CsWRKY21转录因子调控设施黄瓜对低温的适应机制

Low temperature is an important existing problem in greenhouse, and become the bottleneck of the anti-season cultivation. Cucumber as the main facilities vegetable is sensitive to low temperature; its yield and quality have been affected. Transcription factors play an important role in regulating the response to low temperature in plant. WRKY transcription factor family is the hot research point in recent years, Plant WRKY transcription factors (TFs) are trans-regulatory proteins involved in the plant immune response development and senescence, however their roles in abiotic stress are still not well understood, especially in horticultural crop cucumber. The completion of cucumber genome sequencing provides important data for us to do the WRKY research. The project takes the line 9930 cucumber as the research material. We identify the low temperature stress response WRKY gene by the bioinformatics prediction of cucumber WRKY family factors, verify the target gene as transcription factors by transcription activation technology and identify the CsWRKY21 gene function by RNAi, over-expression, gene chip and qRT-PCR technology, it will indicate the regulate patterns of CsWRKY21 response to low temperature. The research will probably indicate the function of CsWRKY21 and the signal pathway response to low temperature. Meanwhile, as this study is closely related with the major issues of vegetable production, it also has important practical significance.

低温是我国设施中普遍存在的问题，成为反季节栽培中的瓶颈。主要的设施蔬菜黄瓜对低温敏感，产量与品质受到严重影响。转录因子在植物低温胁迫的转录调控过程中扮演重要角色。WRKY转录因子是近年来研究较多的转录因子，但非生物胁迫机制尚不清楚，少以高等植物为研究对象。黄瓜基因组测序的完成为我们研究黄瓜WRKY转录因子提供了重要数据，我们之前的研究表明CsWRKY21响应低温表达上调，响应外源ABA表达上调。本项目拟以黄瓜测序品种为试验材料，进一步探索CsWRKY21响应低温胁迫的机理，利用凝胶阻滞及转录激活等实验技术验证目标基因是否具有转录因子活性，并通过RNAi、过表达、基因芯片及qRT-PCR等技术鉴定CsWRKY21的基因功能，初步明确其参与设施黄瓜抵御低温的调控的模式。研究可望阐明低温条件下CsWRKY21转录因子的功能及信号通路，为解决设施瓜类生产中抵御低温提供理论依据，具有重要的应用价值。

为解决低温这一设施普遍存在的，限制作物反季节栽培的问题，本项目以我国设施内栽培面积最大的园艺作物黄瓜为研究材料，利用生物信息学和分子生物学方法，克隆响应低温胁迫的黄瓜WRKY转录因子CsWRKY21，构建转基因等表达载体，转化模式植物拟南芥和黄瓜，进而鉴定基因功能，探讨该转录因子调控植物响应低温的分子机制。获得的实验结果如下：.（1）克隆得到黄瓜CsWRKY21基因，利用生物信息学分析基因序列，其开放读码框948 bp，编码316个氨基酸，蛋白分子量约34.5 kDa，包含4个内含子和5个外显子，也属于WRKY转录因子第II类家族，与拟南芥AtWRKY40高度同源。并且实时荧光定量PCR分析CsWRKY21基因响应低温胁迫和外源ABA，且被诱导上调表达。.（2）CsWRKY21蛋白定位于细胞核中。成功构建CsWRKY21与绿色荧光蛋白(GFP)的融合表达载体，通过检测GFP融合蛋白在拟南芥原生质体中的瞬时表达，揭示CsWRKY21蛋白定位于细胞核中。.（3）构建CsWRKY21过表达载体和干扰表达载体，并成功转化拟南芥。冰冻处理CsWRKY21过表达转基因植株与野生型相比表现稍有抗冻，存活率虽有提高，但并未达显著，低温处理后抗逆相关生理指标EC，MDA和脯氨酸的变化显著，表明CsWRKY21过表达转基因植株较野生型抗冷性增强；此外，我们分别在高盐和干旱胁迫下鉴定了CsWRKY21的基因功能，发现转基因植株在干旱胁迫下出现明显的抗旱表型；对于盐胁迫也有一定的抗性，但效果不明显。.（4）应用实时荧光定量PCR证实转基因过表达CsWRKY21增强了COR47，RD29A，KIN1，CBF1，CBF2和CBF3等冷信号通路基因的表达，推测其可能响应依赖于CBF的冷信号途径。同时，酵母单杂交实验证明CsWRKY21可以结合ABI5的启动子。. 综上所述，黄瓜CsWRKY21转录因子对低温、干旱、高盐这些非生物胁迫有一定的抗性，尤其对低温、干旱有明显的抗性。推测其通过CBF途径参与植物对低温的响应，也可能通过调控其他基因通过ABA途径共同参与植物对低温的响应。