家蚕第二隐性赤蚁基因ch-2的图位克隆-mapping clone of silkworm second recessive red ant gene ch - 2.docxVIP

IV IV 江苏科技大学理学硕士学位论文 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 analysis RT-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. Key words: Silkworm, Bombyx mori, SSR, ch-2 gene, Linkage analysis, Gene Location candidate gene, SiRNA 江苏科技大学学位论文原创性声明 本人郑重声明：所呈交的学位论文，是本人在导师的指导下，独立进 行研究工作所取得的成果。除文中已经注明引用的内容外，本论文不包含 任何其他个人或集体已经发表或撰写过的作品成果。对本文的研究做出重 要贡献的个人和集体，均已在文中以明确方式标明。本人完全意识到本声 明的法律结果由本人承担。 学位论文作者签名： 年 月 日 江苏科技大学学位论文版权使用授权书 本学位论文作者完全了解学校有关保留、使用学位论文的规定，同意 学校保留并向国家有关部门