马银花愈伤组织RoWUS基因及其启动子的克隆与表达分析

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RoWUS cDNA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RoWUS cDNA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：RoWUS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：RoWUS单独形成一个分支，与葡萄、大豆和苜蓿的亲缘关系最近。对RoWUS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。qPCR结果表明：RoWUS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RoWUS基因在该浓度时对赤霉素和脱落酸的响应最强。

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

中图分类号 S685.21 文献标识码 A

Abstract 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（GenBank：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 RoWUS promoter fragment was cloned from the callus of R. ovatum Planch by the genome walking（GenBank：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 RoWUS 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 qPCR 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.

Key words Rhododendron ovatum； Callus； WUSCHEL； Promoter； Cloning； Bioinformatics； qPCR

doi 10.3969/j.issn.1000-2561.2014.10.017

植物干细胞是植物器官发育的源泉，其产生的子细胞具有持续分裂能力并能进行自我更新，是顶端生长过程中必不可少的[1-4]。WUSCHEL（WUS）基因能使它周围的细胞具有干细胞的特征，是植物干细胞的决定基因，该基因的表达与干细胞的形成和器官的发生有密切关系[5]。WUS基因在茎端分生组织中心表达能诱导分生组织细胞增殖，该基因编码一个同源异型结构域（homeodomain）蛋白，此蛋白在多种生物中都存在，参与发育过程并且决定细胞的类型[5]。WUS基因通过与转录阻遏物结合来抑制分化，使茎端分生组织中的干细胞保持未分化状态，既能保持干细胞的多能性特征，又能维持分生组织的形态以及功能的完整，在植物器官和组织的生长过程中对干细胞的维持和分化的调控起到极为重要的作用[6-15]。最近的研究结果表明，WUS基因在番茄花的形态发育、器官鉴别、花离层区和器官脱落过程以及控制番茄小室的数量中起到重要调控作用[16-17]。WUS突变体形成的茎端分生组织不具有正常功能，可能导致叶序混乱、茎和花的分生组织提前终止等现象发生，多数情况下花的大部分器官缺失只形成一个雄蕊，不产生心皮[18-19]。

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