马铃薯热激转录因子HsfA3基因的克隆及其耐热性功能分析

马铃薯在田间生长时常会受到各种不利环境的影响。夏季高温常导致马铃薯块茎产量和质量的下降。因此,阐明马铃薯对热胁迫的响应机制,发掘耐热相关基因,对马铃薯耐热性的提高意义重大。热激转录因子(heat shock transcription factor A3,HsfA3)在植物机体内的活动影响到大量功能基因的表达,在植物响应热胁迫的过程中发挥重要的作用。为了研究马铃薯中HsfA3的结构和功能,本研究通过RT-PCR从马铃薯品种Désirée中克隆到长度为1506 bp的StHsfA3基因,编码501个氨基酸。StHsfA3的相对分子量为55.23 kD,理论等电点为4.9,属于亲水性蛋白。构建StHsfA3-pBA002过表达载体,并转化马铃薯植株,共鉴定得到5个独立的StHsfA3过表达转基因马铃薯株系。通过对转基因植株和非转基因植株叶片中的相对含水量(relative water content,RWC)以及丙二醛(malondialdehyde,MDA)含量的测定发现,在高温胁迫下,转基因植株叶片中的RWC显著高于非转基因植株,MDA含量显著低于非转基因植株,说明StHsfA3在耐热过程中起到正调控作用。对StHsfA3、StHsp26-CP和StHsp70在不同马铃薯植株中的表达分析显示,StHsfA3过量表达诱导了StHsp26-CP和StHsp70的表达,预示着StHsfA3可能协同StHsp26-CP和StHsp70来增强过表达转基因株系的耐热性。

Cloning of potato heat shock transcription factor StHsfA3 gene and its functional analysis in heat tolerance

During potato cultivation in the field,various adverse environmental stresses would affect its growth status.Heat stress in summer always results in the decline of tuber yield and quality.Therefore,it is of great significance to reveal the response mechanism of potato to heat stress and explore heat resistant genes for potato breeding.The activity of HsfA3(heat shock transcription factor A3)affects the expression of numerous functional genes and plays an important role in response to heat stress.In order to investigate the structure and function of potato HsfA3,the StHsfA3 gene was isolated from potato cultivar Désirée by RT-PCR.The full-length cDNA of StHsfA3 was 1506 bp encoding 501 amino acids.StHsfA3 was predicted to be a hydrophilic protein with an estimated molecular weight of 55.23 kD and a theoretical isoelectric point of 4.9.The vector of StHsfA3-pBA002 was constructed and transformed into potato plants.Totally five independent transgenic potato plants overexpressing HsfA3 were obtained.The detection of relative water content(RWC)and malondialdehyde(MDA)content showed that the RWC was significantly increased while MDA content was significantly decreased in the transgenic plants compared with the non-transgenic plants under heat stress,suggesting that StHsfA3 played a positive regulatory role in enhancing the heat resistance of potato.Furthermore,the expression levels of StHsfA3,StHsp26-CP,and StHsp70 were determined by qRT-PCR in different potato plants.The results exhibited that the expression levels of StHsp26-CP and StHsp70 in the transgenic lines overexpressing StHsfA3 were significantly higher than that in non-transgenic plants,indicating that StHsfA3 might act in synergy with StHsp26-CP and StHsp70 to increase the heat tolerance of the transgenic plants.