Analysis Of Gene Expression Patterns In The Metamorphosis Developments Of Silkworm(Bombyx Mori)

The silkworm （Bombyx mori） belongs to holometabolic insect, and its metamorphosis is intricately governed by the neuroendocrine hormones and environment. Two major hormones, ecdyseriod （Ecd） and juvenile hormone （JH）, regulate the molt and metamorphosis synergistically; and diapause hormone （DH） primarily controls the diapause of silkworm eggs. However, it is necessary to do further research on the metamorphosis process of gene expression by omics techniques.The silkworm diapause eggs, the trimolter and scaleless mutant （SL） were selected as the research materials. Based on the next-generation sequencing technology, several important metamorphosis developments were analyzed, including silkworm diapause, the precocious metamorphosis of trimolter and the formation of scales during pupation stage. Adopting RNA-Seq, we found that a large number of genes were still transcribed in diapause eggs （DP_eggs）. The up-regulated genes in DP_eggs were primarily related to DNA replication, transcription and translation, whereas the down-regulated genes in DP_eggs were primarily related to metabolism, signaling pathways, transport and catabolism. We hypothesized that they are probably related to maintain the genome integrity and the minimum metabolism in DP_eggs. Alternative splicing analysis showed that the number of alternative splicing events in DPeggs is more than active tissues （A_tissue）, indicating that the more alternative splicing events is probably the main reason for the abnormal translation of mRNAs in DP_eggs. Moreover, the unevenly transcription of ribosomal proteins was most likely to cause a large amount of inertness of ribosomes in diapause eggs.The prothoracic glands of molting silkworm at the third instar of trimolter （PG₃M）, matured silkworm at the fourth instar of trimolter （PG₃S）, and matured silkworm at the third instar of bimolter （PG₂S） were analyzed by RNA-Seq. Compared with PG₃S, the metabolism of PG₂S was weakened, which may be closely related to the smaller body of bimolter and the lower yield of cocoon. Compared with PG₃M, Cytochrome P450306a1（Phm） was up-regulated in PG₃S, which catalyzed the Ecd synthesis, making its titer reached peak and causing pupation. After anti-juvenile hormone treatment, the trimolter can be induced into bimolter. Compared with PG₃M, juvenile hormone epoxide hydrolase （JHEH） was down-regulated and Basic juvenile hormone-suppressible protein2（BJSP-2） was up-regulated significantly in PG₂S, which were probably closely related to early decrease of juvenile hormone （JH） titer. Meanwhile, up-regulation of Phm in PG₂S catalyzed the Ecd synthesis. From above, we predicted that mRNA changes of JHEH, BJSP-2and Phm reflect the titers change of JH and Ecd, which meets the pupa metamorphosis and leads to the precocious metamorphosis of trimolter.It can be seen from the proteomic analysis that the coaction of imaginal disk growth factor （IDGF）, actin-depolymerizing factor1（ADF1） and profilin （PFN） counteracts the correct organization of the tracheal system in wing disc of SL, and the higher expression of Thiol peroxiredoxin （TPx） in SL is responsible for the apoptosis of scale cells delayed. The results indicated that the SL phenotype is related to the tracheal system developmental deficiency and excessive apoptosis of scale cells. Moreover, on the day before pupation, the wing discs of type wild （WT_S4）, SL （SL_S4） and SL after48h of low temperature treatment （SLC_S4） were further analyzed by DGE （Digital Gene Expression Profiling）. And the results showed that, compared with WT_S4, Tyrosine-protein phosphatase corkscrew （Csw） and Ets DNA-binding protein pokkuri （Aop） were down-regulated in SL_S4, which inhibited the expression of downstream genes through MAPK signaling pathway, making the trachea primary branch and secondary branch blocked and causing the SL phenotype. After low temperature treatment, the number of SL adult scales on the wing was increased. Compared with SL_S4, the up-regulation of Bombyx mori achaete-scute-like protein （Ash1） in SLC_S4might play a vital role. In addition, Csw and Aop were up-regulated, which activated the MAPK signaling pathway and the downstream genes expression, whereas Protein serrate （Ser） was down-regulated, which affects the Notch signal pathway. We speculated their coaction promotes the trachea branches and its extension on the wing surface, making the scales number increased, and the SL phenotype was partially restored to wild phenotype after low temperature treatment.Twenty-nine genes of30K family （Bm30K-1-Bm30K-29） were retrieved from the local Bombyx mori transcriptome database. Phylogenetic analysis revealed that the29genes encode three groups of30K proteins （Typical30KP, S/T-rich30KP and ENF-BP）. The gene duplications in each group might be a major reason for the members increased in the family. Moreover, the differentiation of members in each group was preceding to the differentiation of species in the phylogenetic tree.