Identification And Expression Analysis Of Vitellogenin Receptor From Silk-producing Insect, Bombyx Mandarina

Bombyx mandarina is a kind of wild silk-producing insects, also known as wild silkworm. It is widely perched on China’s sericulture area and found in the silkworm region of Japan, North Korea, South Korea and other countries, also have a small amount of the distribution in Russia and India. Belonging to Lepidoptera Saturniidae. So far, the study about Bombyx mandarina were seldom reported.The vitellogenin receptor play a key role in the embryonic development of oviparous animal, which mediates yolk protein into the oocytes to supply a source of nutrition with ovarian and embryonic development. So far, there are15species 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.Oligonucleotide primers were designed by Primer premier5.0software based on the conserved amino acid sequence of VgR gene reported from Bombyx mori, Actias selene Hubner and other insects. A vitellogenin receptor (VgR) gene was cloned from Bombyx mandarina(B. mandarina) using reverse transcriptase-polymerase chain reaction (RT-PCR). Sequence analysis revealed that this gene was5,861bp long contained a5,433bp open reading frame encoding1,811amino acid residues and had99.7%identity with Actias selene Hubner VgR..Using the ScanProsite software analyzed the functional domains of vitellogenin receptor from Bombyx mandarina, two ligand binding domain of LBD1and LBD2were selected for prokaryotic expression. The fragments of two domains of Bma-VgR were specific amplified by PCR and ligated to pET-28a vector for protein expression. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and western blotting analysis demonstrated that the recombinant proteins were successfully expressed in Escherichia coli(E. coli) cells.The induced cells were harvested and subjected to nickel affinity column chromatography to purify recombinant proteins.In order to obtain polyclonal antibody, the purified recombinant protein was used to immunize a male New Zealand rabbit, the titer of the obtained antibody was measuring by ELISA.Proteins and total RNAs from fat body, ovary, epidermis, malpighian tubule, midgut, head from the first day of just wandering were extracted and analyzed by Western blotting and RT-PCR. The result showed the expression of Bma VgR was detected in fat body and ovary.According to the results of RT-PCR,Proteins and total RNAs from fat body from the first day to seventh day after pupation were extracted and analyzed by Western blotting and Real-time fluorescent quantitative PCR.The expression level of Bma VgR in fat body was significantly changed from3rd day to6th day after pupation.Bombyx mandarina(Lepidoptera, Bombycidae) is one of the major pests of mulberry leaves. At the same time, Bombyx mandarina belongs to the lepidoptera can provide a model for the study of other lepidopteran pests.The study of Bma VgR would be helpful to understand the molecular mechanisms of pests reproduction and provide a theoretical basis for exploring reasonable pest control method in agricultural production.