Function Of Small Peptide Tal-like Gene In Silkworm,Bombyx Mori

The study found that a large number of DNA eukaryotic genomes generate atypical transcripts, but their function is controversial. These transcripts contain many small open reading frame(small open reading frames, sORFs), the sORFs was initially thought to be not translated into protein RNAs. However, increasing evidence suggests that some of the sORFs can be translated into small peptides, and their function has been seriously underestimated.sORFs translated into small peptides may represent a neglected biologically active molecules, recent studies have shown that these small peptides have important function in development, differentiation and other biological processes. The research of tal-like gene related peptide more in-depth, this gene was originally discovered in Drosophila. In Drosophila, tal gene expression and control organization, as a structure and morphogenesis of links, It plays a very important role in the growth and development of fruit flies.Bombyx mori, as a typical model of economic insect,It is similar to Drosophila on the growth and development of many aspects. This study on the basis of cloned Bombyx mori tal-like gene(Bmtal) and related fragment detected tal gene expression levels in the fifth instar silkworm in different tissues and study the effects of over expression of Bmtal and Bmtal on the expression of ovo gene(Bmovo) and some signal pathway genes in Bombyx mori, using RNA-Seq technology to carry out the over expression of BmN cells comparative transcriptomic study control and the Bmtal BmN cells, provides a certain foundation for the tal gene in the silkworm, Bombyx mori on silkworm growth and development. The main results are as follows:1. Cloning and expression profile analysis of tal-like gene of Bombyx moriIn order to study the function of tal-like gene in Bombyx mori, RT-PCR primers were designed according to the sequence of Bmtal was cloned the full-length cDNAsequence of 1661 bp. The structure analysis shows that there are multiple sORFs genes, which the first four sORFs(A1-A4) respectively encoding 11-12 amino acids and small peptides of similar sequence, followed by an encoding 32 amino acid sORFs(B).The structural pattern are very similar to flies, suggesting the function of Bmtal gene may be similar to Drosophila. By fluorescence quantitative PCR(Real-time PCR) on the expression level of Bmtal gene in the silkworm, five three day old all kinds of tissues and the results showed that the Bmtal gene, Malpighian tube in the trachea at the age of five to three days, cluster, high expression in the silk gland, gonad is also expressed.2. subcellular localization of Tal-likeIn Drosophila, more and more evidence shows that tal-like gene on the individual growth and development as vital role, in order to investigate the Bmtal expression rules and cellular localization features, fusion Bmtal function region coding sequence and DsRed gene, was cloned into pet-32a(+), in E.coil successfully expressed recombinant protein. And the immunity of mice to prepare polyclonal antibody. Cellular localization showed that BmTal localization in the nucleus of BmN cells.3.Effect of overexpression and inhibition of Bmtal gene on the ovo gene and other signaling pathway genes in Bombyx moriTo further investigate the function of Bmtal gene. This study through overexpression and RNAi methods survey of up-regulated and down Bmtal gene of bmovo gene and other signaling pathway gene expression level. Results show that overexpression of the gene Bmtal can regulate the ovo gene expression level, the expression level of some genes of other signaling pathways also play significant effects.4. Screening and identification of BmTal interacting proteinsIn order to study the Tal- peptide of silkworm Bm N cell interaction proteins by immuno co precipitation technology from the over expression of Bombyx mori BmN cells in screening interaction proteins, then the interaction of candidate proteins were identified by mass spectrometry, a total of identified six candidate protein(14-3-3 Zeta,cilia and flagella associated protein, the flagellar axoneme associated protein mst101 and poly ubiquitin, U3 small nucleolar RNA protein, cell growth regulator of nucleolarprotein). The results show that these proteins related to signal transduction, cell proliferation and differentiation, protein degradation, cell surface protrusions are formed,nucleotide metabolism and other aspects. The results show that BmTal may regulate biological processes in a variety of ways.5. Comparative study of transcriptome on the overexpression of Bmtal gene in Bombyx mori cell BmNCloning the Bmtal gene into the insect expression vector pIZT-V5/His to construct the recombinant plasmid pIZT/V5-His-tal, then,pIZT/V5-His-tal and pIZT/V5 His plasmid were transfected into BmN cells, through zeocin screening for two months, get2 groups of transformed cells, using high-throughput transcriptome sequencing(RNAseq) for the comparison of the transcriptome studies. Results show, differentially expressed genes mainly concentrated in and cell growth and differentiation and in the proliferation process. The results show that Bmtal gene play an important role in cell growth, differentiation and proliferation.