种子特异启动子At2S3驱动热激转录因子AtHsfA6a提高烟草热抗性

Seed-specific AT2S3 promoter-regulated heat shock factor AtHsfA9 improves plant tolerance in transgenic Nicotiana tabacum

特异启动子指导外源基因在某一特定时空表达,避免个体多余营养的浪费,在作物品质及逆境适应性改良中具有重要应用潜力.采用种子特异启动子联合抗性基因的方法,对拟南芥种子特异启动子At2S3和热激转录因子At Hsf A6a进行克隆,构建p BI121双元表达载体(p At2S3::At Hsf A6a),利用农杆菌菌株转化烟草,获得转基因烟草稳定株系,并进行种子抗高温胁迫萌发实验.结果显示:At Hsf A6a表达量随高温胁迫持续而不断积累,45℃高温处理后,热激转录因子At Hsf A6a的表达量最高上调28倍.转基因烟草株系(L2,L3)与野生型烟草(WT)相比,在45℃高温处理12 h、18 h和24 h后,最终萌发率分别为100%、98%和80%,98%、94%和76%以及97%、92%和70%.根长实验表明转基因株系L2和L3的根长为未处理时的74%和60%,而WT根长为未处理时的52%.上述结果说明超表达烟草株系的萌发和根系生长较之WT对热激处理更不敏感,呈现出更高的抗性;可为种子特异启动子加抗性基因的组合提高转基因植株抗胁迫能力提供直接实验证据,为基因工程育种提供种子特异启动子和抗胁迫新策略.

Gene expression is regulated at multiple levels, and a promoter plays a crucial role in determining the temporal and spatial expression patterns and transcriptional levels of genes. Under the regulation of tissue-specific promoters, genes express in a particular space and time, which can avoid unnecessary energy losses in unexpected organs. This property can be used to improve crop quality and adaptability for surviving under adverse conditions. In this study, tissue-specific promoters were used to drive the expression of resistance genes. First, Arabidopsis seed-specific promoter At2S3 and heat shock transcription factor AtHsfA6a were cloned into pBI121 binary vector （pAt2S3：：AtHsfA6a）. Next, this vector was transformed into tobacco, and stable transgenic tobacco lines were screened. Finally, seed germination experiment was performed to determine the effects on heat stress. The results indicated that the expression of AtHsfA6a increased remarkably under temperature stress. Its expression could be upregulated 28 times after heat treatment at 45 ℃. After 12, 18, and 24 h of the heat treatment, the germination rates of transgenic tobaccos were 100% and 98%; 98% and 94%; and 97% and 92%, respectively, whereas those of wild-type plants were just 80%, 76%, and 70%, respectively. Transgenic plants were found to have acquired thermotolerance at the root elongation stage after heat treatment. These results suggested that the transgenic lines are more tolerant to heat stress than wild-type plants. This study provides direct experimental evidence that the combination of seed-specific promoters and resistant genes can be used to improve plant tolerance against adversity stress, which could be used as a new strategy for genetic engineering breeding.