热激转录因子HsfA4c调控伴矿景天镉耐受性的机理解析

Transcription factors play an important role in regulating the resistance and adaptability of hyperaccumulator plants to heavy metals. In our previous study, a heat shock transcription factor SpHsfA4c from hyperaccumulator Sedum plumbizincicola, could significantly enhance cadmium tolerance in transgenic Arabidopsis thaliana and yeast. However, it remains unclear how SpHsfA4c is involved in cadmium tolerance. In this research, the regulatory domain and key amino acid sites of SpHsfA4c for cadmium tolerance would be found by the PCR mediated recombination and site-directed mutagenesis. Furthermore, SpHsfA4c alternative splicing on cadmium tolerance was also analyzed by western blot and subcellular localization. Finally, the mechanism of cadmium tolerance enhanced by SpHsfA4c would be proved by regulating downstream gene SpPCS2 using transgenic Sedum plants, Arabidopsis thaliana protoplast transient expression system and electrophoresis mobility-shift assay (EMSA). Through this project, it could elucidate the molecular mechanism of SpHsfA4c in regulating cadmium tolerance of Sedum plumbizincicola, and it provides a molecular basis for breeding hyperaccumulator plant germplasms which are more suitable for heavy metal soil pollution remediation by genetic engineering.

在重金属超积累植物中，转录因子在调控超积累植物对重金属的抗性与适应性方面起着重要作用。前期研究发现，重金属超积累植物伴矿景天热激转录因子SpHsfA4c能显著提高转基因拟南芥和酵母的镉耐受性，但是该转录因子如何参与重金属耐受性的分子机制不清楚。本项目将在此基础上，利用PCR介导的重组技术和定点突变分析SpHsfA4c对镉耐受起决定作用的结构域和氨基酸位点；进一步通过Western blot 和亚细胞定位等手段研究SpHsfA4c可变剪切对耐镉能力的影响；最后结合转基因景天植物、拟南芥原生质体瞬时表达技术及凝胶阻滞技术等研究SpHsfA4c对下游基因SpPCS2表达的调控作用，从而阐明伴矿景天SpHsfA4c镉耐受性的分子机理，为利用基因工程培育更适合重金属土壤污染修复的超积累植物新种质提供理论基础。

在重金属超积累植物中，转录因子在调控超积累植物对重金属的抗性与适应性方面起着重要作用。我们从伴矿景天cDNA中分离出热激转录因子SpHsfA4c，ORF全长均为1 302 bp，可编码433个氨基酸，AHA2结构域有转录自激活的功能。SpHsfA4c基因可以发生可变剪切，保留内含子的基因（InSpHsfA4c）DBD结构域不完整。将SpHsfA4c和InSpHsfA4c两个基因分别转化酵母及拟南芥，通过Cd耐性分析发现，只有过表达SpHsfA4c基因的酵母和拟南芥具有较强的Cd耐受性，说明伴矿景天SpHsfA4c基因可能通过内含子的精确剪切响应镉胁迫。. 染色质免疫共沉淀技术（ChIP-seq）结果显示SpHsfA4c能显著的结合到PCS2的启动子的HSE元件。EMSA、酵母单杂交及双荧光素酶实验表明SpHsfA4c能特异性结合SpPCS2的启动子。用镉处理的伴矿景天转录组和RT-qPCR表明SpHsfA4c和SpPCS2在镉胁迫下的表达模式显著正相关。SpPCS2基因在超表达SpHsfA4c的东南景天（NHE）中明显上调。此外，SpPCS2降低了镉胁迫下转基因酵母的死亡率，提高了转基因酵母镉耐受性。SpPCS2提高了转基因东南景天地上部的耐性指数，在转基因东南景天根系中积累了更多的Cd，表明更多的Cd被PCS螯合在根中。以上结果阐明了伴矿景天SpHsfA4c镉耐受性的分子机理，为利用基因工程培育更适合重金属土壤污染修复的超积累植物新种质提供理论基础。