肉鸡MTHFR基因的克隆和表达水平研究

采用RT-PCR方法克隆了鸡MTHFR(methylenetetrahydrofolate reductase, MTHFR)基因,利用生物信息学方法预测蛋白结构,应用定量PCR(qPCR)技术检测组织表达特性。结果表明,鸡MTHFR基因的cDNA序列长度为2 342 bp(GenBank登录号: KU351685),开放阅读框长1 956 bp,编码651个氨基酸;进化树分析表明,鸡MTHFR氨基酸序列与绿头鸭和鸿雁的关系较近。MTHFR氨基酸序列包含保守的MTHFR superfamily结构域,为亲水蛋白;二级结构主要是α-螺旋(38.71%)和无规则卷曲(36.41%);亚细胞定位显示,该蛋白存在于细胞质和细胞核的概率分别为60.9%和30.4%。qPCR分析表明,MTHFR基因在所检测鸡的8种组织中均有表达,其中在心脏中表达水平最高,在腿肌中的表达水平最低;叶酸缺乏试验表明,母鸡叶酸缺乏能极显著增加子代肉鸡MTHFR基因在肝脏中的表达水平(P<0.01)。研究结果可为进一步研究MTHFR基因的结构和功能以及叶酸在家禽生产中的应用提供资料。

Molecular cloning and expression patterns of MTHFR gene in broiler

The broiler methylenetetrahydrofolate reductase (MTHFR) gene was cloned by RT-PCR, and the protein structure of broiler MTHFR was predicted by bioinformatics methods. The expression levels in different tissues were detected by qPCR. The results showed that the broiler MTHFR cDNA (GenBank accession No: KU351685) was 2 342 bp in length, which coded 651 amino acids. The phylogenetic tree analysis indicated the broiler MTHFR was closely related to mallard and swan goose. The broiler MTHFR protein contained conserved MTHFR superfamily domain and was hydrophilicity protein. The secondary structure of MTHFR protein mainly contained α-helix (38.71%) and random coil (36.41%). The residing probability of MTHFR in the cytoplasmic and nuclear was 60.9% and 30.4%, respectively. By qPCR, different mRNA expression levels of broiler MTHFR were detected in all the 8 investigated tissues. Among them, heart showed the highest expression levels and muscle of legs had the lowest expression levels. In addition, the MTHFR expression levels in offspring liver of broiler breeders were significantly increased under folate deficiency in the maternal diets (P<0.01). These results will provide the data for further research of MTHFR gene structure and function, and dietary folic acid supplementation in poultry production.