柑橘GASA4基因在抗逆反应中的作用及其机制

GASA4 is a GA-stimulated gene that plays an important role in GA-induced plant growth and development including flower development. In our previous studies, the citrus GASA4 gene was found to be highly induced by Citrus tristiza virus (CTV), and the virus induction of GASA4 has never been reported before. In the proposed study, the gene will be over-expressed in Arabidopsis and Citrus, and suppressed via RNAi interference in Citrus. The transgenic plants will be analyzed for changes in their phenotypes, and in their responses to different treatments including application of different hormones, abiotic stresses, and biotic stresses of CTV, citrus tatter leaf virus (CTLV), citrus huanglongbing bacteria and citrus canker bacteria. In addition, the expression of marker genes such as PR1 and the content of ROS will be analyzed. It is expected that the results will reveal the exact role of the citrus GASA4 gene in the responses of Citrus to stresses. Our preliminary results also showed that GASA4 protein interacted with several proteins including NPR1, CTLV movement protein and cysteine proteinase, which prompted us that the GASA4 may exert its diverse roles through interacting with a set of diverse proteins. To verify, Fluorescence Resonance Energy Transfer, FRET, will be used to confirm the interactions between GASA4 and its target proteins. Moreover, the cysteine residues conserved in the GASA motif of the protein will be individually mutated and the mutated proteins will be assessed for changes in interaction with the original interactive proteins in a yeast two hybrid system to determine how these cysteine residues contribute to protein-protein interactions. The results obtain from this study will provide a solid basis for a reasonable unitization of the citrus GASA4 gene in improving stress resistance of citrus.

GASA4基因参与GA诱导的植物生长发育如花、果发育。我们在前期研究中发现了GASA4一个未被报道的现象—被病毒（在柑橘中被衰退病）高度诱导。本研究拟在柑橘和拟南芥中超表达该基因，在柑橘中用RNAi抑制该基因的表达，分析过表达和RNAi转基因植株的表型，以及它们对不同激素、逆境、接种衰退病和碎叶病病毒以及溃疡病和黄龙病细菌处理后的表现，结合分析PR1等标志性基因的表达和ROS水平等，探明其在抗病抗逆反应中的确切作用。此外，我们的初步结果表明GASA4蛋白可与NPR1、碎叶病毒移动蛋白、半胱氨酸蛋白酶等互作，这可能是其行使不同功能的分子机制。我们拟用荧光共振能量转移技术来验证GASA4和互作蛋白的互作关系，并拟将GASA结构域中保守半胱氨酸一一突变后，用酵母双杂交检验突变蛋白的互作能力，确定半胱氨酸残基对蛋白互作的贡献。本研究结果有望为在柑橘抗逆性改良中合理利用GASA4打下理论和应用基础。

我们在前期研究中发现了GASA4基因一个未被报道的现象¬¬—被病毒（在柑橘中被衰退病）高度诱导。在此基础上，本项目1）通过qPCR分析了柑橘在GA、ABA、SA、ABA处理以及在细菌、真菌、病毒感染后GASA4基因的时空表达模式。2)克隆了柑橘GASA4基因的启动子序列，测序后用生物信息学软件分析其上的顺式作用元件，推测表达调控因子，并用不同长度的GASA4基因的启动子替换GUS表达载体中的GUS基因启动子，然后通过分析转基因拟南芥中GUS基因的时空表达水平和GUS活性，摸清了决定柑橘GASA4 启动子活性的核心区域以及启动子对各种胁迫的响应规律。3)从克里曼丁橘中克隆了GASA4的全长序列，构建了该基因的超表达载体和CRISPR基因敲除载体，并在柑橘和拟南芥中超表达柑橘GASA4，分析了超表达GASA4的转基因植株的表型及其对各种相关激素处理、非生物胁迫（盐胁迫、干旱、机械损伤等）和生物胁迫（细菌、真菌、病毒侵染）的应答模式。获得了GASA4基因敲除植株，分析了GASA4基因敲除植株的表型。4）通过酵母双杂交筛选和验证了GSAS4 蛋白的互作蛋白，通过点突变推测高度保守的GASA 结构域中12 个半胱氨酸在蛋白互作中起重要作用。通过这些研究较全面地了解了柑橘GASA4的功能、作用及其与其他蛋白的互作，解析了相关分子机制，证明其不仅在柑橘生长发育中起重要作用，在柑橘应答逆境胁迫中也起到重要的作用。这些发现为未来合理利用GASA4打下了理论和应用基础。