香石竹高温胁迫HSF类转录因子功能分析与分子调控机制研究

Carnation (Dianthus caryophyllus L.) is one of the four cut flowers worldwide. Standard carnation varieties are very susceptible to high temperatures which adversely affect the yield and the quality of the cut-flowers. However, the molecular mechanisms underlying the heat stress tolerance are still unclear. It is important to investigate the candidate transcription factor genes and their transcriptional regulation mechanisms involved in response to thermotolerance in carnation. A series of differentially expressed genes were identified by sequencing of transcriptome and high temperature stress profiles of carnation seedlings. 8 DcHSFs, which were closely related to high temperature stress, were identified. Here, we want to isolate the key DcHSFs involved in high temperature stress and to identify candidate gene functions by overexpression of Arabidopsis thaliana and carnation. In addition, the gene biochemical characteristics, transcriptional activation activity and transcriptional binding activity analysis remain to carry out. Moreover, ChIP-seq analysis of transgenic plants with obvious phenotype will be carried out to find the target gene regulated by DcHSF, and to explore its signal regulation network in response to high temperature stress. Therefore, our study will uncover the physiology and molecular mechanism of the DcHSFs regulatory network in the heat stress tolerance in carnation at the transcription level. These analysis of DcHSFs will provide theoretical basis and genetic resources for molecular breeding and genetic improvement of carnation.

香石竹是世界“四大切花”之一。高温胁迫会影响香石竹正常生长和发育，从而影响切花产量和品质。然而，关于香石竹响应高温胁迫过程的转录调节机制尚不明确。因此，鉴定并筛选参与高温胁迫相关的重要转录因子则成为解析转录调控机制的一个重要课题。申请者前期对香石竹幼苗进行了转录组及高温胁迫表达谱的测序，鉴定出了一系列差异表达基因，初步明确了8个热激转录因子DcHSFs参与高温胁迫进程。本项目拟对参与高温胁迫的关键DcHSFs进行分离，通过过表达拟南芥和香石竹对候选基因功能进行鉴定；开展DcHSFs的生化特性和转录激活活性及转录结合活性分析；进一步对有明显表型的转基因植株进行ChIP-seq分析，找到DcHSF调控的靶基因，探索HSF调控高温响应信号的传导网络。本项目拟从转录水平上阐明DcHSFs参与香石竹高温胁迫耐性的转录调节机制，为香石竹的抗逆分子育种及遗传改良提供理论基础和基因资源。

高温胁迫会影响香石竹正常生长和发育，从而影响切花产量和品质。然而，关于香石竹响应高温胁迫过程的转录调节机制尚不明确。本项目结合前期转录组数据，筛选出三个参与响应高温胁迫的差异表达基因DcHsfA2、DcHsfA4和DcHsfB1，并分析了三者在不同非生物胁迫下的表达模式。分离参与高温胁迫过程的关键转录因子DcHsfA2的完整编码序列，该序列全长1080 bp，编码359个氨基酸，生信分析显示其分子量为40.55 KDa，等电点为5.05。氨基酸序列多重比对表明DcHsfA2含有高度保守的DNA结合域，此外，还包含疏水七肽重复区，核定位信号序列和C末端激活域。系统进化分析表明DcHsfA2与拟南芥AtHsfA2同源性最高。qRT-PCR分析表明，热胁迫，冷胁迫，ABA处理和甘露醇处理均可显著诱导DcHsfA2的表达。亚细胞定位结果显示DcHsfA2定位于细胞核中。酵母杂交试验表明，DcHsfA2的激活域位于C端。DcHsfA2在拟南芥中的过表达提高了热胁迫后的存活率。与对照组相比，热胁迫后转基因株系的MDA含量，相对电导率和H2O2和O2-的积累均明显降低，而POD和SOD的含量均高于对照组。 DcHsfA2过表达株系中AtHsp101，AtHsp18.2，AtHsp70，AtHsp90，AtGolS1和AtHsfA1的表达受热胁迫诱导，且显着高于对照植株。此外，DcHsfA2的过表达降低了盐胁迫和ABA处理后转基因拟南芥的发芽率，并增加了根对ABA的敏感性。本项目初步解析了DcHsfA2的转录调控机制，为香石竹的抗逆分子育种及遗传改良提供了理论基础和基因资源。