Analysis On Expression And Function Of Several Diapause Related Genes In Pupal Stage Of Silkworm(Bombyx Mori)

The silkworm,Bombyx mori is a typical insect undergoing diapause at the stage of egg or embryo.According to their voltinism,silkworm strains are divided into univoltine-,bivoltine-,and polyvoltine.They may have one,two,and three or more generations per year,respectively.Diapause of bivoltine offspring is regulated by environment conditions especially temperature and lightness at embryo stage of mother.Temperature,lightness,humidity and other environmental information as diapause-inducing cues are perceived at the embryo period of parental generation.These cues are transferred to central nervous system by sensory nerve,then be converted to semiochemicals mainly in the silkworm diapause hormone(DH)stored temporarily by neurosecretory cells.When meeting corresponding conditions,semiochemicals are released into hemolymph inducing the adjustment of internal environment and establishing physiologic and biochemical responses to adapt change of outside conditions.However,the molecular mechanism of diapause on what participated in parental generation is still remained unknown.Here,we took bivoltin strain Qiufeng as material and analysed the expression level of diapause-related genes by high-throughput sequencing,and carried out functional analysis of severial genes based on RNA interference method.The results are as follows.1.Differential analysis of pupal ovary transcriptome on diapause associated genes of silkworm.We used RNA-seq to sequence the transcriptome of ovary from bivoltine strains Qiu Feng between progeny diapause-destined group(normal incubated in embryo stage)and progeny non-diapause-destined group(incubated at17? under darkness)respectively based on 3-day pupae oocytes being sensitive to diapause hormone.The results of RNA-seq showed that there are 125 differentially expressed genes,in which 46 down-regulated genes and 79 up-regulated genes in progeny non-diapause-destined group compared with the diapause-destined group.GO and KEGG enrichment analysis were performed to annotate differentially expressed genes.The results revealed that these genes were significantly enriched in two pathways related to metabolism and stress response.2.Expressional characterization analysis of differentially expressed genes between progeny diapause-destined group and non-diapause-destined group.Combined with pathway enrichment and differential expression analysis,we selected8 genes,2 up-regulated(BGIBMGA003835,BGIBMGA012335)and 6down-regulated(BGIBMGA008987,BGIBMGA009801,BGIBMGA013317,BGIBMGA010338,BGIBMGA002426,BGIBMGA012996)genes in progeny diapause-destined group to verify the expression level in head and ovary from1-day-old fifth instar larvae to 6-day pupae in diapause and non-diapause group respectively by quantitative real-time reverse transcription polymerase chain reaction(q RT-PCR).The results indicated that expression levels of selected genes have significantly difference at same tissues in different development phases,validating that these genes are diapause-related.3.Functional characterization of diapause-related genes based on RNAi method.We selected four genes based on the results of sequensing as target genes of RNAi,two genes(BGIBMG012996 and BGIBMG002426)up-regulated in non-diapause-destined group,the other two genes(BGIBMGA003835 and BGIBMGA012335)up-regulated in diapause-destined group.Then we synthesized ds RNAs according to principle of RNAi and injected into 2-day pupae females of progeny non-diapause-destined and diapauses-destined,respectively.After they laid eggs,the change of egg colour was observed.We found that pupae of non-diapause-destined group injected with mixture ds RNAs of BGIBMG012996 and BGIBMG002426 genes laid diapause eggs after emergence and copulation.It means these two genes play important role in diapause formation.Our study shows expression profiles of genes in silkworm have significant difference between diapause and non-diapause.Some genes such play important role in diapause formation.These results provide reference to further study on molecular mechanism of diapauses in silkworm,Bombyx mori.