小麦泛素连接酶F-box蛋白基因在调节植物耐旱性中的功能

Drought stress restrains wheat growth and yield. Ubiquitination of proteins through ubiquitin/26S proteasome system (UPS) is an important regulative manner in response to various abiotic stresses in plants. Ubiquitin ligase E3, as a main determinant of substrate specificity in UPS was involved in plant stress adaptability. We had cloned a full length cDNA sequence from wheat, named TaFBA1 (GenBank JN038382), which encoded a F-box protein that was a key component of ubiquitin ligase complex. For analyzing the expression pattern of TaFBA1 transcription, we had created the construct containing the promoter of TaFBA1 and reporter gene (GUS), the recombinant plasmids were transformed to Arabidopsis thaliana, confocal laser scanning microscopy combined with GUS activity dyeing were used to detect the responses of the reporter gene expression to drought stress treatments. We found the expression of TaFBA1 transcription was up-regulated by water stress and ABA treatments.TaFBA1 antibody was produced by immunization of the rabbits. Two kinds of vectors were constructed, one with cauliflower mosaic virus (CaMV) 35S constitutive promoter, and the other with stress-inducible rd29A promoter. The recombinant plasmids with the coding region of TaFBA1 were introduced into model plants Nicotiana tabacum and Arabidopsis thaliana. With the transgenic tobacco and Arabidopsis plants, we found that overexpression of TaFBA1 enhanced the drought resistance of the plants. In this research, we will transform the vectors loading TaFBA1 cDNA with Ubi and rd29A promoters to wheat plant. Meanwhile, we will obtained wheat plants down-regulated TaFBA1 gene expression by gene silencing transformation. Then, the functions of TaFBA1 in wheat tolerance to drought stress will be studied with the transgenic wheat plants above. We will study on the physiological and molecular mechanisms of drought tolerance improved by overexpressed TaFBA1 . For this purpose, the transgenic plants will be treated by drought stress conditions, and the photosynthesis, water status and osmotic adjustment of the transgenic plants will be detected. In particular, we will investigate the antioxidative capability of overexpressed TaFBA1 plants under stress conditions by detecting relative parameters, such as MDA level, protein carbonylation, electrolyte leakage, ROS accumulations, antioxidant enzyme activity, etc. The specifically interaction of TaFBA1 with ASK and CUL1 protein as well the target protein of TaFBA1 will be determined by a yeast two-hybrid analysis combined with GST pull down technique. These results will be helpful for understanding the physiological and molecular mechanisms of drought tolerance of the plants and will be significance in improving the plant tolerance by gene engineering.

干旱严重影响小麦生长和产量。泛素连接酶E3参与植物抗逆性调控。本项目在已经克隆小麦泛素连接酶F-box蛋白基因TaFBA1并发现过表达该基因提高烟草/拟南芥耐旱性的基础上，利用Real-time PCR、免疫印迹和报告基因技术，研究小麦中TaFBA1基因表达对干旱胁迫的响应模式；将TaFBA1基因转化小麦，获得过表达和抑制表达的转基因植株，利用转基因植株研究基因在调控小麦干旱耐性中的功能；从干旱胁迫下转基因植株光合能力和光合机构的稳定性、渗透调节与水分状况保持和抗氧化能力方面，研究基因调控小麦抗旱性的生理生化机制；利用GST pull down 技术体外鉴定TaFBA1互作蛋白；利用酵母双杂交并结合GST pull down 技术寻找和筛选TaFBA1下游靶蛋白，研究基因调控抗旱性的分子基础。研究结果将为揭示小麦抗旱机制提供理论依据，为作物基因工程育种奠定技术基础和储备高效基因资源。

克隆了小麦F-box蛋白基因TaFBA1。该基因编码一个由325个氨基酸残基组成的蛋白，该蛋白N端含有F-box 结构域，C-端含有AMN1结构域。定量PCR检测发现，该基因表达受到高盐、水分胁迫、高温胁迫和ABA处理上调，在根、茎、叶和穗中均有表达。. 构建基因表达载体转化小麦，获得了过量表达TaFBA1基因以及RNAi转基因株系多个。耐旱性研究发现，无论过表达还是抑制TaFBA1基因表达的转基因小麦，在表型和耐旱性方面均有较大改变。RNAi植株的根系干旱胁迫处理后变短较多，抑制较WT大，说明抑制该基因表达导致根系的耐旱性变差。而过量表达该基因的小麦根系，干旱处理后变短较WT小，耐旱性提高。. 利用组成型过表达该基因的转基因烟草研究TaFBA1基因在植物耐旱、耐盐及高温胁迫耐性中的作用。结果发现，组成型过表达该基因不仅提高了转基因烟草的耐旱性，还提高了耐盐性和耐热性。这些逆境耐性提高的共同生理机制均与氧化胁迫能力的提高有关。同时，耐旱性的提高还与水分状况维持有关；耐盐性提高还与离子区域化改善有关；耐热性的提高还与热激蛋白表达高有关。 . 组成型过表达该基因导致转基因烟草前期加速生长，后期生长速度变慢，转基因植株矮化且晚开花。生长发育表型的改变可能与激素代谢修饰有关，特别是GA代谢。. 为解决表型变异给转基因植株的生产利用带来的不利影响，我们利用胁迫诱导型启动子RD29A替代组成型35s启动子。结果表明，胁迫诱导型转基因植株同样可以发挥TaFBA1基因改善植株耐旱性的作用，同时在非胁迫条件下，使转基因植株的表型改变最小化。该策略可以提高转基因植株在农业生产中的利用价值。 . 酵母双杂验证了TaFBA1蛋白与SCF 复合体中其他亚基互作，证明TaFBA1蛋白是SCF型E3连接酶的一部分。筛选小麦DNA文库获得了TaFBA1下游靶蛋白多个。初步证明其中的胁迫响应蛋白（abiotic stress response protein，ASRP1；转录因子蛋白）TaASRP1与植物抗逆性密切相关。推测TaFBA1可能通过调控TaASRP1基因的表达调控植物耐逆性。