红花玉兰MwZFP10基因的克隆及功能分析

Cloning and Function Identification of MwZFP10 Gene from Magnolia wufengensis

该研究选取了湖北五峰地区红花玉兰新品种‘娇红1号’为研究材料,基于实验室前期对红花玉兰转录组测序差异显著表达基因中筛选到的645bp花发育相关的核心片段,采用RACE技术克隆得到1个SUPERMAN类基因MwZFP10(GenBank登录号为MH037314)。生物信息学分析显示,MwZFP10基因全长为1 117bp,开放阅读框726bp,编码241个氨基酸,蛋白分子式为C_(1128)H_(1760)N_(322)O_(383)S_(14),分子量26.4kD,理论等电点4.42,含有一个C2H2型锌指结构域。对比分析发现MwZFP10属于SUPERMAN类锌指蛋白,具有N端的QALGGH和C端富含亮氨酸的L(I)DLXLR(K)L保守结构域。α螺旋(Hh)、延伸链(Ee)和β转角(Bt)构成红花玉兰MwZFP10蛋白的基本结构,蛋白的三级结构具有典型的锌指空间构型。MwZFP10蛋白为亲水性蛋白,可能定位于细胞核中。半定量和实时荧光定量结果显示,MwZFP10在花器官、叶片和苞片中均有表达,且花器官的雌蕊中表达量较高。过表达MwZFP10转基因拟南芥表现为植株矮小且不育,叶片卷缩,花器官发育畸形等特征。研究认为,MwZFP10基因在植物花器官的生长和发育中起重要作用。

In this study,the new species Magnolia wufengensis‘Jiaohong No.1’was selected as the research material in Wufeng,Hubei.A SUPERMAN like gene MwZFP10(GenBank accession MH037314)was cloned with 3′RACE method,based on the 645 bp core fragment related with floral development and the fragment was filtered out from differentially expressed genes based on pre-Laboratory cDNA library of Magnolia wufengensis.Bioinformatics analysis of MwZFP10 gene showed that the full length of MwZFP10 cDNA was 1 117 bp with an open reading frame(ORF)of 726 bp encoding 241 amino acids.The protein molecular formula is C 1128 H 1760 N 322 O 383 S 14,molecular weight is 26.4 kD and isoelectric point is 4.42.The protein contains a conserved domain:the C2H2-type zinc finger.Comparative analysis found that MwZFP10 belongs to the SUPERMAN-like zinc finger protein,with the“QALGGH”in the N terminal and“L(I)DLXLR(K)L”in the C terminal.α-helix(Hh),extended chain(Ee)andβ-turn(Bt)constitute are the basic structure of the protein.The tertiary structure of the protein has a typical zinc finger spatial configuration.The protein is hydrophilic and mainly localized in the nucleus.The semi-quantitative PCR and quantitative real-time PCR results showed that MwZFP10 was expressed in floral organs,leaf buds and bracts,and the pistil expression was higher in floral organs.MwZFP10 transgenic Arabidopsis thaliana is characterized by short and sterile plants,curling leaves,malformation of flower organs,etc.The gene MwZFP10 play an important role in the floral growth and development.