蒙古冰草磷脂酶D基因cDNA3’端的克隆及分析

Cloning and Sequence Analysis of Phospholipase D Gene 3'End cDNA of Agropyron mongolicum Keng

目的:采用3’RACE方法对蒙古冰草磷脂酶D(PLD)基因3’端全序列进行了快速扩增和序列分析,为得到全序列及研究该基因的功能奠定基础。方法:实验以蒙古冰草幼叶为材料,利用RT-PCR技术得到PLD基因的部分cDNA序列,根据此序列设计一条特异性上游引物,反转录引物中的部分序列即M13PrimerM4作为下游引物,按3’RACE试剂盒(TaKaRa)操作流程进行PLD基因3’末端的快速扩增,成功获得993bp的3’RACE反应产物,采用DNAStar软件对扩增的PLD基因的3’RACE产物和中间片段进行拼接,最终获得2113bp的PLD基因3’端cDNA序列。结果:通过BLAST技术,发现该片段与许多植物磷脂酶D基因的同源性为47.0%～80.0%。结论:通过同源性比较,成功克隆了PLD基因cDNA3’端序列。

Objective：To amplify the 3＇-cDNA end of phospholipase D（PLD）gene of Agropyron mongolicum Keng by 3＇RACE technique and to analyze the sequence in the experiment,the 3＇-cDNA end cloning establish foundation in order to obtain the cDNA sequence and research the function of PLD gene.Method：The PLD gene partial cDNA fragment was amplified by RT-PCR from total RNA of Agropyron mongolicum Keng leaves.We designed a special primer on this fragment.With this special primer as up primer and M13primerM4 as down primer,3＇-cDNA end of PLD gene was rapid amplified by following the instruction of 3＇RACE kit（Takara）.3＇RACE product with 993bp was successfully obtained.We spliced 3＇RACE product and intermedial product by DNAStar,the 3＇-cDNA end sequence of PLD gene with 2113bp was obtained.Result：Homology analysis by BLAST showed that homologous with PLD gene of other plants was 47.0%-80.0%.Conclusion：The sequence was thought as the PLD gene 3＇-cDNA end sequence of Agropyron mongolicum Keng,the sequence clone establish foundation in order to obtain the all sequence of the PLD gene.