吉富罗非鱼YY超雄鱼和XY雄鱼消减文库的构建及差异基因分析  

Construction and Analysis of Subtractive Library for Differentially Expressed Genes between YY Super Male Tilapia and XY Male Tilapia

作　　者：陈明 罗洪林 朱佳杰 王瑞 甘西 罗永巨 李莉萍 

机构地区：[1]广西水产科学研究院、广西水产遗传育种与健康养殖重点实验室,广西南宁530021

出　　处：《西南农业学报》2014年第3期1314-1320,共7页Southwest China Journal of Agricultural Sciences

基　　金：广西自然科学基金项目(2011GXNSFB018034);十二五国家罗非鱼产业技术体系南宁综合试验站建设项目(CARS-49)

摘　　要：以吉富罗非鱼YY超雄鱼和XY雄鱼的血液为试验材料,采用抑制性消减杂交技术（SSH）构建YY超雄罗非鱼和XY雄鱼的消减文库.采用PCR方法评价文库的质量后,通过DNA测序技术对204个克隆进行测序,经手动去除载体和引物序列后获得86条Contig,应用BLASTX和BioGPS程序对所获Contigs进行生物信息学分析.成功构建了吉富罗非鱼YY超雄鱼和XY雄鱼消减文库,文库库容量（CFU）为3860,库重组率为95％,所获得的DNA片段集中分布在400～ 650 bp之间.获得31条罗非鱼序列,19条非罗非鱼序列,36条序列未发现同源蛋白,可能为YY罗非鱼的新基因.对罗非鱼种属的25条差异序列进行BioGPS功能分类分析发现这些序列参与12个生物过程,可将其分为至少8个不同的分子功能群.所获序列中编码磷酸甘油酰基转移酶-4样蛋白和N-（-乙酰转移酶-10 X2同种型的基因可能与罗非鱼的性发生发育相关.进一步分析发现血红蛋白、促红细胞生成素受体样蛋白、热休克蛋白、谷光氨肽样蛋白和锌指蛋白Gfi-1B样蛋白可能在YY超雄罗非鱼抗环境温度和应激方面发挥重要作用.所构建的吉富罗非鱼YY超雄鱼和XY雄鱼消减文库质量较高,适合YY超雄罗非鱼与XY雄鱼差异基因的筛选,为今后开发YY超雄罗非鱼性别鉴定方法奠定了坚实基础.The suppression subtractive hybridization （SSH） technique was applied to construct the subtractive library for subduction genes of YY super male tilapia and XY male by using the blood of both fishes.Totally 204 gene sequences were obtained by DNA sequencing post the library quality evaluation by PCR method.86 contigs were obtained after the sequences analysis by deletion of the vector and its primer sequences.All these contigs were used to perform the bioinformatics analysis by using BLASTX and BioGPS online program.Results showed that the library capacity was 3860 CFU,the recombinant rate was 95 % and the DNA segment varied between 400 and 650bp.Among the 86 sequences,31 were highly homologous to tilapia,19 were found to be highly homologous to other animals,and 36 were found no homology in NCBI database,suggesting they might be novel genes.Then the 25 differentially expressed genes were analyzed by BioGPS and results showed these sequences are involved in 12 biological processes,and can be classified into 8 molecular function groups.The genes encoding glycerol-3-phosphate acyltransferase 4-like protein and N-alpha-acetyltransferase 10-like isoform X2 protein might associate with sexal development.The gene encoding hemoglobin subunit alpha-A-like protein,hemoglobin subunit alpha-B-like protein,rythropoietin receptor-like protein,heat shock cognate 71 kDa protein-like protein,glutathione S-transferase theta-1-like protein and zinc finger protein Gfi-1b-like protein,respectively might be relate to the thermal and stress resistance of YY super male tilapia.Our results showed a high quality subtractive library of YY super male tilapia was successfully constructed and it was feasible for the differential gene screening and sexal identification of YY super male tilapia.

关 键 词：吉富罗非鱼 超雄鱼 消减文库 差异基因 

分 类 号：S965.125[农业科学—水产养殖]