Molecular Cloning And Expression Of Bombyx Mori Sterol Carrier Protein X/sterol Carrier Protein 2 (SCPx/SCP2) Gene

In insect, cholesterol is not only required for cellular membrane , but also is involved in the important process of ecdysteroid biosynthesis. However, insects can not produce cholesterol via de novo synthesis because they lack two key enzymes in their bodies. In fact, insects mainly depend on dietary cholesterol to fulfill their growth, development and reproduction. Fundamental studies have determined that midgut of insect is the major site of cholesterol absorption and conversion. After transport by vascular lipoprotein in hemolymph, cholesterol is consumed by the prothoracic glands. On the other hand, cholesterol can be carried to fat body via lipophorin of the hemolymph, and be stored in fat body as free and esterified forms. Therefore, the hallmark of fat metabolism in insects centers on the lipid transport system.Sterol carrier protein2 (SCP2) is a lipid-binding protein, which exist in species broadly. The SCPx/pro-SCP-2 gene is a fusion gene in mammal and the SCP2 protein is considered involved in cholesterol, fatty acid metabolism and their intracellular transport. Protein SCP2 could not only absorb free cholesterol for the construction of cellular membranes, but also provide cholesterol as a precursor to mitochondria for the steroid biosynthesis, such as insects molting hormones.In this work, we predicated the DNA, mRNA and protein of BmSCPx/SCP2 gene by bioinformatics method firstly. Then using a RT-PCR-based cloning strategy, we isolated two types mRNA from a single BmSCPx/SCP2 gene, and analyzed the expression pattern of BmSCPx and BmSCP2 in different stages and tissues. Finally, the BmSCPx and BmSCP2 gene were cloned into the pET28(a) His tag vector to express in Escherichia coli strain BL21.In order to find the candidate of SCPx/SCP2 gene in Bombyx mori, the SCPx amino acid sequence of the Spodoptera littoralis was used to search in the silkworm dbEST and genome sequence of silkworm by TBLASTN program. It was found that BmSCPx/SCP2 gene had 8902 bp in genome, and comprised 2 exons and 1 intron. After assembling all the ESTs, two 'contig' were obtained. Analyzed BmSCPx and BmSCP2 gene by cloned and sequenced, it was found that the results were consistent with the predicted sequences we assembled in silico, which verified our prediction. The BmSCPx/SCP2 gene encodes the two types of transcripts by alternative splicing mechanism. The BmSCPx has 2165 nt and BmSCP2 has 1035 nt.ORF of BmSCPx lies in 197-1804 nt, encoding 536 amino acids. Functional domain from prediction showed that it contains Thiolase-N, Thiolase-C and a SCP2 domain. Compared to BmSCPx, the BmSCP2 contains an ORF of 438 nt coding for 146 amino acids. It only contains a SCP2 domain. Molecular weight and pI of BmSCPx and BmSCP2 are 57.8 kD, 16.2 kD and 6.54, 9.22. The alignment of amino acid sequences showed similarity of the SCP2 gene family members.