Map-based Cloning Of Chocolate 2 (ch-2) Gene In Silkworm,Bombyxmori

Coloration is one of the variegated characteristics for insects.It is quite a value to view and admire for radiantly beautifμL and odd butterflies and some moth. it is interesting and impotant to explore the genetic polymorphisms, adaptive mechanism and biological evolution for insects. Colour mutation are involved in various growth period, including embryonic phase, larval phase, pupa stage, adult stage and cocoon. Different mutants caused the accumulation and interaction of various kinds of melanin, pteridine, xanthommatin in epidermal cells and corium cells, which are resulted in forming different body color. It has been more than 100 mutants which have been found to correlate with coloring model. Based on the study and research on the mechanism of the body color, it can provide knowledge for insect pigmentation and understand the modle of genetic basis, at the same time it can provide the evidence for the analysis of genetic diversity of silkworm.The second recessive chocolate gene (ch-2) is one of the few appeared red color only in newly hatached larvae period, which can distinguish to normal black ones. We used SSR, STS to make map-based cloning this gene and use and siRNA methods to analyze the functional compensation verification, the contents are as follows:1. Initial Linkage Analysis of Gene ch-2 and MolecμLar MappingWe used silkworm strain P50 as wild type and k04 (ch-2/ch-2) variety as parents, both of which are preserved in SericμLture Research Institute, Chinese Academy of AgricμLtural Sciences. Owing to a lack of crossing over in females, reciprocal Backcrossed BC1F progeny were used for Linkage analysis and mapping of the ch-2 gene in chr18 using silkworm stains P50 and k04, which are classified as being black and red larvae. We found seven SSR markers including S1804,S1807,S1808,S1809,S1812,S1814 and S1819, which were identified to be linked to the ch-2 gene. Redesigned microsatellite loci primers, we found 2 makers near S1804 and S1807, all the 9 primers have the same homozygous profile as the parental ch-2 in the chocolate individuals of BC1F, and heterozygous in the wild type individuals as in F1.The recombination fractions were then calculated from the whole dataset using MAPMAKER 3.0 at a LOD score of 5.0 for further confirmation using BC1M, we constructed a linkage map of 70.7cM, with ch-2 mapped at 69.6cM and the genetic distance between ch-2 gene and the nearest marker S1814 and S1819 is 7.9cM and 1.1cM.The order of the SSR markers was ch-2 and S1819. 2. Fine mapping of ch-2 gene and the full sequence of ch-2 S1814 located at the site of 1.526Mb in nscaf2902 after blasting the Bombyx mori genome database, but the sequence of S1819 can't be blast in the genome database, which meant that the sequence of S1819 is located outside the scaffolds of genome, and this is coincident to the result that the ch-2 gene is located in the end of the established linkage map. We screening nscaf2901 and nscaf2902, design new STS makers and CAPs makers using Primer.primer5.0, to map ch-2 gene precisely, at last we had narrowed the ch-2 gene in a region of 300kb. We blasted all of the candidate genes in NCBI and silk worm genome database, and found that the predicted genes BGIBMGA008245-TA (skin protein gene CPG6) and gene BGIBMGA008268-TA might be related to body color. We blast the Bombyx mori genome database to get the function and at last we consider BGIBMGA008245-TA(CPG6)as the important candidate gene. We get the full sequence of ch-2 using DNAMAN after we had a 5'and 3'RACE-cDNA amplification of P50epidermis.3. The candidate gene and function analysisRT-PCR primers were designed based on the EST of these genes, and we found there were 156bp deletions in BGIBMGA008245 -TA in k04's embryo after 9 days incubation, but there was no distinguish between ch-2 and P50 in the embryo after 1day and 4days'incubation. We designed siRNA according to the CPG6, and microinjected into the Nistari eggs which were dealed with different days'incubation. In the end, we had a statistics about the eggs incubation and dissected the eggs which didn't hatch, and ch-2 phenotype newly-hatched larvae had been observed after RNA knock down of CPG6 gene.