Cloning And Expression Analysis Of Vitellogenin Gene From Actias Selene Hubner

Actias selene Hubner is an undeveloped wild silk insect. Few reports about it were found and most of them are on the morphology and biological behavior. Vitellogenin from Actias selene Hubner is composed of two subunits, the big one is 175 KDa and the small one is 45 KDa.Vitellogenin has many other important biological functions in organism as well as nutritional functions. In resent years, researches on insect vitellogenin are very popular, and there are many primary structures of vitellogenin were sequenced continuously, while the vitellogenin gene of Actias selene Hubner has not been analysed yet. Results of this experiment will provide some informations for further study on function and application of vitellogenin in Actias selene Hubner.In this study, ten pairs of specific primers were designed to amplify the DNA sequence of vitellogenin according to the vitellogenin cDNA sequence of Actias selene Hubner and 5′regulation region of the Bombyx mori, Antheraea pernyi and Antheraea yamamai. A sequence of 7329 bp long (GenBank accession number: GU361974) including 6 extrons and 5 introns with an open reading frame encoding a 1774 amino acids peptide was obtained by PCR. A BmDsx binding site (ACATTGT) and some conserved signatures such as CdxA and GATA-X were found in 206 bp long 5′- flanking region of vitellogenin gene in Actias selene Hubner. The size of exons were 2246 bp,205 bp,982 bp,879 bp,184 bp and 863 bp respectively, and introns were 107 bp,84 bp,626 bp,706 bp and 230 bp respectively. Compared with other insects, we discovered that the structure of vitellogenin gene of Actias selene Hubner is more similar to that of Antheraea pernyi. They both have a large intron loss in 31 bp after ATG.Meanwhile, the total RNA of female Actias selene Hubner was extracted to analyse the expression of vitellogenin in different developmental stages and tissues, then reversely transcribed and synthesized the first strand cDNA by M-MLV. Take the 18sRNA of Actias selene Hubner as a reference gene, we carried the semi-quantitative PCR. The result showed that vitellogenin of Actias selene Hubner did not express in the whole larva stages, and its expression reached the maximum in prepupa stage. During the whole pupation, it decreased slightly in the first day and declined a lower level in the forth day. During diapause stage, it kept almost the level of the first day of pupation. In addition, vitellogenin of Actias selene Hubner did not express in midintestine, head and malpighian, while expressed in blood, fat body and ovary with no obvious quantitative difference. It confirmed that the vitellogenin gene of Actias selene Hubner expressed specifically in different stages and tissues.The small subunit was amplified by PCR, ligated to pET- 28a(+) expression vector and transformed into Escherichia coli BL21(DE3), induced by IPTG under different concentrations and different time length. Through SDS-PAGE electrophoresis and Western blot analysis, the induced fusion protein was successfully expressed. This will provide the foundation for further study on eukaryotic expression and function of vitellogenin gene.