脱落酸调控ICE蛋白参与冷敏植物小桐子抗冷性形成的功能分析

Chilling is becoming an important limiting factor for growth and distribution of Jatropha curcas，a sustainable energy plant.ICE-CBF mediated transcriptional regulation network plays important roles in low temperature response in higher plants, whereas the involvement of ABA and molecular machanisn were still barely elucidated. Based on the preliminary results from whole transcriptome sequencing analysis of J. curcas, two potential JcICEs sequences were identified and cloned. Multiple bioinformatic analyses revealed that the two deduced proteins shared 53% identity, and were seperated into different clades of ICEs by phylogenetic analysis. Meanwhile, promoter analysis by PLACE and plantcare revealed the ABA-responsive cis-elements(ABRE) only existed in JcICE1 promoters, who also displayed a chilling-induced expression in leaves. Based on all the previous work,in this study: ①the complete coding sequences of two JcICEs will be cloned, accompanied by further analysis on their expression pattern in response to chilling and exogenous ABA; ②the transactivation activity will be verified by transient expression analysis in yeast system;③construct promoter-GUS fusion vectors,by replacing CaMV 35S promoter in p BI121 binary vector with promoters from JcICE, to validate the transcriptional response to chilling and ABA by GUS activity analysis.Then exact responsive elements to chilling or ABA will be identified by further deletion and mutational analysis;④transgenic engineering will be performed using model plants with different chilling sensitivities, Arabidopsis and tobacco, followed by comprehensive evaluation of chilling tolerance of transgenic plants.This research will primarily elucidate whether ABA involved in ghe ICE-mediated transcription regulation in response to chilling in J. curcas and the corresponding machanism,which not only give us more information on understanding the transcription regulatory network in response to chilling stress in J. curcas, but also provide theoretical bases and important potential genes for improving chilling tolerance of J. curcas by genetic engineering.

低温是限制能源植物小桐子生长和分布的重要环境因子。ICE-CBF介导的转录调控在植物低温响应中起关键作用，植物激素ABA在此过程中的参与性和作用机制尚未阐明。根据前期小桐子全转录组高通量测序工作克隆得到两条JcICE基因全长，二者在系统进化上属不同的分支，仅JcICE1启动子含ABA响应元件，在叶片中的转录受低温诱导。基于此，本项目拟研究：①克隆两个JcICE基因的完整编码区，分析其对低温和ABA响应的表达特性；②用酵母系统进行瞬时表达分析，确认转录激活作用；③构建启动子-GUS融合载体，分析启动子对低温和ABA的转录响应，以删除和突变分析确定响应元件；④将JcICE基因转入不同冷敏感性模式植物，并进行转基因植物的抗冷性评价。研究成果将阐明ABA是否参与JcICE基因在小桐子低温耐受性的形成过程及其分子机制，为小桐子耐冷性的遗传操作提供理论依据和重要的基因资源。

低温是限制能源植物小桐子（Jatropha curcas L.）生长和分布的重要环境因子。到目前为止，小桐子中ICE-CBF低温响应通路中关键元件的鉴定及作用机制、植物激素ABA在低温响应的参与性均尚未阐明。基于此，我们以小桐子幼苗为材料，对JcICE基因参与低温响应的分子机制进行了研究，主要结果如下：.1、小桐子两个JcICE基因对低温和外源ABA处理的差异性响应分析.基因表达模式和启动子活性分析显示，小桐子的两个JcICE基因在转录水平上的调控存在差异，相对于JcICE2，JcICE1基因的启动子具有较高的本底活性，且更易受到低温和外源ABA的诱导，表明植物激素ABA可通过转录调控参与小桐子的低温响应。.2、两个JcICE基因的过表达均赋予转基因烟草一定的耐冷性.低温萌发实验显示转基因烟草的萌发明显快于对照，播种7天时过表达JcICE1和JcICE2的转基因烟草萌发率分别可高达对照的9.91 倍和6.24倍，且耐冷性并不一定与外源基因的表达量成正比。过表达JcICE2的转基因烟草在低温处理24 h后，叶片中的MDA含量、组织中的H2O2积累以及根系电解质渗漏率均显著低于对照，同时在一定程度上正向调节烟草中DREB1/CBF及下游靶基因（NtDREB3、NtERD10C和NtP5CS）的表达，提高了低温耐受性。.3、JcICE基因的作用机理分析.洋葱表皮细胞和拟南芥原生质体瞬时表达系统证实两个JcICE蛋白均定位在细胞核，酵母双杂交验证了二者的转录自激活活性。以酵母单杂交系统验证证实了CAGCTG基序是JcICE2的识别元件之一。最后借助DUAL membrane 酵母双杂交系统体外证实了JcICE2与JcSUMO1（小泛素化修饰蛋白）的直接互作，暗示了JcICE蛋白的泛素化修饰对于其功能的实现可能是至关重要的。.4、小桐子JcERF3基因的克隆及其对植物耐冷、耐盐性形成的功能验证.从小桐子叶片中克隆获得一个新的ERF编码基因JcERF3，该基因与DREB1/CBF同属AP2/ERF转录因子家族，但不含有植物CBF蛋白的保守特征序列。JcERF3的过表达赋予烟草一定的低温和盐胁迫耐受性，表现出较高的种子萌发率、可溶性糖和游离脯氨酸积累，电解质渗漏率以及离体叶盘失绿程度下降，为小桐子的抗逆遗传改良提供一种新的基因资源。