摘要
本研究旨在克隆绵羊YAP1基因CDS序列,构建并鉴定其真核表达质粒pEGFP-C1-YAP1,及其磷酸化位点突变体pEGFP-C1-YAP1S42A。本研究利用RT-PCR技术,从湖羊背最长肌克隆YAP1基因编码区序列,T-A克隆到pMD 19-T载体中,进行测序。利用酶切和T4连接酶连接方法,将YAP1基因编码区克隆到pEGFP-C 1载体中,构建pEGFP-C 1-YAP1真核表达载体,以真核表达载体为模板,构建磷酸化位点突变体pEGFP-C1-YAP1S42A,2种载体均进行PCR、BamHⅠ和XhoⅠ双酶切、BamHⅠ单酶切及测序鉴定。结果克隆出YAP1基因的全长cDNA序列,获得GenBank登录号JQ 714252,其长度为1 712bp,编码区1 212bp,编码403个氨基酸。重组质粒pEGFP-C1-YAP1经PCR、酶切、测序鉴定,证实YAP1基因CDS区已正确克隆入pEGFP-C1表达载体;经测序鉴定,YAP1第42位丝氨酸成功突变为丙氨酸。本研究成功克隆绵羊YAP1基因全长cDNA序列,并构建了重组真核表达质粒pEGFP-C1-YAP1及磷酸化位点突变体pEGFP-C1-YAP1S42A,为进一步研究YAP1蛋白表达及其生物学活性奠定基础。
The study was conducted to clone sheep YAP1 CDS and construct a eukaryotic expression plasmid and a mutant that could not be phosphorylated at Ser42.The CDS of sheep YAP1 was amplified by RT-PCR from healthy sheep longissimus dorsi muscle,and was cloned into pMD19-T simple vector by T/A Ligation to sequence the DNA fragment.After double digestion,YAP1 coding region was subcloned into pEGFP-C1 vector by T4 Ligase to construct a eukaryotic expression plasmid and then make the eukaryotic expression vector as the template to construct the phosphorylation site mutant.PCR,restriction endonucleases analysis and sequencing were used to conform both of the recombinant plasmids.The sheep full-length YAP1 cDNA sequence was 1 712 bp in length encoding 403 amino acids.It was confirmed that the sheep YAP1 CDS was correctly inserted into eukaryotic expression vector and serine had been mutated to alanine at 42ed amino acid by PCR,restriction digestion and sequencing.The results showed that the recombinant plasmid pEGFP-C1-YAP1 and pEGFP-C1-YAP1 S42A was constructed correctly,which paved the foundation for further studies on the YAP1 protein expression and its biological activities.
出处
《畜牧兽医学报》
CAS
CSCD
北大核心
2013年第8期1198-1204,共7页
ACTA VETERINARIA ET ZOOTECHNICA SINICA
基金
中国博士后科学基金特别资助项目(200902154)
科技部家养动物种质资源平台
现代肉羊产业技术体系建设(CARS-39)
江苏高校优势学科建设工程资助项目
江苏省农业科技支撑计划项目(BE2012331)
江苏省六大人才高峰项目
江苏省工程技术研究中心项目(BM2012308)
作者简介
李达（1986-），男，河北保定人，硕士生，主要从事动物遗传资源与分子标记育种研究，Email：4everold@gmail．com
通信作者：孙伟，博士后，教授，主要从事动物遗传资源与分子标记育种研究，E—mail：dkxmsunwei@163．com
