Cloning And Analysis Of Promoters In BmHEⅠ And BmHEⅡ And Primary Identification Of The Possible Transcription Factor Binding Sites

We have already reported the two hatching enzyme(BmHEⅠ and BmHEⅡ)of silkworm,Bombyx mori.The relative expression level of ⑶ and BmHEⅡ gene transcripts increased with the development of embryo and reached their peak at the time of hatching.The transient expression of the hatching enzyme gene was consistent with the hatching progress.However,BmHEⅠ mRNA were detected in the midgut of silkworm at larvae stage,suggesting that it may have other functions.Meanwhile,BmHEⅡ gene transcripts were detected in the silkworm testis,suggesting that BmHEⅡ may be related to the spermatogenesis of silkworm.In this paper,promoters of two BmHE genes were cloned and analyzed,and some of the transcription factor binding sites were identified.The results of these studies will help to elucidate the tissue specificity expression manner of BmHE gene at the molecular level.The present theis mainly included the following three parts.1.Cloning and analysis of the promoters of the BmHEⅠ and BmHEⅡgenes: BmHEⅠp and BmHEⅡp were cloned by PCR methods with the length of 1350 bp and 1240 bp,respectively.The core promoter position of BmHEⅠp and BmHE Ⅱp was predicted by the online software NNPP.By using of online software Alibaba,the possible transcription factor binding sites of BmHEⅠp and BmHEⅡp were analyzed.There were 13 kinds of the specific transcription factor binding sites in total harbored in BmHEⅠp,such as CREB,NF-1,and c-Jun etc.Whereas there were 17 kinds of the specific transcription factor binding sites in total harbored in BmHEⅡp,such as HNF-4α1,Pit-1a,TEF,and MEB-1 etc.The above results provided the basic information for further screening of transcription factor binding sites that regulate the specific expression manner of BmHE gene.2.EMSA analysis of the nuclear proteinof midgut,testis,and eggs of silkworm with the transcription factor binding sites on BmHEⅠp and BmHEⅡp: first use the nuclear protein extraction kit to extract the nuclear protein of midgut,testis and eggs of silkworm.The concentration of nuclear protein of the midgut was 4.73μg/μL,the concentration of nuclear protein of testis was 1.17μg/μL,and the average concentration of nuclear protein silkworm eggs was 4.92μg/μL.The specific transcription factor binding sites of BmHEⅠp and BmHEⅡp were labeled with IR700.The sequence of transcription factor binding sites(Oct-1,CRE-BP1,HNF-4α1,TBP)were subjected to electrophoretic mobility shift assay(EMSA)as a probe with the above extracted nuclear protein.Oct-1 and CRE-BP1 probes could be specific binding to the nuclear protein of midgut,HNF-4α1 and TBP probes could be specific binding to the nuclear protein of testis,however,Oct-1 and CRE-BP1 probes couldn’t be specific binding to the nuclear protein of testis,HNF-4α1 probe couldn’t be specific binding to the nuclear protein of midgut suggesting that the tissue expression specificity of BmHE genes at lavel stage may be related to Oct-1,CRE-BP1,and HNF-4α1.Nuclear protein of 7 to 9 days’ silkworm eggs could be specific binding to TBP probe,nuclear protein of 1 to 9 days’ silkworm could be specific binding to HNF-4α1 probe,suggesting that the expression of BmHE genes at incubation stage may be related to HNF-4α1 and TBP;3.Identification of the blocking bands from EMSA by capillary high performance liquid chromatography(HPLC).The results showed that Oct-1,CRE-BP1,HNF-4α1 and TBP were not detected in the blocking bands,however,other transcription factors were detected.Apoptosis-related protein 1CAD,Cyclic AMP-regμLated protein and FancJ-like protein c can bind to binding site of CRE-BP1 transcription factor;14-3-3 protein zeta,actin-related 2/3 complex subunit 2 and Beadex / dLMO protein can bind to binding site of Oct-1 transcription factor;Fanconi anemia,Complementation groupⅠand p53 can bind to binding site of HNF-4alpha1 transcription factor.Interleukin enhancer binding factor isoform 1,p53 and translation initiation factor 2 gamma subunit can bind to binding site of TBP transcription factor,indicating that tissue expression specificity of BmHE genes was not only related to Oct-1,CRE-BP1,HNF-4α1,TBP,but also to the above transcription factors,which laid the foundation for eluiciting the expression way of BmHE genes.