应用引物退火控制技术(ACP)筛选尼罗罗非鱼雌雄鱼肌肉组织差异表达基因,寻找与雌雄鱼肌肉生长发育相关的候选基因。本实验从同等条件下养殖的尼罗罗非鱼群体中随机选取雌、雄鱼各5尾组成RNA池,采用引物退火控制技术,分析了两组个体肌肉组织差异表达基因。利用20对随机引物差异显示扩增,共获得8条ESTs,其中5个已知的ESTs分别为转录变体3(LOC100691543)、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、肌型肌酸激酶M2-CK和转录因子Sox4,其余3个为未知的ESTs。实时定量PCR分析各差异表达基因在尼罗罗非鱼雌雄肌肉组织中的表达发现,8个差异表达基因中转录变体3与ACP6-Y在尼罗罗非鱼雄鱼肌肉组织中的表达均极显著高于雌鱼(P〈0.01),ACP3-X、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、ACP15-X、肌型肌酸激酶M2.CK与转录因子Sox4在尼罗罗非鱼雌鱼肌肉组织中的表达均极显著高于雄鱼(P〈0.01)。结果表明,应用引物退火控制技术筛选获得了8个可能参与了雌雄鱼肌肉生长发育调控的ESTs,为进一步筛选雌雄鱼肌肉生长发育相关候选基因奠定了基础。
Annealing control primer (ACP) system was applied to find candidate genes related to muscle growth of Nile tilapia by screening differentially expressed genes in the muscle tissues of male and female fish. This study randomly selected five male and five female fish from Nile tilapia populations cultured under the same conditions to build a RNA pool, and differentially expressed genes of two groups were analyzed by annealing control primer system. Eight differentially expressed genes were identified and sequenced by amplification with 20 arbitrary primers. In these genes, five were already known as variant 3 ( LOC100691543 ) ,60S ribosomal protein L3 ( RL3 ), Parvalbumin beta-like, muscle-type creatine kinase M2- CK and transcription factor Sox4 gene, and others, while three were unknown. Base on quantitative real-time PCR, the relative expression levels of transcription variant 3 ( LOC100691543 ) and ACP6-Y in the muscle of male Nile tilapia were significantly higher than those in female fish( P 〈 0.01 ) ,while the relative expression levels of ACP3-X ,60S ribosomal protein L3 ( RL3 ), Parvalbumin beta-like, ACP15-X, muscle-type creatine kinase M2-CK and transcription factor Sox4 in the muscle of female Nile tilapia were significantly higher than those in male fish(P 〈0.01 ). All the 8 ESTs were screened by annealing control primer system,which may have participated in regulating muscle growth of male and female Nile tilapia. This study laid a foundation for further screening of muscle growth related genes.