马银花愈伤组织RoWUS基因及其启动子的克隆与表达分析  

Cloning and qPCR Analysis of RoWUS Gene and Isolation of Promoter from Callus in Rhododendron ovatum Planch.

作　　者：匡华琴[1] 刘生财[1] 陈裕坤[1] 赖钟雄[1] 

机构地区：[1]福建农林大学园艺植物生物工程研究所,福建福州350002

出　　处：《热带作物学报》2014年第10期1984-1991,共8页Chinese Journal of Tropical Crops

基　　金：福建省农业科技平台(No.2008N2001);国家科技支持计划资助项目(No.2007BAD07B03)

摘　　要：WUSCHEL(WUS)基因是植物干细胞的标志基因。采用同源克隆法结合RACE技术从马银花愈伤组织中克隆得到Ro WUS c DNA全长序列,采用染色体步移法克隆得到该基因的启动子序列,登录号分别为KF365488、KF861578。马银花Ro WUS c DNA全长1 123 bp,编码302个氨基酸;DNA序列2 001 bp,包含2个内含子和3个外显子;启动子序列3 122 bp。生物信息学分析表明:Ro WUS亚细胞定位于细胞核,是不稳定的亲水蛋白,无信号肽,具有跨膜结构,包含homeodomain功能位点。系统进化树分析结果表明:Ro WUS单独形成一个分支,与葡萄、大豆和苜蓿的亲缘关系最近。对Ro WUS的启动子进行分析表明,该启动子除了含有丰富的TATA-box和CAAT-box等基本元件以外,还含有多个光响应元件、逆境胁迫响应元件、激素应答元件和其他功能元件。q PCR结果表明:Ro WUS基因在马银花愈伤组织不同生长时期中呈先上升后下降的趋势,在第5个继代周期时表达量最高。外源赤霉素和脱落酸浓度为15 mg/L时表达量最高,说明Ro WUS基因在该浓度时对赤霉素和脱落酸的响应最强。The WUSCHEL（WUS） gene was a plant stem cell marker gene.The RoWUS gene was cloned from the callus of Rhododendron ovatum Planch by the homologous cloning and RACE technology（Gen Bank：KF365488）.The complete cDNA sequence was 1 123 bp,encoding 302 amino acids.The DNA sequence of RoWUS was 2 001 bp which included 3 exons and 2 introns.The Ro WUS promoter fragment was cloned from the callus of R.ovatum Planch by the genome walking（Gen Bank： KF861578）,and the sequence was 3 122 bp.Bioinformatics analysis showed that the protein was likely a kind of hydrophilcity and unstable protein which located in the nucleus.The protein had transmembrane structure without signal peptide,which obtained a homeodomain functional site.The phylogenetic tree analysis showed that Ro WUS has the closest relationship with Vitis vinifera,Glycine max and Medicago truncatula.The promoter software analysis showed that the promoter fragment contained a number of TATA-box,CAAT-box elements and many other promoter elements,such as light response elements,environmental stress response elements,hormone response elements and some function unknown elements.The q PCR results indicated that RoWUS showed a downward trend after the first rising at different culture stages in R.ovatum Planch callus.It expressed at the highest levels in the 5th cycle.The RoWUS gene had strong response under the hormone treatments of GA3 and ABA,and the peak of expression level appeared at 15 mg/L GA3 and ABA.

关 键 词：马银花 愈伤组织 WUS 启动子 基因克隆 生物信息学分析 荧光定量PCR 

分 类 号：S685.21[农业科学—观赏园艺]