毛百合LiGPAT基因3'端与5'端克隆及其序列分析

Cloning and Sequencing Analysis of the 3' and 5' Fragment of GPAT Gene (LiGPAT) from L. pensylvanicum

全长cDNA克隆不仅包含完整的阅读框,还拥有5'和3'端的非编码区,全长cDNA序列的获取是正确地注释基因组序列、进行基因的功能及产物分析的必要基础和前提。本研究以叶片为材料,利用3'和5'末端快速扩增(3'RACE和5'RACE)技术成功克隆了毛百合甘油-3-磷酸酰基转移酶(glycerol-3-phosphate acyltransferase,GPAT)基因3'和5'末端序列,分别得到了长度为777 bp和496 bp的片段。通过与已发表的LiGPAT保守区基因序列(Genbank登录号为HQ654523)拼接,得到全长1544bp的cDNA,其中5'-UTR10bp,ORF区1 233 bp,3'-UTR 311 bp并含有19 bp的poly(A)尾巴,编码410个氨基酸,预测分子量大小为45.9 kD,此序列Genbank登录号为JX524741。氨基酸同源性分析表明克隆得到的序列和其他物种的GPAT基因同源性较高,进一步进化分析表明其与水稻、油棕等单子叶植物的亲缘关系较近,对其进行生物信息学分析结果表明所得基因序列具有典型的GPAT基因的结构特点,包含磷酸酰基转移酶功能域PIsC结构域,由此说明成功克隆了毛百合GPAT基因全长,这将为后续的下游分析及进一步研究GPAT基因的功能奠定基础。

Full length cDNA cloning not only contains the complete reading frame, also has 5＇ and 3＇ end non- coding regions, cDNA sequence is correctly characterizing genome sequence, the function of the gene and essential basis and premise of the product analysis. In This study, we use blade as material, the lily glycerin-3-phosphate acyltransferase （glycerol-3-phosphate acyltransferase, GPAT） gene 3＇ and 5＇ end sequences successfully cloned by rapid amplification （3＇ 5＇RACE and RACE） technology, got a length of 777 bp and 496 bp, respectively. Joining the sequences and published LiGPA T conservative region （Genbank login for HQ654523）, get over 1 544 bp cDNA, which contains a 1 233 bp open reading frame （ORF）, flanked by 10 bp, 5＇-UTR region and 311 bp 3＇-UTR region and contains 19 bp poly（A）, encoding 410 amino acids, its molecular mass is 45.9 kD, this sequence＇ s GenBank re- gistration number is JX524741. Amino acid homology analysis indicated that the sequence and other species of GPAT gene homology are higher and further evolution analysis demonstrated that it has closer relation with the rice and oil palm etc., the bioinformatics analysis results show that the gene sequence has the typical GPAT gene stru- cture characteristics, containing phosphate acyltransferase function domain PIsC structure domain, the lily GPAT gene is cloned successfully, this will lay foundation for subsequent downstream analysis and the further research.