmCho-127,which include 36 early,46 middle and 45 late chorion genes.Two cDNA libraries of egg development stages were used to ensure existence of chorion gene ESTs by alignment with 127 chorion genes,which in turn confirm the annotated chorion genes.The chorion genes are mainly distributed in the genome with the form of gene pairs.Two late chorion genes,BmCho-23 and BmCho-26,are pseudogenes due to multiple stop codons in the CDS region.Phylogenetic analysis showed that the same subfamily members of chorion genes were clustered into one same branch.However,the early B eggs were divided into two branches in the phylogenetic tree.Of which one was clustered into one large branch with middle B.The expression patterns of chorion genes showed that seven early B genes had the same expression patterns with the middle B genes.The phylogenetic tree analysis revealed that the early chorion genes underwent a faster evolution,and the late chorion genes were relatively conserved in evolution.Two pseudogenes,BmCho-23 and BmCho-26,also underwent rapid evolutionary rates.The identification of complete chorion locus is an important foundation for comprehensively and systematically studies of chorion genes.2.Expression patterns analysis of chorion genesIn order to analyze the expression patterns of chorion genes during egg development period,we constructed the expression pattern maps of chorion genes.Depends on the different development,eggs in 8-day after pupation can be divided into vitellogenesis and choriogenesis.No chorion gene was expressed in vitellogenesis,so the eggs show yellowish.Chorion proteins were synthesized and secreted to form the chorion during choriogenesis,so that white and shiny eggs can be observed.According to the expression characteristics of chorion genes,eggs in choriogenesis were subdivided into 11 development periods of I ~ XI.RT-PCR was performed to determine the expression patterns of 127 chorion genes,the results showed that the early and late chorion genes were expressed in early stage(I ~ III)and late(IX ~ XI)of choriogenesis,respectively.The middle chorion genes were expressed throughout the choriogenesis.BmCho-1 and BmCho-96 were identified by screen the cDNA libraries of silkworm testis.For BmCho-1 gene,the transcripts in follicle cells and testis have the same second exon,and different first exon.The first exon of testis transcript was located in intron region of follicle cells transcript.The transcripts of BmCho-96 in the testis have three exons,and its CDS sequences in the testis and follicular epithelial cells are the same,which means they encode the same proteins.We identified the transcript of BmCho-11 in the cDNA library of embryos on 200 hours after fertilization.The CDS sequences in the embryos contain 27 bases more than the transcripts in follicle cells,and additional 9 amino acids could be encoded.Except the mature transcripts,splicing intermediate was presented in almost late chorion genes.The splicing intermediate consists of 5'UTR,exons,introns,3'UTR and polyA sequences.3.Proteomics analysis of chorion260 proteins were identified in the chorion by LC-MS/MS,which including 88 chorion proteins,28 ovary-specific proteins(non-chorion protein)and 144 other proteins.Chorion genes are mainly distributed in the genome in form of gene pairs,and each gene pair includes single A and single B gene which sharing bidirectional promoter,A and B genes are transcribed in the opposite direction.The probability identified of the B-type proteins are bigger than A in chorion proteins encoded by gene pairs,suggesting that the bidirectional promoter may have a strong transcriptional activity for the B genes.Ovary-specific proteins were also identified by proteomics analysis.Genes encoding these proteins were clustered on chromosomes 2,10,15 and 16.Majority of those are unknown genes but specific for silkworm.On chromosomes 2,there are 12 ovary-specific genes located on the upstream sequences of chorion locus,which were distributed in a range of 100 kb.However,10 of these genes encode proteins were identified in chorion.We speculated that those unknown genes might be play important roles in regulating the expression of chorion genes or contributing to construction of chorion.4.Preliminary studies on mutantion mechanism of Kei's grey egg(Grk)The Grk is the grey egg mutant caused by the mutation of chorion genes,and the mutant gene is located in the chorion locus(2-7.2).Phenotype: homozygote(Grk/Grk)eggs are spherical and low fertilization rate;heterozygote(Grk/+)eggs are grey and nomal shape;wild type(+/+)eggs are puce.SEM observation showed that there were no significant differences exhibited on the surface of the chorion of Grk,however,differences existed between chorion lamellae and micropyle.In the chorion lamellae,chorion of Grk wild is composed of lamellae that were parallel to the oocyte.However,the middle chorion lamellae of the heterozygote are perpendicular to the surface of the oocyte.For homozygote,there is a greater damage in chorion lamellae with disorder state.In the micropyle structure,micropyle of Grk wild is surrounded by flower-shaped markings where the inner-stripes protruded from the surface of the chorion,and the micropyle has four micropyle tube gates.A smaller flower-shaped marking in Grk heterozygote than Grk wild,as well as a milder protrusion of inner-stripes in Grk heterozygote.The number of micropyle tube gates is normal.There is no protrusion of inner-stripes in homozygote egg with two micropyle tube gates.The number of petals was decreased in Grk wild,heterozygote and homozygote according to priority.By observing and comparing the structure of the three genotypes of Grk,we deduced that the change of chorion llamellae resulted in mutant phenotypes,and the decrease of micropyle tube gates caused low fertilization rate of eggs.The results showed that the solubility of Grk wild,heterozygous and homozygous chorion in 8 M urea/35 mM DTT lysis was decreased according to priority.The solubility will be improved if adding the 0.2% into 8 M urea/35 mM DTT lysis,however,the chorion of Grk mutant could not be dissolved completely in the new lysis.These results indicated that the structure change of Grk chorion may affect the dissolution of the chorion proteins.Label-free quantitative proteomics analysis of Grk mutant chorion showed that BmCho-20,BmCho-41,BmCho-57,BmCho-62,BmCho-73 and BmCho-115 were up-regulated in Grk homozygote,which BmCho-20 and BmCho-115 were early chorion proteins,and BmCho-73 was a middle chorion protein,and BmCho-41,BmCho-57 and BmCho-62 were late chorion proteins.Three of 6 up-regulated proteins are cysteine-rich late chorion proteins mainly distributed in the outer layer of chorion,where the intermolecular disulfide makes chorion form a hard shell.Therefore,up-regulated Hc proteins in Grk mutant increased the hardness of the chorion,making it more difficult to dissolve.Hc proteins are mainly expressed in the late stage of choriogenesis.If the three Hc proteins are expressed in advance,they will disrupt the process of chorion formation and cause the change of chorion structure.Label-free quantitative proteomics analysis also revealed that BmCho-17 was only lost SEM in homozygous chorion.As a middle chorion protein BmCho-17 should involve in the construction of middle chorion lamllaes.So the lost BmCho-17 in homozygote could effect the construction of middle chorion lamllaes.5.Analysis of expression regulation of chorion genes by miRNAThe characteristic of strictly spatiotemporal expression pattern makes the chorion genes an excellent model for gene regulation study.However,expression regulation mechanism of chorion genes could not be explained clearly by reported regulatory factors so far.In present study,we analyzed the role of miRNAs in the regulation of chorion gene expressions.Totally,847 miRNAs include 399 known miRNAs and 448 novel miRNAs in follicle cells were identified using high-throughput sequencing.The results of qPCR showed that 33 miRNAs were specific in follicle cells.Target prediction showed that miRNAs target all types of chorion genes and their transcription factors.At the same time,it has also been found that one miRNA based on the same target sites can regulate a class of chorion genes.Integrated analysis of miRNA and proteomics revealed that miRNAs were also involved in the regulation of genes encoding other structural proteins in chorion.Dual luciferase assay showed that bantam-3p directly regulated BmC/EBP expression in choriogenesis.In summary,the results showed that miRNAs are involved in the expression regulation of chorion genes.