Studies On Hippo Signaling Pathway Major Genes Of Silkworm, Bombyx Mori

Hippo signaling pathway, an inhibiting signaling pathway of cell growth, is very conservative in the process of biological evolution. In Drosophila, accepting extracellular inhibition signal, the upstream membrane protein receptors of the signaling pathway phosphorylate the transcriptional coactivator YKI through a series of kinase cascades. The phosphorylated YKI, interacted with the cytoskeleton, was stranded in the cytoplasm and unable to transcript target genes in the nucleus so as to regulate of organ size. The silkworm is the typespecies of Lepidoptera and the research reports of genes and molecular mechanism relative to Hippo signaling pathway are limited. This research obtained BmHpo, BmSav, BmWts, BmMats and BmYki genes, detected expression level of these genes in different tissues and diverse developmental stages of the silkworm, investigated the impact of overexpression BmYki1and its mutant on the size and cycle of cultured cells as well as the upstream and downstream genes of Hippo signaling pathway and observed the development of wing when downregulated the expression of BmYki1, expectating to clarify the mechanism of the Hippo signaling pathway in silkworm and provide new ideas for the regulation of the size of ovaries, silk glands and wings. The main results obtained in this study are as follows:1. Cloning and sequencing analysis of the Hippo pathway major genes of silkwormIn order to study the function of major genes in Hippo signaling pathway of silkworm, Hpo, Sav, Wts, Mats and Yki gene sequences were obtained by silico cloning. The primers were designed and RT-PCR was performed. The results showed that the open reading frame of BmHpo was1479, encoding492aa, GenBank accession number was KF904333. The open reading frame of BmSav was1209, encoding402aa, GenBank accession number was KF904337. The open reading frame of BmWts was1535, encoding510aa, GenBank accession number was KF904338. There were three alternatively spliced isoforms of BmMats gene, designated as BmMatsl, BmMats2and BmMatsS. The open reading frame of them were570,564and570, encoding189,187and189aa, GenBank accession number were KF904334, KF904335and KF904336, respectively. Compared with BmMatsl, there was a lack of six bases in the fourth exon in BmMats2and in BmMats3the245nt was changed as T, the513nt was changed as G. Three alternatively spliced isoforms of BmYki gene were required, designated as BmYkil, BmYki2and BmYki3. The open reading frame of them were1314,1173and1335, encoding437,390and444aa, GenBank accession number were KF904339, KF904310and KF904311, respectively. Compared with BmYkil, BmYki2had a deletion of the third exon, and15and6bases were increased to the3’end of the fifth exon and51end of the sixth exon in BmYki3, respectively.Structural analysis showed that the secondary structure of BmHPO, BmWTS and BmMATS1protein were mainly a helix and random coil, while BmSAV and BmYKI1protein were random coil primarily. The kinase domains were detected in BmHPO and BmWTS, suggesting that they were endowed with kinase activity. The WW domains were detected in BmSAV and BmYKI1, suggesting that they were involved in intracellular signaling pathways and a variety of biochemical processes. There showed high homology between silkworm and fly in WTS and MATS1proteins, indicating that the two kinds of proteins were very conservative in both insects and may play a similar role. The molecular phylogenetic analysis based on YKI showed that YKI was aggregated according to vertebrate and invertebrate respectively and it had some homology in different species.2. The expression profiling of the Hippo pathway major genes of silkwormIn order to investigate the expression of Hippo signaling pathway major genes, real-time quantitative PCR was performed to analysis the expression levels of BmHpo, BmSav, BmWts, BmMats and BmYki genes in diverse tissues in the3rd day of5th instar and in gonads in diverse developmental stages. in the3rd day of5th instar different tissues, the expression of BmHpo, BmSav and BmYki2genes were in a low volume in nearly all tissues. The expression levels of BmWts and BmMats genes in the head and stomatal plexus was higher in various tissues and BmYkil/3gene was expressed highest in the intestine while lowest in blood cells. The detected in diverse developmental stages showed that each gene expressed highest in ovaries in the7th day of5th instar and the2nd day of pupal stage. Compared to several other genes, BmYkil/3expressed much higher and in ovaries in the7th day of5th instar, BmYkil/3expressed highest than other stages. The results also showed that BmYki2gene exbited higher expression in the2nd day of pupal stage, suggesting that different spliced isoforms of BmYki genes may play different functions at different development stages.3. The subcellular localization and protein detection of BmYKI1and BmYKI1S97AThe transcriptional coactivator YKI is the most important component in Hippo signaling pathway. In order to investigate the expression pattern and cellular localization features of BmYKI, BmYki1gene was cloned into the prokaryotic expression vector pET-28a (+).BmYKI1recombinant was successfully expressed in E.coli and the polyclonal anti BmYKI1was obtained. The western blotting results showed that BmYKI protein can be detected in testis, ovary, midgut, silk gland, fat body and stomatal plexus, but in the head and malpighian tube, no clear signal was observed. Cellular localization results showed that BmYKI1protein was mainly localized in the cytoplasm of cells, while BmYKI1S97A protein was transferred into the nucleus when the important phosphorylation sites, BmYKI1S97, mutated into A (BmYKI1S97A), indicating that phosphorylation of BmYKI1is vital for cellular localization.4. The effects on the wing disc and cultured cells of silkworm after regulation of expression on Yki geneTo investigate the function of BmYkil gene, we worked on the upregulation of BmYki1and BmYki1S97A genes and downregulation BmYkil gene in order to discover the effects on cultured cells, wing disc and expression levels of upstream and downward in Hippo signaling pathway. The results showed that when overexpression BmYki1and BmYkilS97A gene, the volume of cultured cells increased by23%and27%and the percentage of dividing cells were increased by14.7%and10%compared with control BmN cells. After overexpression BmYkil gene, the expression level of fj, crb, serr, wnt, cat, bmpr and dpp genes increased significantly compared with control cells while the expression level of the ex, kibra and fj genes increased after overexpression BmYkilS97A genes, indicating that cell cycle and volume can be regulated by adjustable the expression of BmYkil and BmYkilS97A genes. After RNA interference on Yki gene, the expression levels of ex and kibra genes decreased by2.25and1.67times, respectively. After injection yki-siRNA-298(1μg/head) to the right wing disc of pupa, the atrophy ratio of right wing of the moth reached35%. The results showed that the atrophy ratio of hind wing was greater than the front wing, reaching the decline of58.3%in extream. At the same time, the expression levels of ex and crb genes decreased by1.95and8.20times while the expression levels upstream components of Hippo signaling pathway like wts, fj and serr genes were significantly upregulated, indicating that regulation of expression of Yki gene may influence upstream and downstream genes in Hippo signaling pathway expression and the wing development of silkworm.In summary, we cloned Hippo signaling pathway major genes of silkworm, crystalized the main features and expression patterns of each gene and figured out that BmYkil gene was essential to regulate the expression of upstream and downstream genes in Hippo signaling pathway, which may influence the volum and cycle of cultured cells as well as the development of wing disc. The research consequences laid a foundation to elucidate the mechanism of Hippo signaling pathway in silkworm and provide new ideas for the regulation of the size of ovaries, silk glands and wings.