Expression Analysis And Functional Identification Of Diapause-related Genes In Bombyx Mori

Most of animals will delay development and enter dormancy,when they encounter unfavorable living conditions,which provides a guarantee for their life continuity,such as polar bears,frogs,hedgehogs,etc.through dormancy for winter;and sea cucumbers,african pneumocarps,swordpigs,etc.through dormancy for hot summer.Diapause as a form of dormancy is a strategy for organisms to overcome the periodic disadvantage of their living environment and an important physiological characteristic for insects to overcome the disadvantageous living conditions.According to the time of insect diapause,insect diapause can be divided: egg diapause(embryo diapause),larval diapause,pupae diapause and adult diapause.Due to its unique historical culture and economic value,silkworm has long been concerned about an experimental model for researchers.In this study,the bivoltine strain of the silkworm Dazao was used as the experimental object to study the diapause of silkworm embryos.The phenomenon of diapause in offspring was effected by theambient temperature and light of bivoltion silkworm.The diapause eggs were produced under the 25? with long light and the non-diapause eggs were produced under the 15? within dark.The study of molecular mechanism of silkworm diapause helps to expand sericulture production and improve industrial efficiency,and it is great significance in the prevention and control of agricultural and forestry pests.In the present study,we sequenced the transcriptome of bivoltine silkworm Dazao's embryo including the blastokinesis phase and head pigmentation phase at different conditions which resulted in laying the diapause or non-diapause eggs,using high-throughput RNA-Seq technology.The diapause related genes were identified,and the expression patterns of these diapause-related genes were executed in the embryonic stage of univoltine or bivoltine silkworm,and the functions of diapause related genes were verified by RNAi technology.The main results are follows: 1.Transcriptome sequencing analysis of Dazao embryo at the blastokinesis phase and the head pigmentation phaseIn order to explore how the parental embryo experiences external stimulation and ultimately affect the diapause of the progeny.we sequenced the transcriptome of bivoltine silkworm Dazao's embryo that under the 25? with long light or the 15? within dark at blastokinesis phase and head pigmentation phase using high-throughput RNA-Seq technology.The results showed that 812 differential genes identified were up-regulated and 65 differential genes were down-regulated at embryo blastokinesis phase(BK25 vs BK15).The 201 differential genes identified were up-regulated and 232 differential genes were down-regulated at embryo head pigmentation phase(HP25 vs HP15).GO and KEGG analysis revealed that all of these differential genes were involved several functional terms related to Phototransduction-fly,Amino sugar and nucleotide sugar metabolism,Glycosaminoglycan degradation.In additionally,the cluster analysis of blastokinesis phase and head pigmentation phase indicated 44 common differentially expressed genes were obtained,including 32 co-upregulated genes and 12 co-downregulated genes.And the function of these genes participated membrane structural components,catalytic activity,binding proteins and metabolic processes.Sixteen differentially expressed genes were randomly selected to verify the accuracy of RNA-Seq using qRT-PCR.The results showed that the significant change of fifteen differential genes was consistent with the transcriptome results,which proved the reliability of sequencing data.Referring to the transcriptome analysis results and previous studies,we identified seven diapause candidate genes,including three heat shock protein genes BGIBMGA014536,BGIBMGA014618,BGIBMGA004614;two rhythm genes BGIBMGA00048(period),BGIBMGA003345(takeout);two TRP superfamily genes BGIBMGA009272,BGIBMGA009273.2.Expression patterns of diapause-related genes in embryonic stage of univoltine and bivoltine strains of silkwormIn this experiment,the univoltine silkworm strain C-4,the bivoltine strains Haoyue(HY) and Qiufeng(QF) were selected as experimental subjects,and the non-diapause eggs were incubated at 25 ? in long light and 15 ? in the dark.And take the material until the silkworm larva are out.The expression patterns of seven diapause-related genes were detected using qRT-PCR in the embryonic stages of these three strains.The results showed that the three heat shock protein genes BGIBMGA014536,BGIBMGA014618 and BGIBMGA004614 in the bivoltine strains(HY)and Qiufeng(QF)have similar embryonic expression patterns: they have significant differential expression under the conditions of 25 ? and 15 ?,which are expressed under the condition of darkness at 15 ? significantly higher than 25 ?.But there were no significant difference in the univoltine strains C-4.The embryonic expression pattern of two TRP family genes BGIBMGA009272,BGIBMGA009273 in the bivoltine strains of Hayue(HY) and Qiufeng(QF) were 25? significantly higher than 15? in the early stage of embryonic development.And there was no difference in the univoltine strains C-4.The expression levels of rhythm genes BGIBMGA000486 and BGIBMGA003345 in the bivoltine strains of Hayue(HY)and Qiufeng(QF)were 25? were significantly higher than 15? in the middle and late stage of embryonic development.There was no difference in the univoltine strain C-4.Based on the differential expression patterns of the seven diapause-related genes in the embryonic stage of univoltine and bivoltine strains,we speculate that these genes may be involved in the regulation of diapause in silkworm through the expression level.The rhythm genes and TRP family genes were expressed highly could cause to diapause of silkworm;otherwise,heat shock protein genes induced to produce the non-diapause eggs.3.Functional identification of diapause-related genesBased on the analysis of transcriptome results analysis and the differential expression patterns of differentially expressed genes in univoltine and bivoltine strains silkworm,we speculated that the high expression of rhythm gene BGIBMGA00048(period),BGIBMGA003345(takeout) and TRP family gene BGIBMGA009272,BGIBMGA009273 at 25 ? might be the reason for their regulation of silkworm diapause,In addition,significant up-regulation of heat shock protein genes BGIBMGA014536,BGIBMGA014618,BGIBMGA004614 at low temperatures(15?) may be responsible for the production of non-diapause eggs.so we further utilized RNAi technology to verify the function of these genes at the individual level.Non-diapause eggs of bivoltine silkworm Dazao were injected dsRNA of diapause-related genes.Silkworm eggs injected with 14536-dsRNA,14618-dsRNA and 4614-dsRNA were incubated in dark at 15?.Silkworm eggs injected with 3345-dsRNA,486-dsRNA,9272-dsRNA and 9273-dsRNA were incubated in the light condition at 25?.Silkworm eggs injected with dsRed and untreated eggs were incubated under two conditions as control group.After the moth lays eggs,the diapause status of the progeny was counted.At present,this part of the experiment is in progress,and the results need to be counted.