PmACRE基因的克隆及遗传转化拟南芥
Isolation of PmACRE gene and its transformait on in Arabidopsis thaliana
李慧敏 1谢婉凤 2冯丽贞 1陈慧洁 1刘宏毅 1叶小真2
作者信息
- 1. 福建农林大学林学院, 福建福州350002
- 2. 福建农林大学林学院, 福建福州350002;福建农林大学金山学院, 福建福州350002
- 折叠
摘要
Avr9/Cf-9 rapidly elicited(ACRE)gene是富含亮氨酸重复单元,参与蛋白与蛋白互作,特异性识别病原物激发子的关键基因,在植物抗病过程中有重要意义.利用RT-PCR从马尾松中克隆出Avr9/Cf-9 rapidly elicited gene,命名为PmACRE(GenBank登录号:MF630966).测序结果显示,PmACRE cDNA序列全长862 bp,其中完整编码区长度624 bp,编码207个氨基酸;以PFGFP Bar 137质粒为基本载体,构建了由组成型CaMV35S启动子驱动PmACRE基因的植物表达载体PFGFP Bar 137-PmACRE;采用冻融法转入农杆菌AGL1菌株,通过花序浸染法对拟南芥进行了遗传转化,并获得了4株阳性苗,经目的基因ACRE和GFP基因双重PCR检测,证实目的基因已整合到拟南芥基因组中.研究结果为进一步分析马尾松ACRE基因的功能和深入揭示R基因调控马尾松响应松材线虫侵染的作用机制奠定基础.
Abstract
The Avr9/Cf-9 rapidly elicited ( ACRE) gene belongs to leucine rich repeats ( LRR) , which is the key gene involved in the process of protein-protein interaction and specific identification of pathogen elicitors and it plays a significant role in disease risistance.The Avr9/Cf-9 rapidly elicited gene from Pinus massoniana ( GenBank accession number: MF630966 ) was cloned through RT-PCR and named as PmACRE.The ACRE of P.massoniana, whose cDNA length is 862 bp, contained an open reading frame of 624 bp which encoded a polypeptide of 207 amino acids.The plant expression vector PFGFP Bar 137 was constructed, in which the PmACRE gene was driven by the constitutive promoter CaMV35S.The constructed expression vector was transformed into Agrobacterium tumefaciens AGL1 strain by the floral dip method.Leaf segments of Arabidopsis thaliana were inoculated with A.tumefaciens AGL1 containing plasmid PFGFP Bar 137-PmACRE.The transgenic lines were selected by ACRE and GFP gene PCR detection and the result showed that the target gene had been integrated into the genome of A.thaliana and the 4 positive plants have been obtained.The results of this study gave a foundation for the further analysis of the function of ACRE and revealing the mechanism on the P.massoniana in response to Bursaphelenchus xylophilus infestation.
关键词
马尾松/PmACRE/基因克隆/载体构建/遗传转化Key words
Pinus massoniana Lamb./PmACRE/gene cloning/expression vector construction/genetic transformation引用本文复制引用
基金项目
福建省财政厅资助项目(K81139238K8113001A)
出版年
2018
国家科技期刊平台
NSTL
维普
万方数据