| The silkworm(Bombyx mori)is an important economic insect,and the hatching rate(HR)is an important index of the quality of silkworm eggs.This paper investigates the correlation between the egg shape index(ESI)and HR,the genes related to HR at the late embryonic development stages.The experimental methods of the dual luciferase reporter system,yeast one-hybrid,and RNAi were used to investigate the effect of candidate transcription factor Period on transcriptional regulation of the silkworm hatching enzyme gene.The main findings are as follows:1.The ESI,HR,fertilization rate(FR),and mortality rate in egg with pigmentary body of embryo(MR)was investigated between the wild type normal egg variety(NISTAR~+)and its new mutant spindle egg variety(NISTARI~-),and the correlation of ESI with HR,FR and MR were analyzed,respectively.The ESI of NISTAR~+was1.26,which was significantly lower than that of NISTARI~-(1.73)(P<0.01);the HR of NISTAR~+was 96.22%,which was significantly higher than that of NISTARI~-silkworm eggs(51.33%)(P<0.01).When the ESI of NISTARI~-was1.77?1.81,the HR and FR of its eggs were the highest;while the ESI of NISTAR~+was1.24?1.25,the HR and FR were the highest and the MR was the lowest.The results of correlation analysis showed that there was a certain correlation between the ESI and the HR,and it would affect the FR and MR.In the later stages of silkworm egg embryo development,the expression levels of Bm HE I,Bm HE II,Chitinase and Period genes of NISTARI~-silkworm eggs were extremely significantly lower than those of NISTAR~+silkworm eggs(P<0.01),which suggested that the differences in the expression of the four genes are involved in the molecular basis for the differences in HR between the silkworm varieties of NISTAR~-and NISTAR~+.2.Reporter vectors with different lengths of Bm HE II promoter driving luciferase gene were constructed to transfect silkworm Bm N cells.The result showed that the activity of the full-length promoter fragment was relatively high.After deleting the region from-834 to-1240 upstream of the transcription start point,the activity of the promoter was significantly reduced,which indicated this region may contain a specific binding site for positive transcription regulatory elements.3.The bait strains Y1HGold[Ip-A-Ab Ai]and Y1HGold[Ip-C-Ab Ai]were screened with the midgut c DNA library by yeast one-hybrid,respectively.As a result,2 or 6 possible candidate transcription factors were selected.The bait strains Y1HGold[IIp-A-Ab Ai]and Y1HGold[IIp-C-Ab Ai]were screened with the testis c DNA library by yeast one-hybrid,respectively,and 3 or 7 possible candidate transcription factors were selected.4.Using RT-PCR method to detect the expression level of the candidate Period gene in the tissues of the third day of fifth instar and embryonic development stage,the results showed that it was expressed in all tested tissues at larval stage,especially the highest in the testis,and it was expressed at different embryonic development stage,reached to highest level before hatching.5.Transfection of silkworm Bm N cells by si Per showed that the decrement of Per expression level resulted in the decrement of Bm HE transcription expression level,implying that Per has a certain positive regulatory effect on the transcription of the Bm HE.In summary,there is a certain correlation between the ESI and HR,and Bm HE I,Bm HE II,Chitinase,Period genes are involved in the molecular mechanism of the difference in HR among different egg shapes,which provides some basic information for practice in increasing the HR of silkworm eggs in sericulture.The function of Bm HE II promoter was analyzed,and the candidate binding protein of transcription factor Period was selected through the yeast one-hybrid experiment.The RNAi experiment showed that Period has a positive regulatory effect on the transcription of the Bm HE,which provides a basis for further elucidation of sophisticated transcriptional regulation of BmHE. |
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