龙眼GPX基因的克隆、原核表达及其在体胚发生过程中的表达分析  

Cloning of GPX,Prokaryotic Expression and Its Expression During Longan Somatic Embryogenesis of Dimocarpus longan Lour.

作　　者：陈义挺[1,2] 赖钟雄[1] 方智振[1] 蔡英卿[1] 林玉玲[1] 李焕苓[1] 陈登云[1] 

机构地区：[1]福建农林大学亚热带果树研究所,福建福州350002 [2]福建省农业科学院果树研究所,福建福州350013

出　　处：《热带作物学报》2013年第8期1483-1489,共7页Chinese Journal of Tropical Crops

基　　金：国家自然科学基金项目(No.31071787;31201588;30471204);高等学校博士学科点专项科研基金项目(No.20093515110005);福建省科技平台建设项目(No.2008N2001)

摘　　要：采用RT-PCR与RACE相结合的方法,从龙眼胚性愈伤组织中克隆了长947 bp含有完整开放阅读框的龙眼DlGPX cDNA序列(GenBank登录号为EU364813)和长度为1 736 bp的DNA序列(GenBank登录号为EU680970)。其编码1个含有168个氨基酸的蛋白质。DlGPX基因中含有5个内含子,均符合真核生物内含子通用的GT-AG法则。生物信息学分析显示:该蛋白为亲水的酸性蛋白质,定位于叶绿体;与其他植物的GPX有较高的同源性。将该基因构建成原核表达载体,经IPTG诱导表达了1个分子量约为23 ku的蛋白。利用实时荧光定量PCR(qPCR)技术研究该基因在龙眼体胚发生发育过程中的表达情况,结果显示,从松散型胚性愈伤组织到不完全胚性紧实球形结构阶段,DlGPX mRNA的转录水平逐渐升高,在胚性紧实球形结构阶段降到最低水平,子叶形胚阶段又上升到与不完全胚性紧实球形结构阶段相当的水平。Using RT-PCR and RACE, DIGPX, a 947 bp cDNA with an ORF was isolated from the embryogenic calli of Dimocarpus longan. The DlGPX gene, encoding a protein with 168 amino acids, contained 5 introns consistent with the GT-AG dogma for eukaryotic introns. Bioinformatic analysis indicated that DlGPX was a hydrophilic acidic protein and located in the chloroplast. Its amino acid sequence showed high similarity with D1GPX from other plant~ Prokaryotic expression vector fused with DIGPX was constructed and a protein about 23 ku was expressed. qPCR results indicated that the transcription level of DlGPX increased gradually from embryogenic calli to incomplete compact pro-embryogenic culture stage and decreased to the lowest level at compact pro-embryogenic culture stage, then upregulated to a comparable level with that at incomplete compact pro-embryogenic culture stage.

关 键 词：龙眼 体细胞胚胎发生 GPX 克隆 生物信息学 实时荧光定量PCR 

分 类 号：S667.2[农业科学—果树学] Q78[农业科学—园艺学]