核桃JrWOX4b基因的克隆和功能分析

Cloning and Functional Analysis of JrWOX4b in Juglans hindsii×regia’Linzaoxiang’

[目的]为探索WOX4b基因在核桃不定根发生过程中的调控作用提供理论支撑。[方法]以核桃品种‘林早香’(Juglans regia‘Linzaoxiang’)的cDNA为模板克隆该基因CDS全长序列;利用生物信息学技术对其进行多重序列比对和系统进化分析;构建该基因与黄色荧光蛋白(Yellow Fluorescent Protein,YFP)标签的融合表达载体进行亚细胞定位分析;通过对银腺杨‘84K’的遗传转化,分析其功能。[结果]JrWOX4b基因的编码序列(Coding sequence,CDS)长度为678 bp,编码226个氨基酸,分子量为25.42 KDa。所编码氨基酸序列的多重比对及系统进化分析结果显示:核桃JrWOX4b与山核桃属的长山核桃同源蛋白遗传关系最近,与壳斗科栎属白栎次之,而与禾本科的黍稷和二穗短柄草的亲缘关系最远。JrWOX4b基因过表达植株的表型分析结果显示:JrWOX4b的过表达和对照植株生长发育均正常,但在相同培养条件下,JrWOX4b基因的过表达能够显著增加杨树的不定根数量,2周龄过表达植株不定根数量为对照植株的3~4倍,不定根长度是对照植株的1/2。[结论]从核桃中成功克隆了WOX基因家族成员JrWOX4b基因,其过表达可显著增加转基因植株的不定根数量。研究结果不仅能够为不定根发生调控机制的研究提供理论支撑,同时也为其他难生根木本植物的快速繁殖提供优良基因资源。

[Objective]To provide theoretical support for exploring the regulatory function of WOX4b gene in walnut adventitious rooting.[Method]Using the cDNA of Juglans regia’Linzaoxiang’as template,the fulllength CDS of JrWOX4b was cloned.The multiple sequence alignments and evolution analysis were applied by using bioinformatics technology to analyze its molecular characteristics.The fusion expression vector of JrWOX4b and yellow fluorescent protein(YFP)tag were constructed for subcellular localization analysis.After completing the genetic transformation and phenotype analysis of Populus alba×P.glandulosa,the function of this gene was determined.[Result]The CDS of JrWOX4b is 678 bp in length,encoding 226 amino acids,and molecular weight is 25.42 KDa.The results of multiple alignment and phylogenetic analysis of the amino acid sequences showed that walnut JrWOX4b has the closest genetic relationship with the homologous protein of Carya illinoensis,followed by Quercus lobata,while has the farthest genetic relationship with Panicum miliaceum and Brachypodium distachyon.Phenotypic analysis of JrWOX4b overexpression plants showed that both the transgenic and control plants grew normally.However,under the same culture conditions,JrWOX4b could significantly increase the number of adventitious roots.The number of adventitious roots of 2-week-old overexpression plants is 3~4 times that of the control plants,and the length of adventitious roots is 1/2 of that of control plants.[Conclusion]JrWOX4b,the member of WOX gene family,was successfully cloned from walnut,and its overexpression can significantly promote the number of adventitious roots.The results provide theoretical support for the research on adventitious root regulation mechanism,and also provide excellent genetic resources for the rapid propagation of other difficult-to-root plant species.