同源异型盒基因kn1提高柑橘转基因效率的分子机理研究

Citrus juvenile tissues (epicotyls of germinating seedlings) were used as primary explants in genetic transformation, although the transformation is efficient in general, the juvenile phase of juvenile citrus is excessively long, making identify the function of genes and promoters related to characters of flowers and fruit highly difficult. While using mature citrus as explants can reduce juvenile phase drastically, the transformation efficiency is extremely low. Our previous research showed that overexpression maize homeobox gene knotted 1(kn1) drastically improves genetic transformation efficiencies of juvenile citrus, however, the molecular mechanism of kn1 gene enhances transformation efficiency has not been determined. In this project, the expression level changed genes were analysis after kn1 gene integrating into ‘Sucarri’ orange genome, and cloning the genes of regulated CK, IAA and GA biosynthesizing and degrading in citrus, then to test whether these genes have a function to improve transformation efficiency of citrus; In addition, exploring the molecular mechanism of kn1 gene improving citrus genetic transformation efficiency via studying the interaction between KN1 and downstream target genes and KN1 binding sites, thereby finding 3-5 genes that can increase the efficiency of citrus, and also providing a theoretical basis for improving the transformation efficiency of mature citrus and other plants.

柑橘转基因研究主要以实生态材料为外植体，虽转化效率较高，但过长的童期使与花器官发育、果实发育及品质相关基因及启动子的功能验证很难在短期内完成。而以成年态柑橘为材料虽可克服童期，但转化效率极低。前期研究发现应用玉米同源异型盒基因knotted1（kn1）可大幅度提高实生态柑橘转基因效率，而kn1如何提高转基因效率的分子机理并未研究。本项目拟从kn1转化实生态埃及糖橙后与生长素、细胞分裂素、赤霉素合成及代谢相关基因表达量的变化入手，分析并克隆目标基因并对其进行转基因验证；同时，研究KN1与下游目标基因的互作以及具体结合位点来探索kn1提高转基因效率的分子机制，发现3-5个能提高转基因效率的基因，为提高成年态柑橘及其他植物转基因效率的研究提供理论基础。

柑橘转基因研究主要以实生态材料为外植体，虽部分品种转化效率较高但大部分转化效率低，同时过长的童期使与花器官发育、果实发育及品质相关基因及启动子的功能验证很难在短期内完成。而以成年态柑橘为材料虽可克服童期，但转化效率极低。前期研究发现应用玉米同源异型盒基因knotted1（KN1）可大幅度提高实生态柑橘转基因效率，而KN1如何提高转基因效率的分子机理并未研究。本项目通过转录组测序筛选出转KN1基因后外植体愈伤组织差异表达基因主要富集在植物激素信号转导通路上，其中生长素信号转导相关表达差异基因最多并且多富集在降低生长素生物活性通路，其次是细胞分裂素相关基因以及乙烯、赤霉素、油菜素内酯、茉莉酸相关基因，最后分析结果表明KN1基因通过调控效相关基因表达减弱生长素、乙烯和茉莉酸生物活性，同时增强油菜素内酯生物活性增加细胞再生能力，从而提高转基因效率。并且通过酵母单杂交技术筛选出转录因子KN1与下游基因启动子结合的motif序列。因此本项目研究明确了KN1基因提高柑橘转基因效率的分子机理及其转录因子结合特性，为提高成年态柑橘及其他植物转基因效率的研究提供理论基础。