Ran结合蛋白（RanBP1)在小麦春季冻胁迫中的生理功能研究

Spring freeze stress has a strong impact on wheat production. The yield loss caused by spring freeze stress may reach 14–85%. The research on molecular mechanism is currently less than that on physiological mechanism on spring freeze stress in wheat. Following three-day exposure to –5℃ simulated spring freeze stress, wheat plants at anther connective tissue formation phase of spike development were analysed freeze stress responsive proteins in leaves at 1 and 3 days after following freeze stress exposure, using two-dimensional electrophoresis and matrix-assisted laser desorption/ionisation time-of-flight mass spectrometry in the previous study. 75 protein spots were successfully identified under freeze stress conditions. We found that Ran-binding protein 1 homolog c-like（RanBP1）involved in signal transduction was closely related to freezing resistance in wheat under spring freeze stress condition. In order to prove this speculation, the cDNA sequences of TaRanBP1 gene are to be cloned. Using gene overexpression and Virus-induced gene silencing, the vectors of overexpression and silencing of TaRanBP1 are to be constructed. And two kinds of wheat plants should be obtained. The function on RanBP1 in wheat under spring stress condition should be proved by testing the freezing resistance of two kinds of wheat plants. The target proteins regulated by RanBP1 should be researched by the proteomic analysis of spring freeze stress responsive proteins in two kinds of wheat plants. The pathway of RanBP1 should be built by using Pathway Studio software. The molecular mechanism on RanBP1 in plant under stress condition is to be tested in-depth study .

春季冻胁迫对小麦生产造成了严重影响，产量损失可达14–85%，而有关小麦春季冻胁迫的分子机理研究较少。在前期研究中利用蛋白质组学技术，研究了春季冻胁迫对小麦叶片蛋白质的影响，辨析出75个差异蛋白点，进一步发现其中Ran-binding protein 1 homolog c-like (RanBP1)与小麦春季抗冻性密切相关。为证明这一推测，本项目拟克隆TaRanBP1基因cDNA全长，并利用基因过表达和病毒诱导的基因沉默等技术，构建TaRanBP1基因过表达和沉默载体，并获得这两类小麦植株。通过对这两类植株的抗冻性检测，以证明RanBP1在小麦春季冻胁迫中的功能。进而通过检测两类植株在冻胁迫期间蛋白质组学的变化，以研究RanBP1所调控的目标蛋白，并利用Pathway Studio software软件，构建其pathway通路，以深入研究RanBP1在植物抗逆过程中作用的分子机理。

在前期研究中，项目主持人发现一个蛋白Ran-binding protein homolog c-like （RanBP1）在小麦春季冻胁迫中表达量显著提高1.79-2.42倍(图1)，推测它可能在小麦响应春季冻胁迫中发挥了重要功能。为证明这一推测，本项目从小麦中克隆出RanBP1基因的cDNA和DNA序列全长，其开放阅读框(ORF)内不包含内含子，编码一个由207个氨基酸组成的亲水性蛋白质(图2)，该基因定位在4DL染色体上。为检测该蛋白在细胞器内的定位，将重组质粒转化到水稻原生质体中，激光共聚焦显微镜观察检测GFP融合蛋白荧光信号，结果显示其定位在细胞膜和核膜边缘(图4)。qRT-PCR检测结果表明，TaRanBP1受非生物胁迫和激素诱导显著上调表达(图5)。.项目利用基因过表达和病毒诱导的基因沉默技术(VIGS)，分别从正反两个不同的途径验证了Ran结合蛋白（RanBP1）的功能：（1）构建了TaRanBP1基因的沉默载体，获得了此基因的BSMV-VIGS小麦植株。qRT-PCR检测结果表明，该类植株TaRanBP1基因显著下调(图8)。对病毒接种后30天生长发育到拔节初期的小麦植株进行-5℃冻胁迫处理后，利用iTRAQ技术进行蛋白质组学分析结果表明，以冻胁迫处理2d的BSMV-GFP小麦植株为对照，BSMV-TaRanBP1沉默植株差异蛋白总数为140个，其中上调蛋白94个，下调蛋白46个(图9，表1)。辨析了TaRanBP1调控的目标蛋白(基因）并构建了该蛋白的KEGG通路。（2）构建了TaRanBP1基因的过表达载体，转至拟南芥，以野生型拟南芥为对照，对生长25 d的TaRanBP1转基因植株进行了外源脱落酸处理，结果表明转基因拟南芥对脱落酸处理的敏感性增加(图13)。研究还表明TaRanBP1过表达抑制了根和莲座叶原基的生长，阻碍了营养生长，增加了顶端优势，使转基因拟南芥开花提前(图14-15)。研究表明TaRanBP1在植物抗逆性及生长发育方面发挥了重要作用。