| Because of its many excellent properties such as light, soft, smooth and good hygroscopicity, silk is deeply loved by people. Health and natural colored fabric is becoming more and more popular along with the improvement of people’s living standard and the growth of awareness of environmental protection. There are abundant natural colored cocoon resources in our country, and they produce brilliantly colored yellow, golden-yellow, pink, flesh, red, rusty and green cocoons. Compared with white silk, color silk contains lutein, β-carotene and flavonoids, so it has many excellent properties, such as the better resistance to ultraviolet, antioxidant, antibacterial. Therefore, the development and utilization of the colored cocoon resources has important economic value and broad application prospects.Due to the formation of color of the colored cocoon and the pigment transfer regulatory mechanism are not clear, we must first study the transport regulation mechanism of pigments in silkworm before the development and utilization of natural colored cocoon resources. The previous research results showed that the Cameo2 and SCRB15 gene both belong to the scavenger receptor family, but their products can be specific to transport different kinds of pigments in the silkgland of silkworm. Cameo2 and SCRB15 determine the selectivity for transport of lutein and β-carotene, respectively, to the silk gland.In this study, we choose Cameo2 and SCRB15 as the research object, because they both play a key role in the formation pathways of cocoon color of Bombyx mori. Through bioinformatics analysis, combining the related molecular biology techniques, we has carried on the preliminary study to study the regulation mechanism of the two genes. The main results are as follows:1. Cloning and sequence analysis of SCRB15 and Cameo2With the cDNA of rusty color cocoon strain 10-200 and the golden cocoon strain 03-210, Jianpuzhai, the complete CDS sequence of SCRB15 and Cameo2 were amplified. We found the CDS sequence of SCRB15 from strain 10-200 has only one base difference compared with strain c44. However, the CDS sequence of Cameo2 from strain 03-210 and Jianpuzhai are completely consistent with the yellow cocoon breeds N4.2. Bioinformatics analysis and speculation of function mechanism of SCRB15 and Cameo2Using amino acid sequence alignment and structure domain prediction, we found that SCRB15 consists of 504 amino acids while Cameo2 consists of 494 amino acids. And they all have a big extracellular domain.Then we infer the structure of Cameo2 and SCRB15 by homology modelling. The striking feature of their structure is the presence of interconnected cavities that form a tunnel through the entire length of the ectodomain. And the nature of some amino acid residues in the channel have big differences in the two proteins, which may play a important role as to the selective recognition of pigments.3. The prokaryotic expression SCRB15 and Cameo2 genes, and the polyclonal antibody preparation of SCRB15 geneThe sequence corresponding to the extracellular domain of SCRB15 and Cameo2 genes was subcloned into expression plasmid pET28-a followed by identification of sequenceing. Then the recombinant plasmid was transformed into BL21. We finally get a small amount of soluble protein by affinity chromatography. Then we immune rabbit with the purified SCRB15 to obtain polyclonal antibodies.4. The expression pattern analysis of SCRB15 geneUsing the anti-SCRB15 antibody and Western blot, we have analyzed the expression pattern of SCRB15 gene. We found that the contents of SCRB15 were very high in the three rusty cocoon color strains 10-200,03-510 and 03-520. And the temporal profile of SCRB 15 from day 1 to day 6 of 5th instar stage showed that the content of SCRB 15 was highest in the mid-stage of the final instar.5. The preliminary functional verification of SCRB15 geneUsing the purified SCRB15-GST fusion protein, we conducted the experiment to identify the functional domains of SCRB 15 in vitro. Howerer, we didn’t achieve the desired result. So it is necessary to further validate the functional domains responsible for the pigment transport of SCRB 15 on a cellular level. |
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