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目的:研究转化酶抑制子基因对番茄果实糖转运与积累的影响,并初步探讨其调控机制。创新点:首次在番茄果实瞬时表达系统中证明转化酶抑制子可明显抑制细胞壁转化酶活性,并且此抑制作用是通过翻译后水平发挥作用。方法:将Micro-Tom番茄分为对照组(果实未注射农杆菌及注射不含载体的空白农杆菌)和实验组(果实注射含有过表达转化酶抑制子基因的农杆菌),番茄果实分为萼片、中果肉、胶质胎座和心室隔壁四个部位,用实时定量聚合酶链式反应(qR T-PCR)技术检测转化酶抑制子和转化酶基因家族的表达变化,利用比色法分别检测了蔗糖代谢关键酶活性的变化,用高效液相色谱检测果实各部位果糖、葡萄糖和蔗糖的含量,利用高氯酸水解法测定淀粉的含量。结论:利用农杆菌注射进行果实瞬时表达后果实各部位的细胞壁转化酶活性受到明显抑制,但转化酶基因家族的转录水平表达变化不大,果糖和葡萄糖含量下降,蔗糖含量有所升高。这一结果表明Sly-INH基因主要是通过在翻译后水平对番茄细胞壁转化酶进行调控,进而影响番茄果实糖的组成与含量。本研究从分子水平对转化酶抑制子及其调控的转化酶基因家族进行了系统性研究,这为利用栽培手段调控植物体内转化酶的表达和活性,调节库组织的蔗糖的输入速率以及同化产物的分配以改善品质提供依据与技术支持。
Objective: To study the effect of invertase repressor gene on sugar transport and accumulation in tomato fruit and its regulation mechanism. Innovation: For the first time in the tomato fruit transient expression system showed that invertase inhibitors can significantly inhibit cell wall invertase activity, and this inhibition through post-translational levels play a role. Methods: Micro-Tom tomato was divided into control group (Agrobacterium without Agrobacterium injection and Agrobacterium without Agrobacterium injection) and experimental group (Agrobacterium containing gene with over-expression of invertase inhibitor) Sepals, midrib, placenta and ventricular septum. The qR T-PCR was used to detect the expression of invertase inhibitor and invertase gene family, respectively. The changes of key enzyme activities of sucrose metabolism were detected. The contents of fructose, glucose and sucrose were determined by HPLC. The content of starch was determined by perchloric acid hydrolysis. CONCLUSION: The activity of cell wall invertase in various parts of the fruit after Agrobacterium tumefaciens injection was significantly inhibited. However, the expression level of invertase gene family changed little, the content of fructose and glucose decreased, and the content of sucrose increased. This result indicates that the Sly-INH gene mainly affects the composition and content of sugar in tomato fruit through the regulation of tomato cell wall invertase at post-translational level. In the present study, we investigated the expression of invertase gene and its invertase gene family at the molecular level. This is a method to regulate the expression and activity of invertase in plants, and to regulate the sucrose input rate and assimilation products Distribution to improve the quality to provide the basis and technical support.