小热激蛋白HSP21响应光敏型黄化茶树返绿的分子机制研究

The etiolated tea plant is a rare kind of tea germplasm resources, The yellow color of shoot leaf returned to the green during shading. Although a lot of progress of the molecular mechanism of etiolation has been made, it is not yet clear. Recently, some significant discovery about the mechanism of small heat shock protein HSP21 had been established. The sHSP21 took part in chloroplast development though regulating the PEP transcriptional activity under the condition of heat stress. The previous studies indicated CPsHSP21 was significantly expressed during the course of the tea plant shoot from yellow to green. Using CPsHSP21 as entry point, this project aims at studying the mechanism of light-sensitive tea plant “Huangkui” during the shoot color from yellow to green by ranscriptome sequencing technique, protein-protein interactions and protein- nucleotide interactions.In this project, we first have to clarify the screening of pTAC which is the target gene of CPsHSP21, the formation mechanism of CPsHSP21/pTAC protein complex and if this protein complex can regulate the PEP transcriptional activity. Finally analyze the regulation molecular mechanism of CPsHSP21 in the etiolated tea plant.It will extend the function of chloroplast small molecule heat shock protein, and provide molecular foundation for the development of high quality tea plant germplasm resources.

光敏型黄化茶树是一类珍稀茶树种质资源，在遮阴过程中，茶树新稍叶色由黄白返绿，有关其黄化机理的研究获得了较多进展，但遮阴后叶色由黄白返绿的机理并不清晰。近期国际上有关拟南芥叶绿体小热激蛋白HSP21在热胁迫条件下通过调节PEP转录活性参与叶绿体发育的机制取得重要进展。本课题组前期研究发现，叶绿体小热激蛋白HSP21（CPsHSP21）在黄化茶树新梢返绿中显著表达。本项目以CPsHSP21为切入点，利用转录组测序、蛋白互作、核酸-蛋白互作等技术，探究光敏型黄化茶树返绿的分子机理。本项目首先明晰CPsHSP21的靶蛋白pTAC，探明CPsHSP21与pTAC复合体(CPsHSP21/pTAC)形成机理，再进一步探究CPsHSP21/pTAC对PEP转录活性的调控，最终解析CPsHSP21参与黄化茶树返绿的分子调控机理。本研究拓展了叶绿体小分子热激蛋白的功能，同时也为开发优质茶树资源提供分子基础。

茶树作为多年生乔木植物，喜光怕晒，喜温怕寒，在自然生态条件下，常常会有黄化或白化的症状。光敏型黄化茶树黄魁新稍叶片色泽，会随着光照强度的变化而出现可逆的“黄化-返绿”现象。本论文通过基因表达筛选、基因功能验证，发现叶绿体定位的小热激蛋白CsHSP24.6和质体类核蛋白CspTAC5，通过蛋白互作，在“黄化-返绿”现象中起重要作用。主要结果如下：.1..本论文首先采用转录组测序和qRT-PCR技术，对基因表达的品种特异性和遮荫处理诱导表达特异性进行分析，并从中发现CsHSP24.6和CspTAC5的基因表达可能与茶树“黄化-返绿”现象有关。与舒茶早相比较，CsHSP24.6和CspTAC5基因在叶色特异的安吉白茶和黄魁品种中高表达；遮荫处理后，黄魁茶树新稍叶片由黄返绿，叶绿素及类胡萝卜素含量增加的同时，CsHSP24.6和CspTAC5基因及其叶绿素和类胡萝卜素合成相关基因表达均上调。.2..本论文将CsHSP24.6和CspTAC5基因超表达在拟南芥中进行功能验证。结果发现，超表达CsHSP24.6和CspTAC5的拟南芥，它们的表型与野生型拟南芥差异不大；但是，与野生型相比较，在高温和强光胁迫条件下，超表达CsHSP24.6和CspTAC5拟南芥的光合系统PSII的电子传递受损程度低，表明CsHSP24.6和CspTAC5的表达与光合机构的保护有关。.3..本氏烟瞬时表达分析发现，CsHSP24.6和CspTAC5蛋白共定位于叶绿体内；GST-Pull down和BiFC实验结果表明，CsHSP24.6和CspTAC5蛋白具有互作关系。