苹果ACC合成酶基因MdACS6在果实乙烯合成中的调控机制研究

Ethylene biosynthesis in plant is divided into two systems, system 1 and system 2. ACC (1-Aminocyclopropane-1-carboxylic acid) synthase (ACS) is the most important enzyme in ethylene signaling pathway regulating the ripening of climacteric fruit. A new member of apple ACS family, named MdACS6, was found by searching the apple genome database. MdACS6 is expressed differentially in apple fruit development and ripening, which started from 60 d before commercial harvest and decreased during the storage at room temperature. We propose that MdACS6 might work in system 1 ethylene synthesis. In this proposal, the following experiments are designed to study the role of MdACS6 in regulating system 1 and system 2 ethylene synthesis in apple fruit based upon these findings. Firstly, the full length of MdACS6 will be obtained by RACE. Its expression during apple fruit development and in different organisms will be detected by real time PCR. Fruits will be treated with 1-MCP (1-Methylcyclopropene, an ethylene inhibitor) and ethylene, which will be used to check the expression of MdACS6 to determine whether it is affected by ethylene. The protein of MdACS6 will also be detected by Western blot assay in different developmental stage and during storage in air. An assay of Agrobacterium infiltration will be carried out on unripe apple fruit to verify if over-expression of MdACS6 is able to activate system 2 ethylene synthesis. In addition, MdACS6 will be introduced into tomato to further determine its function in ethylene synthesis and fruit ripening. The predicted results of this proposal will be greatly helpful in clarifying the role of MdACS6 in regulating apple fruit ripening and in integrating the research on fruit ripening, as well as in supporting the technology of fruit storage.

植物体中乙烯合成分为系统1和系统2。ACC合成酶（ACS）是乙烯信号途径中调控呼吸跃变型果实成熟的关键酶，从苹果全基因组数据库中获得果实成熟相关的新ACS基因，命名为MdACS6。MdACS6从果实采收前60天开始表达，并在室温贮藏过程中逐渐减弱，初步推断MdACS6可能作用于系统1乙烯合成。本项目在此基础上，首先通过RACE法克隆MdACS6基因的全长序列，明确其组织特异性和时空表达情况，结合MdACS6在室温贮藏和1-MCP（乙烯抑制剂）及乙烯处理的苹果果实中的表达情况，确定其是否为乙烯合成系统。通过Western blot 检测其在果实不同发育阶段的蛋白水平，通过农杆菌注射实验在未成熟苹果果实中过表达MdACS6以及番茄转基因实验，研究MdACS6在苹果果实乙烯合成系统1和系统2中的功能；阐明MdACS6在调控苹果果实乙烯合成和成熟中的作用，完善果实成熟理论，为果实贮藏提供理论依据。

植物激素乙烯与果实的发育成熟及贮藏有着必然的联系。在乙烯合成通路中，SAM在ACC合成酶（ACS）的作用下生成ACC，此步是乙烯合成途径中的限速步骤，ACS是限速酶。目前对苹果果实中的ACS基因的研究主要集中在果实贮藏过程中，而在果实发育过程中起作用的ACS基因尚未报到。我们分析鉴定了一个新的苹果ACS基因（MdACS6），其主要作用于果实发育过程，负责该时期微量乙烯的合成。通过农杆菌注射方法分别过表达和沉默MdACS6后发现，MdACS6过表达果实乙烯产量显著增加，并且诱导了在果实转熟过程中起作用的MdACS3a的表达；而沉默MdACS6的果实乙烯生产量显著降低。通过酵母单杂交实验和GUS报告基因实验发现，乙烯应答转录因子MdERF2可以结合MdACS3a的启动子并正调控其表达，并且该转录因子受乙烯调控。我们认为MdACS6主要催化合成了苹果幼果期的乙烯，该过程中的乙烯促进了MdERF2的表达，MdERF2作为转录因子通过转录调控启动了MdACS3a的表达，进而使果实进入转熟期。