Cloning, Expression And Functional Analysis Of The Gene CDNA Encoding BmZFP16-like In Silkworm, Bombyx Mori

The silkworm Bombyx mori is the model insect of Lepidoptera with important economic value in food,textile,medicine,chemical industry.The silk gland of silkworm is a specifically differentiated silk-producing organ.Previous studies suggested that the expression of silk protein genesis mainly regulated at the level of transcription,the expression regulation of silk protein in transcription is a hot topic in current research.Zinc Finger Protein is a sequence-specific transcriptional factor which involved in special intracellular nucleic acids binding,controlling of gene expression and play important role in cell division and differentiation,embryo development and individuals growth.A BmZFP16-like gene which can special expression in silk gland was found and studied by using clone,expression,qRT-PCR,western blot technology.The role of BmZFP16-like in transcriptional regulation of the heavy chain protein gene of Bombyx Mori was discussed.The main research results are as follows:1?The special primers were designed according to conserved sequences with the other lepidoptera insects in GenBank.Sequence analysis revealed that it contained an ORF of984 bp and encoded a polypeptide with 327 amino acids.The theoretical isoelectric point and molecular mass for the protein are estimated as 6.15 and 37.4 kDa,respectively,and shared six highly conserved ZFP domains at its C-terminus,which indicated it belonged to ZFP family.Phylogenic analysis showed that this sequence was highly homologous to zinc finger protein?ZFP16?of Danaus plexippus,so we named it as BmZFP16-like.2?The prokaryotic expression vector involved pET-28a-BmZFP16-like was constructed and transformed to E.coli Transetta?DE3?,then recombinant vector was expressed in Escherichia coli induced by IPTG in different concentrations.SDS–PAGE and western blot analysis demonstrated that the recombinant protein was successfully expressed in Escherichia coli cells.The induced cells were harvested and subjected to nickel affinity column chromatography for purifying the recombinant protein.Then,the purified recombinant protein was used to immunize a New Zealand rabbit to obtain polyclonal antibody.3?Proteins and total RNAs from silk gland,etc in different tissues and from silk gland in different periods were extracted to determine the Bm ZFP16-like transcription and protein expression level by using western blotting and qRT-PCR.The results showed the expression level of BmZFP16-like existed relative specificity of time and space,and it was highest in silk gland and lowest in fatbody.The expression of BmZFP16-like from the late 4^th instar gradually increased and reached the highest at 7^th day of 5^th instar,and then gradually reduced in the spinning stage.The expression pattern of BmZFP16-like is similarity to that of fib-H.4?Proteins and total RNAs of silk gland in 3^rd day of 5^th instar from P50 and H9 were extracted,and then analyse the transcription level and protein expression situation of BmZFP16-like by using western blotting and qRT-PCR.The results indicated that the expression of BmZFP16-like from H9 is higher than that of P50.The BmZFP16-like probably involved in the transcriptional regulation of fib-H.5?Analysis about the expression regulation region between BmZFP16-like and fib-H indicated that the three amino acids in the second zinc finger domain's?helix areas can identify and interact with the TATA box in 5'upstream region of the fib-H promoter,which indicated that BmZFP16-like may play an important role in the transcriptional regulation of fib-H.