rol基因在枳橙矮化性状建成中的分子作用机制

Plants transferred rol gene display dwarfing effects and full hairy-root syndrome. These agronomic traits play important roles in agricultural production. rol genes' function had been interpreted by transgenic method in past investigation, however, the molecular network which induced by rol genes expression is still unknown. In a previous study, we found that rol genes' expression broke the balance of plant hormones, CTK was dramatically increased, but indole acetic acid (IAA) and gibberellic acids (GAs) were remarkably decreased in transgenic citrange. Moreover, peroxidase (POD) activity was significantly increased in transgenic citrange with rol genes. rol gene expression could suppress the pathway of GA53/GA12 transform to GA1/GA4 by down-regulating the expression of GA20ox1 gene in bud. we suppose various genes involved in plant hormone synthesis play an important role in the morphogenesis of dwarf traits in transgenic citrange. In this study, comparative transcript profiling of spring shoots by RNA-seq after self-pruning perform on transgenic citrange with rol genes, citrange treated by spraying GA3 and wild-type citrange, focusing on analysis of RNA splice, SNPs, and the differential expressing genes in the pathway of plant hormones synthesis and signal transduction, selecting. We hope to check key dwarfing genes by qRT PCR, then identify these genes by genetic transformation system of seedling of trifoliate orange, which was established in our library. We will interpret the molecular processes of rol genes regulating dwarf character in citrus.

rol基因转入可诱导植物矮化和大量毛状根产生，这些农艺性状在生产中具有重要的应用价值。目前单个rol基因的功能已经得到阐释，但其表达启动的下游分子网络仍不清楚。我们前期对转rol基因枳橙的矮化研究发现：rol基因表达促使CTK大量累积、IAA和GAs含量相对减少，植物激素平衡被打破，并且过氧化物酶（POD）活性显著增强。同时，rol基因表达下调GA20ox1基因表达，影响惰性赤霉素GA53/GA12向活性GA1/GA4的催化途径，认为其可能通过作用植物激素合成关键基因来推动矮化性状建成。本研究以转rol基因Toryer枳橙及其喷施GA3的处理和野生型为材料，利用RNA-Seq分析三个样本转录组RNA剪接、SNPs和植物激素合成代谢途径的差异表达基因，结合qRT-PCR验证筛选出矮化关键基因，并通过实生态枳遗传转化系统验证基因功能，探索rol基因在柑橘矮化性状建成中的分子作用机制。

果树矮化密植具有早产丰产、优质高效的优点，果树矮化机理是矮化育种的基础，近年来成为果树分子育种研究的一个热点。柑橘缺乏矮化资源，矮化机理研究相对滞后。本研究以转rol基因矮化枳橙（Citrus sinensis (L.) Osb × Poncirus trifoliate (L.) Raf.）B、D、E系及野生型侧生嫩芽为材料，利用RNA-Seq进行转录组比较分析，寻找到664个差异基因，通过RT-PCR结合RACE技术克隆到CPMAX2、CPMAX1、CPpin1、CPBES 1、CPPAL、CPCAD等6个矮化关键基因的cDNA全长，并构建其植物表达载体，通过植物遗传转化对矮化关键基因的功能进行验证。.qRT-PCR分析证实在转rol基因枳橙侧生嫩芽中：赤霉素合成基因PtGA20ox1、PtGA20ox2下调表达，生长素运输蛋白基因CPpin1上调表达，油菜素内脂转录因子CPBES1和独脚金内脂信号转导因子CPMAX2下调表达，细胞分裂素合成基因CPipt5上调表达，五种植物激素信号互作协同调控侧芽萌发和生长，并且发现转rolABC基因枳橙细胞中木质素合成基因CPPAL、CPCAD基因均上调表达，引起木质素大量累积，造成细胞膨大机械障碍，在转rolABC基因枳橙枝梢腋芽萌发和生长发育中发挥重要作用。在此条件下，提出五种植物激素和木质素协同推动转rolABC基因枳橙簇生分枝和矮化性状的建成的分子机制模式图。