镁离子转运蛋白基因CsNIPA4调控茶树高温响应的机制研究

Heat stress is one of the main meteorological factors affecting the growth and development of tea plant (Camellia sinensis) in summer and autumn, which directly affects the yield and quality of spring tea in the coming year. Hence, it is important to study the mechanisms of high temperature responses in Camellia sinensis, which will ensure the safety production of tea industry. NIPAs (nonimprinted in Prader-Willi/Angelman syndromes) are an important kind of Mg2+ transporters, but their functions in regulating plant heat stress responses have not been reported yet. In our previous study, we cloned a heat-responsive gene CsNIPA4 from C. sinensis using suppression subtractive hybridization technology, and overexpression of CsNIPA4 enhanced the thermotolerance of transgenic Arabidopsis thaliana. In this study, we propose to clarify the temporal/spatial expression characteristics of CsNIPA4 and the relationship among gene expression, promoter sequence polymorphism and tea plant thermotolerance using qRT-PCR, transient expression and genome walking techniques; to identify the functions of CsNIPA4 involved in high temperature responses using transcriptomics and metabolomics in transgenic yeast and Arabidopsis thaliana; to screen upstream regulators of CsNIPA4 using yeast one-hybrid; to verify the binding of transcription factors and CsNIPA4 promoter using ChIP and EMSA. The success of this project will help to elucidate the molecular mechanism of CsNIPA4 in response to heat stress, and provides a novel strategy for heat resistance breeding as well as tea cultivation.

夏、秋季高温是制约茶树生长、发育的重要因素，直接影响来年春茶产量和品质，研究茶树高温响应机制对茶叶安全生产具有重要意义。NIPAs是一类重要的镁离子转运蛋白，但在调控植物响应高温胁迫中的作用机制尚未见报道。申报者前期通过抑制消减杂交技术筛选得到茶树响应高温胁迫的差异表达基因CsNIPA4，转基因功能分析发现，过表达CsNIPA4增加了转基因拟南芥在高温胁迫下的存活率。本项目拟通过qRT-PCR、瞬时表达和染色体步移技术，分析CsNIPA4的时空表达特征，明确基因表达、启动子序列多态性和茶树耐热性三者的关系；通过转基因酵母和拟南芥，借助转录组和代谢组手段，验证CsNIPA4的基因功能；利用酵母单杂交筛选CsNIPA4上游调控因子，并通过ChIP和EMSA体内外联合验证转录因子和启动子的结合情况。研究结果有助于阐明CsNIPA4响应高温胁迫的分子机制，为茶树抗热育种和抗热栽培提供理论依据。

茶树喜温怕热，当温度上升到茶树本身所能耐受的临界温度时，茶树不能正常生长发育，甚至死亡，谓之热害。热害常常被人们所忽视，认为热害就是旱害：旱害是由于水分亏缺影响茶树生理活动；而热害是旱害的一种特殊表现形式，是由于临界高温造成茶树体内蛋白凝固，酶失去功能活性。随着全球气候变暖的加剧，夏秋季节，茶树更易遭受高温炎热天气的影响，这直接影响来年春茶产量和品质，因此，研究茶树高温响应机制对茶叶安全生产具有重要意义。项目申请人前期通过抑制消减杂交技术有针对性地分离得到一个茶树叶部响应高温的差异表达基因：镁离子转运蛋白基因CsNIPA4（nonimprinted in Prader-Willi/Angelman syndrome 4）；实时荧光定量PCR分析发现，高温（38 °C）强烈诱导了CsNIPA4基因的表达。项目组以此为切入点，对CsNIPA4参与调控茶树热响应机理进行深入研究，取得以下主要研究进展：（1）CsNIPA4的转录受到高温胁迫的诱导，其编码蛋白定位于叶绿体；（2）异源表达CsNIPA4降低了转基因酵母细胞对热激（45 °C 30 min）的耐受性；（3）超表达CsNIPA4降低了转基因拟南芥幼苗对高温的耐受性，同时CsNIPA4回补拟南芥突变体atnipa4株系的耐热性显著低于突变体atnipa4，这表明CsNIPA4负调控拟南芥对高温的耐受性；（4）以CsNIPA4启动子为诱饵，利用酵母单杂交筛选到一个茶树热激转录因子CsHsfa，后续还有待进一步验证两者之间的互作；（5）对茶树热激转录因子家族基因CsHsfs进行了全面鉴定，初步明确了其基因特征、进化关系、CsHsfs家族关键基因CsHsfA2的定位和在酵母中的功能。本研究对揭示CsNIPA4参与调控茶树高温响应机制方向取得了新认识，对茶树抗热新品种选育具有重要的理论意义和潜在的实践价值。