The Cloning And Functional Analysis Of Vitellogenin Receptor In Silkworm, Bombyx Mori

Silkworm, Bombyx mori as a modal in Lepidoptera insect is also an important economic insect. We have completed the detail genome map of silkworm, combining with the more than 600 mutants of genetic resource in our laboratory, which provided a wealth of resource with the functional research in silkworm. Vitellogenin receptor is an important protein to ovary and embryonic development in oviparous animals, which mediates vitellogenin into the ovary to supply nutrition with oocytes through endocytosis. So far. there are 11 species of insects have been reported in the VgR cDNA and genomic sequences, all of them are belong to the low-density lipoprotein receptor which have a typical conserved functional domains. In insects, the expression of VgR has begun in early stage of oocyte differentiation, and expressed through the whole period of vitellogenesis. VgRs are ovary-specific receptors of the LDLR gene family. Drosophila and Chicken can not lay eggs when absent of VgR/YPR. When applied technology of dsRNA-mediated interference(RNAi) in Dermacentor variabilis,Haemaphysalis longicornis and Solenopsis invicta, the ovarian development have been affected or the egg formation or development have been disrupted. The results indicated that VgR gene play an important role in the ovary development.We have reported the 3'terminal part sequence of BmVgR in previous study. In this study, we study on the full-length cDNA of BmVgR and expression localization analysis by using RT-PCR, gene cloning, in situ hybridization, Northern hybridization technique. Additionally, we explore the gene function of BmVgR by combining vit mutant and RNAi technology. The results are as follows:1. The bioinformatic analysis and cloning of the full-length cDNA of BmVgR gene in the silkwormBased on the silkworm genome database, the bioinformatic analysis reveals BmVgR located in nscaf481 on chromosome 20 in silkworm. We isolated and cloned the full-length cDNA of BmVgR from silkworm, BmVgR cDNA sequences are 5746bp in length and spans over 70 kb in genome with 35 exons containing a complete open reading frame. The deduced mature protein contains a signal peptide (MKVVLLAIVLCTTSCVG) with a cleavage site between amino acids 17 and 18 at the amino acid junction CVG-QF. The molecular mass and isoelectric point of BmVgR was predicted to be 202.5 kDa and 5.54 before removal of the signal peptide. The Amino acid multiple sequence alignment indicated BmVgR as a member of low-density lipoprotein receptor family have highly conserved domains. Phylogenetic analysis indicated BmVgR evolved slower than other insects.2. The transcriptional expression and localization of BmVgRThe transcriptional expression characteristics of BmVgR were analyzed by RT-PCR. The tissue expression profile shows BmVgR was specifically expressed in ovarian tissue and restricted in the eggs, but not in tissues such as antennae, head, thorax, abdomen, wings, tail, and tubal. Further analysis in situ hybridization, the results revealed BmVgR only expressed in oocytes of ovarian cells. The transcriptional signals revealed that BmVgR was expressed during the entire life span of silkworm. The expression level of BmVgR was low from the embryo to day 5 of the fifth instar larvae and began to increase on day 6-7 of the fifth instar, and reached to the highest on day 4-5 day after pupation, while vitellogenin began to express after 24 hr post wandering. These results illustrated that BmVgR was upregulated at the time of egg maturation in silkworm.3. The mutational analysis and expression analysis of BmVgR in vit mutationIn this study, we cloned the full length cNDA of BmVgR in vit mutant. The BmVgR in vit had lacked two fragment when compared to BmVgR of Dazao. The first fragment was located between 1680bp-1832 bp, and this deletion caused a loss of amino acid sequence between 560 to 609 aa. Structural analysis showed that this deletion was in the third Class B region of the first epidermal growth factor precursor domain. The second fragment was located between 3879bp-3941 bp which was the first Class B region of the second epidermal growth factor precursor domain. Northern hybridization in BmVgR showed there are no clear differences in expression or size between dazao and vit mutant. The first fragment has important role in dimerization of the receptor and ligand dissociation. We speculated it is because of the lack of the first fragments, the vit mutation can not take up Vg normally. But to completely clear whether VgR gene caused vit mutations, it needs further experimental verification.4. RNAi of BmVgR By dsRNA-mediated RNAi experiments, we choose the 1^st female pupae as injected object. In order to achieved good effect, we continuously injected when the pupae developed to 4^th and 7^th.The RNAi results showed that the pupae injected dsVgR developed normally and can mate of moths, but the laid eggs has many small egg with white colour, almost less half of the normal and can not hatch. The pupae injected elution buffer and dsRed develop normally, whose laid normal eggs. The fluorescence quantitative results showed small eggs had low expression in VgR in relative to the control. ELISA test was performed to measure the amount of BmVg in eggs and found the amount of BmVg in small egg was much less than the control. The result indicated that vit mutant was most likely caused by the mutation of BmVgR. which can not transport BmVg into oocyte. It has been reported that vit mutant is homogeneous lethal. Combined with the results of our experiment, it has been concluded that BmVgR is important in the egg formation and BmVg is essential in embryonic development in silkworm.