Functional Analysis Of Hatching Enzyme Gene Promoter In Silkworm, Bombyx Mori And Priliminary Screening Of Transcription Factors

Hatching Enzyme?HE?is a kind of protease that digests the egg shell or egg membrane and plays an important role in the hatching progress.At present,two hatching enzyme genes?BmHE I and BmHE II?had been reported by our research group,both of which are expressed in the late stage of silkworm embryo development,and the relative expression of them reached peak at the time of hatching.In addition,BmHE I was only detected in the midgut of the larval stage,while the BmHE II was detected in the testis from the fifth larval stage to the moth stage.For the study of tissue specific expression of two hatching enzyme genes,two hatching enzyme gene promoters?BmHE Ip and BmHE IIp?function analysis were performed,and the method of Yeast one hybrid system were used to preliminarily screen the normalizationed cDNA library for its potential transcription factors in this study.The main results of this study are the following.1 The activity analysis of hatching enzyme promoter of silkworm in vitro.Different lengths of promoters were generated by a 5'terminal truncation PCR,Then,the fragments were constructed into pGL3.0-Basic vector respectively,and then were transfected into BmN cell with pRL-CMV vector.Results show that the 1.3 kb upstream of BmHE I promoter can drive the firefly luciferase expression,but the overall activity is not high,The fragment between-40bp upstream and the transcription start site is the core promoter region,The deletion of-97^-40 bp significantly increased its promoter activity,suggesting that there maybe an inhibitory factor binding site in this area.2.The activity analysis of hatching enzyme promoter of silkworm in vivo.Different lengths of BmHE I and BmHE II promoters were constructed into pFBDLuc-P10Rluc transfer vector which was derived from pFBD-Dual vector.Rluc gene was driven by P10 promoter,while Luc was driven by turncation of 5'terminal hatching enzyme gene promoter.The recombinant baculovirus with dual luciferase quantitative assay system were prepared by Bac-to-Bac expression system,and injected fifth instar silkworm larvae.The luciferase activity of different length promoter fragments in the midgut and the testis were determined.The results showed that there was an important binding site responsible for tissue specific transcription factors between-886^-509bp in BmHE I promoter,and there was a transcription factor binding site between-1226^-819bp in the BmHE II promoter.3.Normalizationed full length cDNA library construction.In combination with DSN enzyme and SMART technology,normalizationed full length cDNA expression library of midgut and testis from 3^rd days fifth instar silkworm were constructed respectively.Normalizationed cDNA library was inserted into pGADT7-Sfi shuttle plasmid vector of yeast and E.coli.The shuttle library plasmids were transformed into E.coli for amplification,and the library capacity of the primary library from the midgut was 1.9×10⁶ cfu.The average fragment size insertion was more than 1kb.recombination rate was 100%,and redundancy rate was 0%.While the primary library from the testis tissue was 1.8×10⁶ cfu,recombination rate was 100%,and redundancy rate was 0%.Those results indicated that two library are of high qulity.Two library plasmids were extracted,and the total quantity of good quality library plasmid was 4 mg respectively.4.Construction of bait yeast strains.Based on the promoter activity of identification and prediction of bioinformatics,the two segments of BmHE I promoter,Ip-A?-543^-90bp?and Ip-B?-990^-486bp?were anplified.A DNA fragment containing three times tandem repeat of two prediction cis-elements?HNF-1and GATA-1?was synthesized,which was named as Ip-C.The two segments of BmHE II promoter,IIp-A?-455^-83bp?and IIp-B?-866^-398bp?,were amplified.A DNA fragment containing three times tandem repeat of three prediction cis-elements?WT1,PEA3,Ftz?was synthesized,which was named as IIp-C.These fragments were subcloned into pABAi bait vector,the resulted recombinant vectors were transformed into Y1H yeast strains,respectively.Through SD/-Ura defect type medium selection and colony PCR identification,six bait yeast strains containing BmHE I or BmHE II promoter fragments or elements were successfully constructed respectively,which were named as Y1HGold[Ip-A-AbAi],Y1HGold[Ip-B-AbAi],Y1HGold[Ip-C-AbAi],Y1HGold[IIp-A-AbAi],Y1HGold[IIp-B-AbAi],and Y1HGold[IIp-C-AbAi].5.Preliminary screening of potential transcription factors in BmHE I and BmHE II promoter.Y1HGold[Ip-B-AbAi]and Y1HGold[IIp-B-AbAi]bait yeast strain was choosed to screen against midgut cDNA library and testis cDNA library respectively.The result suggest that there were five possible transcription factors or binding proteins may contribute to BmHE I promoter.,which were aspartyl/asparaginyl beta-hydroxylase,leucine-rich repeat-containing protein 70,TBC1 domain family member 16,KRR1 small subunit processome component homolog and regucalcin.There were seven possible transcription factors or binding proteins may contribute to BmHE II promoter,which were period?Per??mimitin,mitochondrial?39S ribosomal protein L51?ribosomal protein L8?UPF0545 protein C22orf39 homolog?zinc finger CCCH domain-containing protein?nucleoporin NUP53.In summary,this study had found some important area responsible for tissue specificity regulation,Yeast one hybrid system was used to preliminarily screen several possible transcription factors which may combined with BmHE promoter.The results of this study lay a foundation for further study on transcriptional expression regulation of silkworm hatching enzyme gene.