细胞壁蔗糖转化酶（CWIN）在番茄果实耐热性中的功能分析

Heat stress causes tomato flower and fruit abortion and impairment of fruit development including color-turning, which results in severe yield losses. However, the underlying mechanisms of heat tolerance or susceptibility of tomato fruit are poorly understood, and the key regulating factors in heat tolerance remain unclarified. Upon heat stress, a network of physiological and biochemical processes in tomato fruit development are affected, in which carbohydrate metabolism, especially sucrose cleavage to hexoses catalyzed by invertase and sucrose synthase, may play an important role in regulating heat tolerance of tomato fruit. Our previous research indicates that cell wall invertase (CWIN), particularly one of its members, Lin7, is likely key enzyme regulating heat tolerance of young tomato fruit (Li et al., 2012). In this proposal, we aim to isolate the Lin7 gene (including its native promoter) from the heat-tolerant (HT) tomato line, and express the HT Lin7 gene in the heat-sensitive (HS) line by transgenic method to test if this will confer heat tolerance in the HS line. On the other hand, we attempt to silence Lin7 expression in the HT line by using RNAi technology to further understand the function of Lin7 in tomato fruit development and heat tolerance. In addition, a transgenic tomato line with elevated CWIN activity (by silencing the CWIN inhibitor LeINVINH1, Jin et al., 2009) will be used in this research to analyze the performance of heat tolerance of transgenic tomato and its crossed lines under heat stress conditions. The overall goal of this project is to acquire molecular evidence that high CWIN activity is required for heat tolerance of tomato young fruit.

高温逆境可造成番茄落花落果、果实膨大与转色不良，给番茄生产带来重大的产量损失。因此，研究番茄果实耐高温的分子机理，解析调控番茄果实耐高温的关键因素具有重要意义。高温胁迫会影响到番茄果实发育的许多生理生化过程，其中糖代谢，特别是蔗糖转化为己糖，可能是调控番茄果实耐热性的关键因素。本项目的前期研究发现，细胞壁蔗糖转化酶（CWIN）是番茄果实耐高温的关键酶，编码CWIN的Lin7基因是与番茄果实耐热性相关的重要基因。以此为基础，本项目拟从耐热番茄中克隆Lin7基因（含启动子），并在热敏感番茄中进行基因的功能互补验证。同时，利用RNAi技术在耐热番茄中抑制Lin7基因的表达，以进一步解析该基因在果实发育与耐热性中的功能。此外，利用已有的CWIN酶活性提高的转基因番茄为材料，在高温胁迫下进行系统的分析研究，目的是获得CWIN调控番茄果实发育与耐热性的分子证据，为解析番茄果实耐高温的分子机理奠定基础。

在非生物胁迫条件下，细胞壁蔗糖转化酶（CWIN）酶活性的降低常常导致植物座果率的降低和种子的败育。以CWIN酶活性升高的转基因番茄为材料，本项目研究了长期中等高温胁迫（long-term moderate heat stress, LMHS）条件下，CWIN调控番茄坐果的分子机制。研究发现，CWIN酶活性的升高可以抑制番茄果实中由LMHS引发的程序性细胞死亡（programmed cell death, PCD）。对H2O2和丙二醛含量以及一系列抗氧化酶活性的测定表明，CWIN是通过不依赖于活性氧（ROS-Independent）的方式对程序性细胞死亡产生抑制作用的。在LMHS胁迫的条件下， CWIN酶活性的升高可以维持蔗糖向幼果的运输，并且可以提高胚珠中己糖激酶和果糖激酶的酶活性。此外，与野生型番茄相比，转基因番茄胚珠中热激蛋白基因Hsp90和Hsp100，以及幼果中HspII17.6基因和生长素合成基因ToFZY6的表达量升高，而幼果中生长素响应基因IAA9的表达量下降。以上结果表明，在LMHS胁迫的条件下，CWIN促进番茄坐果是通过抑制不依赖于活性氧的程序性细胞死亡的方式进行的，这一过程包括蔗糖运输与代谢，热激蛋白表达，生长素响应和合成等途径。