冷胁迫下采后哈密瓜膜劣变的分子机制研究

Hami melon is characteristic amphisarca in Xinjiang, it is highly effected by chilling injury during low temperature storage, and the main characteristics of chilling injury are surface sag, browning, etc.. The quality change of postharvest melon during low temperature storage is closely related to the membrane deterioration. The project prepares to systematically study on transcriptional expression of postharvest hami melon fruits which are stored under different conditions using digital gene expression spectrum technology, and high-throughput screening for the different expression genes of membrane lipid metabolism which are closely related to the membrane deterioration. The change of the plasma membrane proteomics of postharvest hami melon fruits will be study using iTRAQ technology on basis of the transcriptome studies, and different membrane proteins which are closely related to the membrane deterioration will be identified. The function of different expression genes of membrane lipid metabolism and plasma membrane proteomics will be analyzed and clustered through bioinformatics means, we will reveal the correlation of membrane deterioration, different expression genes of membrane lipid metabolism and the plasma membrane proteomics under the condition of different cold stress, and with the help of KEGG database, the main biochemical pathways and signal transduction pathways which different expression genes and plasma membrane proteomics involved in will be identified. The results will help us to clarify the mechanism of membrane deterioration of postharvest hami melon fruit which are stored under different cold stress on the molecular level, establish a foundation for regulation of different expression genes and plasma membrane proteomics, and provide the molecular basis of genes mining which related to quality deterioration of postharvest hami melon fruit, then provide the theory basis for different storage measures to extend hami melon fruit freshness.

哈密瓜是新疆特色瓜果，在采后低温贮藏过程中易发生冷害，其主要特征包括表面凹陷、褐变等，这些品质变化与膜劣变密切相关。本研究拟采用数字基因表达谱技术对不同条件贮藏下的哈密瓜果实转录表达进行系统研究，高通量筛选获得与膜劣变密切相关的差异表达膜脂代谢基因；在转录组学研究的基础上，利用iTRAQ技术，研究质膜蛋白组学的变化，鉴定与膜劣变相关的重要差异质膜蛋白；通过生物信息学手段对差异表达膜脂代谢相关基因和质膜蛋白进行聚类、功能解析，揭示不同冷胁迫条件下哈密瓜果实膜劣变与膜脂代谢基因、质膜蛋白表达的关联性；借助KEGG数据库确定差异表达膜脂代谢基因和质膜蛋白参与的主要生化代谢途径和信号转导途径。研究结果将从分子水平阐明采后哈密瓜果实冷胁迫中的膜劣变机制，为开展相关膜脂代谢基因和质膜蛋白调控奠定基础，同时为哈密瓜品质劣变相关基因的挖掘提供分子基础，进而为采用不同贮藏措施延长哈密瓜保鲜期提供理论依据。

哈密瓜是新疆特色瓜果，在采后低温贮藏过程中易发生冷害，明晰哈密瓜冷胁迫下分子响应机制，可以为延长哈密瓜货架期和提高储藏品质提供重要的理论依据。本研究通过对采后金皇后和伽师瓜冷胁迫下生理生化指标、抗性酶活性变化研究；差异表达基因，差异蛋白组学分析等，较为深入的揭示了哈密瓜果实对冷胁迫的应答机制，研究结果表明：.1、利用BGISEQ-500测序平台分别对0.5℃、3℃和21℃贮藏下的金皇后和伽师瓜果实进行转录组测序，各组样品获得原始数据1.1G左右；金皇后哈密瓜分别鉴定到17491、18131、18968个差异表达基因，伽师瓜分别鉴定到18363、18419、19107个差异表达基因。.2、将鉴定到的差异表达基因进行GO功能注释分析，其中金皇后和伽师瓜差异表达基因被分别注释到生物过程、细胞组份和分子功能的23、16和13条GO-term中。金皇后哈密瓜得到注释最多的条目分别是：代谢过程、单一生物过程、细胞、细胞膜以及结合和转运活性；伽师瓜得到注释最多的条目分别是：代谢过程、对刺激的响应、单一生物过程、细胞器、细胞膜、以及结合和转运活性。.3、利用iTRAQ技术对金皇后和伽师瓜蛋白进行定量分析，分别鉴定到5250、5291个差异蛋白，对其进行GO注释，分别注释到47、49个GO-term上；与COG数据库比对，金皇后共有4532个蛋白获得注释，其中筛选获得77个差异质膜蛋白；伽师瓜共有4582个蛋白获得注释，其中筛选获得60个差异质膜蛋白。.4、将筛选获得77和60个差异质膜蛋白对其进行GO功能注释分析，其中金皇后和伽师瓜差异质膜蛋白被分别注释到生物过程、细胞组份和分子功能3个GO本体。.5、将筛选获得的部分差异质膜蛋白进行实时荧光定量PCR验证，结果表明:V-ATPase和HmHSP70蛋白在哈密瓜受到低温胁迫后表达量上调显著。