转录因子SlWRKY6和SlWRKY41在硅诱导番茄抗旱性中的功能分析

Water deficit is one of the main factors limiting the stable and high tomato production in dry-farming areas. Application of exogenous silicon (Si) can effectively improve the drought resistance of tomato, but the molecular mechanism of Si-induced resistance has not been explored yet. WRKY transcription factors are closely related to the stress responses in plants. However, the regulatory function of tomato WRKY genes during water deficit has not been fully elucidated. Based on the transcriptome sequencing results, tomato cultivar ‘Zhongza No.9’ will be used as experimental material in this project. By means of RACE, real-time PCR and Southern blot, SlWRKY6 and SlWRKY41 genes will be cloned and then performed the sequence characteristics and gene family analysis, subcellular location and transactivation identification. Tomato plants with different SlWRKY6 and SlWRKY41 gene expressions will be constructed through VIGS and transgenic techniques. Moreover, the temporal and spatial changes of SlWRKY6 and SlWRKY41 genes expressions, and their relationship with exogenous Si-induced drought resistance in tomato will be investigated. The present study aims to clarify the response mechanisms of SlWRKY6 and SlWRKY41 transcription factors in the Si-mediated resistance of tomato to water deficit stress. All those will help to provide theoretical basis for tomato breeding concerned with drought resistance and water saving, and explore a new way based on Si fertilizer in the anti-drought and high-efficiency tomato cultivation .

水分亏缺是旱作区番茄高产稳产的主要限制因素之一。通过使用外源硅（Si）可有效提高番茄的耐旱能力，但Si诱导番茄抗旱性的分子机制还不够清楚。WRKY转录因子与植物的抗逆应答反应密切相关，但番茄WRKY转录因子在水分亏缺下的调控功能尚未完全阐明。本项目以转录组测序结果为基础，以‘中杂9号’番茄为试材，基于RACE、real-time PCR、Southern blot、VIGS和转基因技术，克隆SlWRKY6和SlWRKY41基因并对其进行序列特性分析、基因家族分析、亚细胞定位与转录激活活性验证，构建SlWRKY6和SlWRKY41基因的不同表达活性植株，探讨这两个基因的时空表达特征及其与外源Si诱导番茄耐旱性的关系。本研究可望阐明外源Si诱导下SlWRKY6和SlWRKY41基因表达对水分亏缺的响应机制，为抗旱节水番茄育种提供理论依据，并探索一条基于Si肥的番茄抗旱高效栽培的新途径。

水分亏缺是限制旱作区作物高产稳产的主要因素之一，提高作物抗旱机制的研究备受关注。WRKY转录因子是植物界中存在的一类较大的基因家族，参与植物在逆境胁迫中的多种应答反应。外源硅（Si）可在一定程度上缓解水分胁迫对植物生长发育的抑制效应。但迄今为止，大多数番茄WRKY（SlWRKY）基因的生物学功能尚不清楚，并且外源Si调控SlWRKY基因的表达诱导番茄抗旱性的分子机理仍不明确。本项目基于外源Si对番茄水分胁迫的缓解效应的转录组测序结果，以表达量变化较大的两个转录因子SlWRKY6和SlWRKY41为研究对象，对其进行了基因全长克隆，并进行了功能验证。初步研究结果如下：利用RACE技术克隆了SlWRKY6和SlWRKY41基因，其开放阅读框分别为1653bp和1011bp，分别编码550个和336个氨基酸；生物信息学分析显示，SlWRKY6和SlWRKY41基因分子量分别为59.66 kD和37.73 kD，理论等电点分别为7.25和5.55；系统进化树表明SlWRKY6和SlWRKY41结构域在进化过程中保守程度较高，与茄属植物的亲缘关系较近；亚细胞定位结果显示，两者均定位于细胞核；转录激活活性分析结果表明，两者在酵母体内均具有转录激活活性。利用VIGS和农杆菌介导叶盘转化法，获得SlWRKY6和SlWRKY41基因不同表达量的番茄植株。以野生型番茄为对照，发现过量表达SlWRKY6和SlWRKY41基因的转基因植株的电导率、丙二醛含量显著下降，脯氨酸含量显著提高；沉默表达SlWRKY6和SlWRKY41基因的转基因植株的电导率、丙二醛含量显著升高，脯氨酸含量显著下降。水分胁迫下，以野生型番茄为对照，过表达SlWRKY6、SlWRKY41基因的转基因植株的抗旱能力明显提高，并且外源Si处理有助于进一步增强其抗旱性。通过本项目的实施，丰富了SlWRKY6和SlWRKY41基因的生物学功能和调控机理，有助于阐明外源Si诱导下SlWRKY6和SlWRKY41基因表达对水分亏缺的响应机制，可为抗旱节水番茄育种提供理论依据，同时创造一条基于Si肥的番茄抗旱高效栽培的新途径，具有较高的理论价值和较强的现实意义。