桃Pp4CL2基因的克隆及抗寒功能验证

Cloning and Cold Resistance Function Verification of Pp4CL2 Gene in Peach

基于‘丁家坝李光桃’抗寒转录组数据,采用RT-PCR技术克隆桃4-香豆酸辅酶A连接酶基因(Pp4CL 2),并对其进行生物信息学及转化模式植物拟南芥和烟草的抗寒分析,以解析‘丁家坝李光桃’的抗寒机制。结果显示:(1)成功克隆获得桃Pp4CL 2基因(登录号:LOC18792923),其cDNA序列为1635 bp,编码544个氨基酸残基,具有4CL基因家族保守结构域。(2)二级结构分析显示,Pp4CL2蛋白由4种状态的二级结构组成,其中α螺旋占30.51%、β-折叠占7.35%、不规则卷曲及延伸链分别占41.54%和20.59%。(3)顺式作用元件分析发现,Pp4CL 2基因上游启动子区含有光、低温以及多种激素响应元件。(4)序列系统进化分析显示,桃Pp4CL2与杏(Prunus armeniaca)、欧洲甜樱桃(Prunus avium)和梅(Prunus mume)的蛋白相似性最高,分别为99.08%、97.98%和96.14%。(5)成功构建转化载体Pp4CL2-pRI101,对拟南芥和烟草进行遗传转化并通过PCR鉴定获得转基因拟南芥和烟草。(6)与野生型相比,低温胁迫下转基因拟南芥和烟草的Pp4CL 2基因相对表达量高,受冷害程度轻,具有更高的渗透调节物质含量和抗氧化酶活性,对低温具有更强的耐受性。研究表明,过表达Pp4CL 2基因可增强植物对低温的耐受性,推测Pp4CL 2基因在‘丁家坝李光桃’响应低温胁迫过程中具有重要作用。

Based on the cold-resistant transcriptome data of‘Dingjiaba Liguangtao’,we cloned peach 4-coumarate-CoA ligase gene(Pp4CL 2)by RT-PCR and analyzed by bioinformatics and transformation model plants Arabidopsis thaliana and tobacco,so as to analyze the cold resistance mechanism of‘Dingjiaba Liguangtao’.The results showed that:(1)Pp4CL 2 gene(accession number:LOC18792923)of peach was successfully cloned,and its cDNA sequence was 1635 bp,encoding 544 amino acid residues,and has the conserved domain of 4CL gene family.(2)The secondary structure analysis showed that Pp4CL2 protein consists of four secondary structures,of which alpha helix accounts for 30.51%,beta turn accounts for 7.35%,random coil and extended strand account for 41.54%and 20.59%respectively.(3)Analysis of cis-acting elements showed that the upstream promoter region of Pp4CL 2 gene contained light,low temperature and many hormone response elements.(4)Phylogenetic analysis of Pp4CL2 sequence showed that Pp4CL2 of peach has the highest protein similarity with apricot(Prunus armeniaca),sweet cherry(Prunus avium)and plum(Prunus mume),which are 99.08%,97.98%and 96.14%respectively.(5)The transformation vector Pp4CL 2-PRI101 was successfully constructed and transgenic A.thaliana and tobacco plants were identified by PCR.(6)Compared with wild type,transgenic A.thaliana and tobacco had higher relative expression level Pp4CL 2 gene,lighter degree of cold injury,higher osmoregulation substance content and antioxidant enzyme activity,and stronger tolerance to low temperature.The study has shown that overexpression of Pp4CL2 gene can enhance the tolerance of plants to low temperature,and it is speculated that Pp4CL2 gene plays an important role in the response of‘Dingjiaba Liguangtao’to low temperature stress.