Identification Of BmJHBP Gene Family With Juvenile Hormone Binding Protein Domain And Function Of BmJHBPd2 In Bombyx Mori

Silkworm(Bombyx mori)is the ancient wild silkworm by long-term domestication of secrete silk insect,is a classic model organism in the field of biology,since ancient times has very high value in the economy.The Juvenile hormone(JH)synthesized by corpora allata and 20-hydroxyecdysone(20E)synthesized by prothoracic gland are two important hormones that regulate the growth and development of silkworm.Among them,JH was secreted into the haemolymph after synthesized in the corpora allata,bound by juvenile hormone binding protein(JHBP),which serves as a carrier to release the hormone to various target tissues and cells,and then JH regulate the growth and development of silkworm.The economic benefits of silkworms are related to the high efficient synthesis ability of silk protein.It is reported that the silkworm can increase the production of silk protein added with JH,however,JHBP of Bombyx mori(Bm JHBP)as the first key molecule of JH signaling pathway,and the relationship with the increase of silk protein production is unclear.At present,researchers have identified some of the Bm JHBP genes,which are expressed in various tissues and organs.The aim of this study was to identify and analyze the members of silkworm JHBP gene family by bioinformatics.The expression pattern and the function of Bm JHBP were expressed by RT-PCR,cloning,Western blot and immunofluorescence.The main results are as follows:1.Identification and expression pattern analysis of silkworm JHBP gene familyIn order to comprehensively identify and analyze the Bm JHBP gene family that contains the juvenile hormone binding protein domain,this study was based on the silkworm genome database and NCBI.First,Using bioinformatics to identify the members of the Bm JHBP gene family members.In results,a total of 41 Bm JHBP genes were identified,which were distributed on 8 chromosomes,mainly on chromosome 15 and 23.Gene structure analysis showed that the structure of the family JHBP genes were diverse,and were significant differences in gene length and exons.Signal peptide prediction showed that 33 Bm JHBP genes encode a signal peptide,and the 8 Bm JHBP genes do not encode signal peptides.After analysis of the protein structural domain,the Bm JHBP genes contained at least one JHBP domain,except some Bm JHBP proteins contained both JHBP and Grp7_allergen domain.Through the evolution analysis of the Bm JHBP with Px JHBP,Dm JHBP and Am JHBP,the results showed that the JHBP gene family can be divided into two branches and four subfamilies.Microarray data and RT-PCR experiments showed that Bm JHBP were mainly expressed in the gonad,head,epidermis,Malpighian tubules,and identified that Bm JHBPd2 is the only high expression gene in the posterior silk gland of the whole family JHBP gene family members.2.Expression pattern of Bm JHBPd2 and identification of JHI in posterior of silk glandIn order to analyze the expression pattern of Bm JHBPd2,first of all,bioinformatics analysis of Bm JHBPd2 gene showed that the length of this gene was 8053 bp,including 5 exons and 4 introns.The ORF(Open reading frame,ORF)is 732 bp in length,encoding 243 amino acids,the former 18 amino acids encoding a signal peptide,with a typical JHBP domain,the isoelectric point was 4.80,predicted molecular weight of 27.3k D and located in nscaf3027 on chromosome 23.Using Western blot method to detect the protein expression of Bm JHBPd2 in tissues of the 3 day of 5th instar larvae,and found that the expression of Bm JHBP were higher in the posterior silk gland,which was consistent with the microarray data and RT-PCR.Then,RT-PCR was used to analyze the expression of Bm JHBPd2 from the 3 day of the 4th instar to the pupal stage.The Results showed that Bm JHBPd2 was highly expressed at 1 day to 5 day in the 5th instar,almost no expression in the 4th molting period and the latest of 5th instar.In order to further verify the expression site of Bm JHBP in the cell,we constructed the over-expression vector of p SLfa1180-GFP-Bm JHBPd2 and then transfected into Bm E cells.The results showed that Bm JHBPd2 was expressed in cell cytoplasm.Using immunofluorescence method of paraffin section,positional analysis showed that Bm JHBPd2 highly expressed in cytoplasmic layer in the posterior of silk gland,consistent with the previous RT-PCR,Western blot,and cellular overexpression of subcellular localization.Finally,q RT-PCR was used to analyze the expression of Bm JHBPd2 in the low silk of Dazao and the high silk of 872.The results showed that in 3 day or 5 day of 5th instar larvae in 872,Bm JHBPd2 expression was significantly higher than that of Dazao,suggesting that it may have relationship with silk protein synthesis.The JH of the silk gland in the 3 day of the 5th instar larvae was qualitatively detected by GC-MS.The results showed that JH peak was detected at the same time compared with the standard,indicating the presence of JH in the posterior silk glands of silkworm.3.Research on the relationship between BmJHBPd2 protein and JH in silkwormIn order to study the relationship between BmJHBPd2 and JH,we first selected the 1 day of the 5th instar larvae,smeared back line with 1ug JHA.The results showed that the silkworm smeared with JHA,the wandering and pupation time were delayed 1 to 2 days.In addition,after applying JHA,the economic traits of silkworm were significantly improved.The expression of Bm JHBPd2 was detected by q RT-PCR at 12 h,24h,48 and 72 h after smearing JHA.It was found that the expression of Bm JHBPd2 was up-regulated after applying JHA,indicating that exogenous JHA could promote the expression of Bm JHBPd2.In order to further study the function of Bm JHBPd2,constructed the p SLfa1180-Bm JHBPd2 cell expression vector and then transfected into Bm E cells,QRT-PCR showed that the genes of Bm JHBPd2 and JH signaling pathway genes did not change significantly,indicating that Bm JHBPd2 can not activate the JH signaling pathway,fib-H expression also did not be affected.When the JHA,JH signaling pathway was significantly up-regulated,indicating that JHA activates the JH signaling pathway.After overexpression of Bm JHBPd2 and JHA,the expression of Bm JHBPd2 can inhibit the transmission of JH pathway signal,but it has no significant effect on the expression of fib-H,indicating that Bm JHBPd2 can not act directly on fib-H.The expression of Bm JHBPd2 and the addition of JHA were compared with those of JHA alone.The JH signal pathway of the former showed a down-regulated expression,indicating that overexpression of Bm JHBPd2 could inhibit the transmission of JH pathway signal.