摘要
在白色莱航鸡孵化第13d^21d期间,每天随机选取7个-8个胚胎,从中随机选取3个胚胎,分别采肝脏和骨骼肌样品(n=3),用Primer Express2.0(Applied Biosystem)和BLAST(NCBI)软件设计引物,从采取的肝脏和骨骼肌样品中分离出总RNA,采用反转录聚合酶链式反应(RT-PCR)法,合成cDNA。并应用限制性内切酶产物琼脂糖凝胶电泳法和核酸序列分析法对其进行同源性分析和鉴定目的片段。分别对其肝脏和骨骼肌中葡萄糖激酶、己糖激酶-I和己糖激酶-II的mRNA进行定量分析,旨在了解鸡胚胎发育过程中糖酵解关键酶的mRNA表达规律。结果表明:在孵化期的第13d^21d,鸡胚胎肝脏中葡萄糖激酶、己糖激酶-I和己糖激酶-II的mRNA含量均高于骨骼肌中的含量,但差异不显著(p>0.05)。孵化第17d时肝脏中葡萄糖激酶mRNA的含量明显减少(p<0.05),之后缓慢增加,到第21d时其含量仍低于第13d^15d时的含量;肝脏中己糖激酶-I mRNA在孵化第15d明显减少,之后缓慢增加,到孵化第19d时与第13d的含量接近;在孵化第13d^17d期间,肝脏中己糖激酶-II mRNA含量基本没有变化,到孵化第19d时略有升高,但差异均无显著(p>0.05)。骨骼肌中的3个关键酶的mRNA表达,在整个胚胎发育期均无明显的变化(p>0.05)。
In this study, 7 -8 chicken embryos were selected from 13d - 21d incubation period everyday, and selected 3 embryos randomly, then sampled the liver and the skeletal muscle（ n = 3 ） respectively. According to the BLAST （NCBI） , and using the Primer Express2.0 （ Applied Biosystems） the primers were designed. Then isolated the total RNA from the liver and the skeletal muscle of chicken embryos, and using RT- PCR, synthesized the cDNA. Then analyzed the cDNA fragments by the restriction enzyme analysis and DNA sequencing analysis. Then analyzed the changes in mRNA expression of glucokinase, hexokinase - Ⅰ, and hexokinase - Ⅱ with quantitive PCR method, in order to investigate the glycolysis process within chicken fetation. The results showed that： Within the incubation period of 13d- 21d, the expression of glucokinase mRNA, hexokinase -Ⅰ mRNA and hexokinase-Ⅱ mRNA in the liver was higher than that of in the skeletal muscle （p 〉 0.05）. On the day 17 of incubation glucokinase mRNA was decreased, and then increased gradually, until on day 21, the expression was still less than that of on 13d - 15d; On day 15 hexokinase - Ⅰ mRNA was decreased, and then increased gradually, until on 19d, it was close to the expression of 13d＇s in liver; Between the 13d and 17d of incubation the hexokinase - Ⅱ mRNA was nearly not changed, until 19d the expression was increased a little, but there was no significant difference （p 〉 0.05 ）. In the case of skeletal muscle, there were no significant changes in glucokinase mRNA, hexokinase - Ⅰ mRNA and hexokinase- Ⅱ mRNA （p 〉0.05 ） within the entire experimental periods.
出处
《内蒙古农业大学学报（自然科学版）》
CAS
北大核心
2011年第1期23-27,共5页
Journal of Inner Mongolia Agricultural University(Natural Science Edition)
