过表达紫花含笑McMYB52基因增强拟南芥耐热性

Overexpression of Michelia crassipes McMYB52 gene in Arabidposis thaliana enhances its heat tolerance

本文以紫花含笑花被为材料,克隆得到一个受热胁迫诱导显著上调的R2R3-MYB转录因子McMYB52,全长717 bp,蛋白分子量为27.95 kDa,等电点为8.86。亚细胞定位结果显示其编码蛋白定位在细胞核中。构建35S::McMYB52过表达载体并利用花序浸染法转化拟南芥,通过潮霉素抗性筛选以及PCR检测得到T3代纯合转基因拟南芥株系,用于研究McMYB52耐热性的功能。结果表明:与野生型相比,转基因拟南芥株系幼苗基础抗热及获得性抗热存活率分别高达78.57%、95.65%,成苗经高温胁迫后能够复绿。叶片解剖结构说明过表达McMYB52促进维管束横截面面积增大,特别是木质部面积占比,高温后叶片增厚从而调节细胞内渗透平衡。生理指标测定表明,转基因植株内游离脯氨酸含量增高、SOD和POD活性增强以清除热胁迫活性氧的积累从而维持细胞膜的完整。实时荧光定量PCR表明,过量表达McMYB52基因显著提高AtHSFA2及其靶基因AtHSFB2a、HSPs及AtAPX2上调表达,推测过表达McMYB52可能促进高温下AtHSFA2转录调控,协同抗氧化途径,利用维管系统疏导来提高拟南芥耐热性。综上说明, McMYB52可能作为正调控因子参与植物的高温响应。

A R2R3-MYB transcription factor gene McMYB52 was cloned from Michelia crassipes,which was significantly up-regulated under heat stress.The gene had 717 bp,encoding 239 amino acids with 27.95 kDa and 8.86 pI.Subcellular localization analysis revealed that McMYB52 protein is localized in nucleus.The expression vector 35S::McMYB52 was constructed and transformed into wild-type(WT)Arabidposis thaliana plants by the floral dip method.T3 transformed plants were confirmed and selected to study the function of McMYB52 on the heat resistance.The results showed that compared to WT plants,the ectopic expression of McMYB52 in transgenic Arabidposis plants exhibited the survival rates of 78.57% and 95.65% in basal and acquiredheat resistance, respectively. The adult transgenic plants could successfully regreen after the high temperaturetreatment. Leaf anatomy indicated that overexpression of McMYB52 promoted the increase of vascularbundle cross-sectional area, especially the xylem area ratio, and leaf thickening after high temperatureregulated the intracellular osmotic balance. The free proline content, SOD and POD activities were enhanced toremove the accumulation of reactive oxygen and maintain the integrity of cell membrane. Quantitative real-timePCR analysis indicated that overexpression of McMYB52 gene significantly increased the up-regulated expressionof AtHSFA2 and its target genes AtHSFB2a, AtAPX2 and the heat shock proteins AtHSP70, AtHSP90,AtHSP101. Taken together, these results demonstrated that McMYB52 played an important role in improvingheat tolerance, possibly by promoting vascular tissue development, working on the antioxidant pathway andtargeting on the regulation of AtHSFA2 to enhance the expression of some stress-associated genes.