葡萄VvBAP1调控糖代谢参与抗冷机制的研究

Low temperature is one of the bottlenecks restricting the growth of the grape industry. It is very important for researching on the mechanism of cold tolerance in grape. BAP1 is a phospholipids binding protein and it was induced expression by cold. This study will focus on the the cold tolerant gene VvBAP1 that we had cloned from grape. By using real time PCR, the VvBAP1 experssion mode will be studied after the grape seedlings are treated with cold (4℃), phytohormone (ABA, SA, JA) and signal molecules (Ca2+, H2S, NO, H2O2). The subcellular localization of VvBAP1 will also be observed with transient expression assays. Using Vitis vinifera and Arabidopsis as material, we will obtain the VvBAP1-overexpression and VvBAP1-knockdown plants by transgene and RNAi technique. We will detect the physiological indicators in these transgenic plants, including total soluble sugar content, the content of malondialdehyde, activities of superoxide dismutase, the relative permeability of cell membrane under cold stress. Moreover, we will detect the expression and activity of invertase and G6PDH which could increase soluble sugar content. These evidence will prove that VvBAP1 could regulate soluble sugar content for cold tolerance in grape. This project will lay the foundation for characterization and enhancement of cold tolerance in grapes.

低温是制约葡萄产业发展的“瓶颈”问题之一，探明葡萄抗寒机制一直备受关注。BAP1是具有C2结构域的磷脂结合蛋白，其表达受到低温诱导。本课题拟在实验室前期克隆的葡萄抗冷相关基因VvBAP1基础上，利用荧光定量PCR技术研究VvBAP1在低温和与低温相关的植物激素和信号分子处理后的表达模式，利用瞬时表达技术观察其亚细胞定位；以敏感葡萄品种和抗性葡萄品种为材料，低温处理后检测VvBAP1的表达量差异，同时以VvBAP1基因过表达植株和VvBAP1基因缺失植株为材料，检测低温处理后各项抗冷相关生理指标变化，从而探明VvBAP1的生物学功能；检测过表达植株和缺失体植株中糖代谢相关酶的表达量和活性变化，探索VvBAP1是否通过调节可溶性糖含量参与葡萄的抗冷过程，为阐明VvBAP1的作用机制打下基础。本项目为深入研究葡萄抗冷基因特性，进一步利用基因工程提高葡萄的抗冷性提供理论基础和技术支持。

葡萄是对低温比较敏感的植物，我国主要栽培的品种多为欧亚种，抗寒性较差，而且冻害已成为制约葡萄产业发展的“瓶颈”问题之一，因此探明葡萄抗寒机制具有重要的理论和实际意义。BAP1（BON association protein 1）是近年来发现的一个具有C2结构域的磷脂结合蛋白。但关于其功能和作用机制研究较少。前期工作克隆了葡萄VvBAP1基因，生物信息学分析显示其含有保守的钙离子依赖性的C2结构域。本实验利用低温处理抗冷葡萄品种‘F-242’和‘左优红’与冷敏感品种‘赤霞珠’和‘霞多丽’，检测VvBAP1的表达量，发现抗冷品种要明显高于冷敏感品种，推测VvBAP1可能与葡萄低温应答过程相关。进一步以抗性葡萄品种‘F-242’为材料，对其表达特性进行了分析，结果发现，低温以及低温相关的信号分子均可诱导VvBAP1的表达。亚细胞定位结果表明，VvBAP1位于细胞质和细胞膜上。为进一步研究其功能，将VvBAP1转入拟南芥，获得异源过表达植株，检测了低温下（4 ℃和-8 ℃）抗冷相关生理和相关基因的表达变化。结果显示，低温条件下，VvBAP1异源过表达植株超氧化物歧化酶（superoxide dismutase，SOD）活性、过氧化物酶（peroxidase，POD）活性和相关基因表达高于野生型植株，细胞膜透性和丙二醛含量均低于野生型。同时，还检测到转基因植株的可溶性糖含量以及糖代谢相关基因BAM4-7、SS4、G6PD5表达量明显高于野生型。此外，检测了低温应答标志基因CBF1、CBF3、COR15a、COR6.6、COR27和KIN1，发现以上基因表达量都有不同程度的升高。推测，VvBAP1能够通过调控可溶性糖含量、激活保护酶活性等途径增强葡萄的抗冷性。本项目解析了VvBAP1参与葡萄抗冷的作用机制，为葡萄抗冷分子育种提供优质的基因资源。