The Function Of The Receptor Tyrosine Kinase-like Orphan Receptor 2 Gene Ror2in Silkworm, Bombyx Mori

The receptor tyrosine kinase-like orphan receptor Ror2 is a member of the receptor tyrosine kinase orphan receptor（ROR） family, which belongs to the receptor tyrosine kinase（RTK） superfamily. It is a single-transmembrane receptor, only a tyrosine kinase domain. RTKs are a large family of glycoproteins that regulate cell proliferation,polarity, differentiation, migration, metabolism and survival. In mammalian like mouse,Ror2 mutant mouse show respiratory dysfunction and lead to neonatal lethality. Xror2 and its mutant also inhibit convergent extension. So Ror2 may play important roles in early development stages. Ror2 is a highly conserved receptor tyrosine kinase with homologs in many metazoans from Aplysia to Drosophila to humans. The silkworm is the typespecies of Lepidoptera and the research reports of genes and molecular mechanism relative to Ror2 are limited. This research obtained Bm Ror2 gene, detected expression level of these genes in different tissues and diverse developmental stages of the silkworm, investigated the impact of overexpression Bm Ror2 on the size and cycle of cultured cells as well as part of signaling pathways genes in silkworm.In this study, we cloned the open reading frame（ORF） of silkworm Ror2（HOP）.Sequence analysis showed that the ORF was 1914 bp in size and encodes 638 amino acid residues. The gene homology comparison analysis showed that this protein and other species Ror2 proteins have high homology. Bm HOP protein has a singnal peptide,a transmembrane region, an typical extracellular domain FZ, KR tricyclic domain, a transmembrane region and the tyrosine kinase domain Tyr Kc. The results of q PCR indicated that the abundance of Bm HOP m RNA in the head was the highest, was lower in the hemocyte.1 Cloning and sequencing analysis of the Ror2 gene of silkwormIn order to study the function of Ror2 gene in silkworm, Ror2 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 Bm Ror2 was 1924, encoding 638 aa,Gen Bank accession number was NP₀01186538. Structural analysis showed that the secondary structure of Bm Ror2 protein were mainly α helix and random coil. Silkworm Bm Ror2 protein structure is similar with the other receptor tyrosine kinases,consists of extracellular regions: the FZ domain（CRD）, and the Kringle domain; transmembrane regions; cytoplasmic regions: the tyrosine kinase（TK） domain and C-terminal to the kinase domain rich in serine and threonine residues. Ror2 associates via its CRD domain to the ligands. Shown Ror2 protein has receptor function. The kinase domains were detected in Bm Ror2 suggesting that they were endowed with kinase activity.2 The subcellular localization and the expression profiling of the Ror2 gene of silkwormIn order to investigate the expression of Ror2 gene, real-time quantitative PCR was performed to analysis the expression levels of Bm Ror2 gene in diverse tissues in the 3rd day of 5thinstar and in gonads in diverse developmental stages. In the 3rdday of 5th instar different tissues, the expression of Bm Ror2 gene were in a low volume in nearly all tissues. The abundance of Bm Ror2 m RNA in the head was the highest, was lower in the hemocyte. Ror2 in the 4rdday of 4thinstar were the highest expression levels both in ovarian tissue and testis tissue, in other diverse developmental stages were in a low volume.In order to investigate the expression pattern and cellular localization features of Bm Ror2,Bm Ror2 protein antibodies were prepared. The Western blotting results showed that Bm Ror2 protein can be detected in head. Immunofluorescence showed that Bm Ror2 in ovarian sources of Bm N cells can express, and are mainly distributed in cell membranes. It is consistent with its receptor function. The 4rdday of 4thinstar silkworm major organs immunohistochemistry showed, Bm Ror2 proteins were expressed in various tissues, but the highest expression is in the head, consistent with the Real-Time PCR and Western blotting results.3 Construction of transgenic common carrier p BGRNFor more efficient and convenient screening transgenic silkworm, we constructed transgenic common carrier p BGRN. The carrier is based on the piggy Bac transposonadded to neo, ds Red, and EGFP gene express components. The neo gene use the promoter ie-1 from Bm NPV to control, so the transgenic silkworm you can get G418 resistance, can be used G418 screening in the early stages of the transgenic silkworm.The ds Red gene use artificial promoter 3 × P3 control, so the transgenic silkworm eye with red fluorescence, can screening in pre-incubation and pupae stage. So we constructed of transgenic common carrier contains multiple genetic screening, can be more convenient and efficient screening transgenic silkworm.4 The effects on the cultured cells of silkworm after regulation of expression on Ror2 geneIn order to investigate the usefulness of transgenic common carrier p BGRN, We constructed a recombinant vector PBGRN-Ror2 to overexpression silkworm Ror2 gene.We use it transfected the silkworm ovary cells, obtained Ror2 overexpression transgenic cell lines, after transfection48 h green fluorescence can be observed. Detecting the expression level of Bm Ror2 gene by quantitative PCR, it was found the gene upregulated 5.14-fold. Description of the transgenic common carrier p BGRN we build can be used, and can more easily obtain transgenic silkworm.In order to research the function of Ror2 gene, we selected some signal pathway genes for quantitative detection. The results showed that when overexpression Bm Ror2 gene the expression level of wnt, socs6, stat and pr have no changes, socs2 genes increased 1.56-fold and crb, wts, cat, fj, dpp, kibra, serr, myc genes significantly decreased compared with control cells. After Ror2 gene silence, the expression level of dpp raised 1.88-fold and stat, socs2, socs6 gene have no obvious change compared with control cells. The crb, wts, cat, fj, kibra, myc there have also been decreased compared with control cells. And the volume of cultured cells were decreased compared with control Bm N cells. Description Bm Ror2 gene have a moderating effect on the cell size.