大葱AfCER1基因的克隆及表达分析  

Cloning and Expression Analysis of Af CER1 Gene in Welsh Onion

作　　者：奥娜 赵泓[2] 裴雁曦[1] 王永勤[2] 

机构地区：[1]山西大学生命科学学院,山西太原030006 [2]北京市农林科学院蔬菜研究中心,农业部华北地区园艺作物生物学与种质创制重点实验室,蔬菜种质改良北京市重点实验室,北京100097

出　　处：《山西农业科学》2016年第5期569-574,582,共7页Journal of Shanxi Agricultural Sciences

基　　金：国家自然科学基金项目(31372066);北京市农林科学院科技创新能力建设专项(KJCX201101010)

摘　　要：基于大葱RNA-seq转录组数据库中蜡质相关基因Af CER1的序列设计引物,利用RT-PCR(Reverse transcription-polymerase chain reaction)和RACE(Rapid amplification of c DNA ends)技术获得Af CER1(Allium fistulisum CER1)的全长c DNA序列,并通过生物信息学手段分析基因的序列;利用RT-PCR和Real-time PCR分析Af CER1基因在大葱野生型和突变型植株中的空间表达模式。结果表明,Af CER1基因全长为2 144 bp,包含一个1 857 bp完整的开放阅读框(ORF),编码618个氨基酸。蛋白序列比对分析表明,Af CER1与其他物种的CER1具有相似的结构域:脂肪酸羟化酶结构域和WAX2-C结构域。由RT-PCR的结果可知,Af CER1基因在大葱的根、叶鞘、叶片等部位均有表达,且在野生型和突变型植株中的表达量有差异。Real-time PCR分析表明,在大葱的根和叶鞘及刚见光的叶片中,Af CER1在突变型植株中的表达量相对野生型少;而在见光较多的叶片部位,Af CER1在突变型植株中的表达量却比野生型多。研究可为大葱蜡质合成的分子机制提供理论基础。The wax-related gene CER1 primer design was based on RNA-seq transcriptome database of welsh onion, the Af CER1 gene full sequence length was obtained by RT-PCR（Reverse transcription-polymerase chain reaction）and RACE（Rapid amplification of c DNA ends）technology, and then analyzed its basic structure in NCBI. The differentially expression in wild and mutant plants was analyzed using RT-PCR and Real-time PCR technology. The results showed that the Af CER1 full-length c DNA was 2 144 bp, and contained an 1 857 bp open reading frame（ORF）which encoded 618 amino acids. The protein sequence alignment result showed that Af CER1 had a fatty acid hydroxylase region and a WAX2-C domain, which was similar to the CER1 functional domains of other species.RT-PCR results indicated that Af CER1 gene was expressed in root, leaf sheath and leaf, there were differences in the expression of wild and mutant plants. Real-time PCR results indicated that Af CER1 less expressed in mutant compare to wild in root, leaf sheaths and leaves. However, Af CER1 gene higher expressed in the lighted leaves of mutant plants. This study provides a theoretical basis for the molecular mechanism of wax synthesis of welsh onion.

关 键 词：大葱 蜡质 AF CER1 基因克隆 表达分析 

分 类 号：S633.1[农业科学—蔬菜学]