花花柴热形态发生基因KcPIF4克隆及耐热性分析

Cloning and heat tolerance analysis of the thermomorphogenesis gene KcPIF4 in Karelinia caspica

【目的】荒漠植物花花柴具有极强的耐高温性,克隆并研究其耐高温基因,将其用到棉花等经济作物上以期提高其耐热性,为作物耐高温性分子育种提供理论基础和基因资源。【方法】克隆花花柴KcPIF4基因并进行生物信息学分析;烟草瞬时表达分析其蛋白亚细胞定位;用qRT-PCR分析45℃处理5,30,120,240min时KcPIF4在花花柴幼苗根、茎和叶中的表达量;观察45℃处理下过表达KcPIF4拟南芥表型变化并测定相关生理生化指标。【结果】花花柴KcPIF4CDS全长为1593bp,编码530个氨基酸,含1个bHLH保守结构域,基序与多种植物的同源蛋白间存在差异;其蛋白定位在叶片气孔保卫细胞膜上;KcPIF4在花花柴叶中表达量随处理时间延长先上调再下调随后又上调,240min时达最高,为对照组的4.4倍;过表达KcPIF4拟南芥叶片45℃下黄化萎蔫程度低于野生型,其CAT、SOD、POD、叶绿素a、叶绿素b和类胡萝卜素显著高于野生型,MDA含量和相对电导率显著低于野生型。【结论】花花柴KcPIF4对高温胁迫表现出耐受—响应—适应的变化,能增强拟南芥对高温的耐受能力,表明该蛋白具有耐高温性。

[Objective]The desert plant Karelinia caspica has strong heat tolerance.The aim is to clone and study the heat-tolerant genes of K.caspica,and apply them to economic crops such as cotton to enhance heat resistance.This provides a theoretical basis and genetic resources for molecular breeding of heat-tolerant crops.[Methods]KcPIF4 gene was cloned and bioinformatics analysis was performed.Subcellular localization of the protein was analyzed through transient expression in tobacco.qRT-PCR was used to analyze the expression of KcPIF4 in roots,stems,and leaves of K.caspica seedlings at different time under 45℃treatment.Changes in phenotype of Arabidopsis overexpressing KcPIF4 at 45℃were observed,and relevant physiological and biochemical indicators were measured.[Results]The full length CDS of KcPIF4 is 1593 bp,encoding 530 amino acids and containing a bHLH conserved domain.The motifs showed differences compared to homologous proteins from various plants.The protein was localized at the membrane of the guard cell of stomata.qRT-PCR showed that the expression of KcPIF4 in K.caspica leaves was first increased and then decreased,and increased again with prolonged treatment time,reaching its peak at 240 min,4.4 times higher than the control.Overexpression of KcPIF4 in Arabidop-sis produced less chlorosis and wilting in the leaves compared to the wild type at 45℃,with higher level of CAT,SOD,POD,chlorophyll a,chlorophyll b,and carotenoids,as well as lower level of MDA content and relative conductivity compared to the wild type.[Conclusion]KcPIF4 functions in tolerance-response-adaptation to high temperature stress,enhancing Arabidopsis tolerance to high temperature.This result suggests that the protein has high temperature resistance.