水稻锌指蛋白转录因子HST在花期高温胁迫应答中的功能与调控研究

Heat stress has become a threat to crop production. It is the important base on the rice heat tolerant breeding to illuminate the mechanism of rice heat stress tolerance, and to clone, identify the genes related to the heat stress tolerance. A heat-resistant mutant of the rice ZH11（hst，heat stress telorance）acquired before was utilized to clone the related genes. With map-based cloning and sequencing, it was found that 4 basic groups were inserted into the zinc finger transcription factor HST, which resulted in the frameshift mutation. According to the above evidences, it can be indicated that this transcription factor was a negative regulator of rice heat stress tolerance. Some technologies and anatomy methods, such as the transgenosis, ChIP-seq, RNA-Seq and so on, will be used to study the spatial and temporal expression pattern of HST, to exploit the downstream target genes of HST at flowering under the condition of 40℃. Meanwhile, the pollen viability, the developmental structure of the anther, the physiological and biochemical indexes and the agronomic traits including the setting rate, the production and so on will be studied, too. All of these will verify the functions and the regulatory network of HST taking part in at the rice flowering under heat stress, illuminate the molecular mechanism of the heat stress in rice and provide theoretical basis of molecular breeding under heat stress.

高温胁迫已成为作物生产的威胁,水稻耐高温机制的阐明和耐高温相关基因的克隆、鉴定及功能解析是水稻耐高温育种的重要基础。本研究以前期获得的一份抗高温能力明显增强的水稻中花11突变体（hst，heat stress tolerance）为材料，经图位克隆与测序发现该突变体中的目的基因发生了移码突变，且为锌指蛋白转录因子，在花期高温胁迫中起负调控作用。为此，本项目采用转基因、ChIP-seq、RNA-Seq等技术和解剖学手段，在花期高温（40℃）胁迫下，研究该锌指蛋白转录因子HST在水稻花期的时空表达模式，寻找HST下游的直接调控靶基因,分析花粉活力、花药结构与结实率、产量等农艺性状及其生理生化（激素、氧化胁迫）指标，探明锌指蛋白转录因子HST在水稻花期高温胁迫响应中的功能与调控网络，为阐明水稻高温热害的分子机理提供科学依据，为作物耐高温分子育种提供理论指导。

高温严重威胁水稻的生产安全，水稻开花期遭遇高温是减产的主要原因。水稻耐高温品种的筛选和培育，耐高温基因克隆、鉴定和解析以及耐高温机制的研究显得尤为重要。本项目基于前期获得的水稻中花11耐高温突变体hst，并构建了其互补突变体HST-hst，探明HST转录因子在水稻花期高温胁迫应答中的功能。对HST-GUS-hst水稻转基因植株进行GUS染色，探究HST基因在水稻不同组织器官中的表达模式，发现HST基因在水稻的根部，叶片，节，花药中都有表达。荧光定量PCR结果表明HST基因在叶中的表达量远高于其它组织和器官。在高温胁迫下，水稻花器官中HST表达水平较对照明显升高，表明HST可对高温胁迫做出响应。对DR5-GUS-ZH11、DR5-GUS-hst、Tcsn-GUS-ZH11、Tcsn-GUS-hst进行高温处理。对花期高温处理后0 h，12 h，24 h，48 h，72 h的根部、叶片及花药进行染色，发现花期高温胁迫条件下HST参与生长素和细胞分裂素激素响应通路。 利用RNA-Seq技术分析hst突变体花药中表达差异基因，寻找HST调控的靶基因。相比较野生型ZH11，突变体中上调基因657个，下调基因809个。GO富集和pathway富集图可看出，突变体中表达变化的基因参与胁迫响应、氧还反应、糖代谢、激素合成等多种生物代谢过程。对野生型ZH11、突变体hst以及互补突变体HST-hst扬花期进行高温胁迫，发现野生型ZH11和互补突变体HST-hst在花粉活力和柱头上花粉粒数性状上，没有显著差异。相较野生型ZH11和互补突变体HST-hst，突变体hst具有更高的花粉活力和柱头上花粉粒数，这可能是其具有较强的花期高温耐性的关键因素。对耐高温突变体hst花器官在高温胁迫下的生理指标研究可知，高温胁迫导致水稻颖花中可溶性蛋白和可溶性糖含量均显著下降，但突变体hst降幅小于野生型ZH11；而颖花中的MDA、H2O2及游离脯氨酸含量均显著提升，突变体hst的增幅低于野生型ZH11。高温胁迫下hst突变体颖花中保持较多的渗透调节物质及较低的MDA和H2O2含量，使得水稻花器官具备较强的渗透调节能力和生物膜保护能力。以上研究结果为进一步揭示水稻高温热害的生理分子机制提供科学依据。