野生毛花猕猴桃酚类物质积累差异及合成相关基因表达与功能分析

Actinidia eriantha (A. eriantha Benth) is a wild and potential berry fruit resource in our country. However, the fruit of A. eriantha usually tastes astringent and its flavor quality is poor with rich phenolic compounds in fruit. Thus, there is no commercial cultivation of A. eriantha as far. The A. eriantha nonastringent line 'MM-13' and astringent line 'MM-16', which were found in Jiangxi province and differed in contents of total phenolic compounds of fruit, were selected as materials. They located in closed area with high genetic similarity coefficient. During the fruit development, the variation of each phenolic component content will be detected using liquid chromatography-mass spectrometry (HPLC-MS). The 'MM-13'and 'MM-16' fruits at differnet stages with significant difference in phenolic compounds contents will be collected to analyze the transcriptome using the Illumina high-throughput sequencing method. Furthermore, the phenolic compounds synthesis pathways of other species will be referenced to screen differentially expressed genes (DGEs). Using Real-time PCR technology, the accuracy of DGEs will be verified and their expression levels will be detected during the fruit development. Then, the genetic transformation will be used to identify DGEs function to screen the key genes controlling the phenolic compounds synthesis. This study aims to lay the foundation for revealing the molecular mechanism of phenolic compounds synthesis and to provide a theoretical basis for ameliorating the astringent flavor of A. eriantha fruit.

毛花猕猴桃(Actinidia eriantha Benth)是我国特有的极具开发潜力的野生浆果资源，但因其果实酚类物质含量变异丰富，鲜食口感普遍带涩，目前尚无商业化栽培。本研究拟以江西境内发掘的果实总酚含量差异显著、地理分布位置近、遗传相似性系数高的野生毛花猕猴桃无涩型优株'MM-13'和涩型单株'MM-16'为试材，采用液质联用(HPLC-MS)技术对其果实发育进程中酚类物质组分及含量动态变化与积累差异进行检测分析。进而对积累差异时期的果实进行Illumina高通量转录组测序，并参考其它物种酚类物质合成代谢途径，筛选出差异表达候选基因。应用Real-time PCR技术验证差异表达基因的准确性并检测其在果实发育进程中的表达水平，经转基因鉴定其功能，明确酚类物质合成相关的关键基因。本研究为揭示毛花猕猴桃果实酚类物质积累的分子机制奠定基础，亦为毛花猕猴桃果实口感呈涩的合理调控提供理论依据。

毛花猕猴桃（Actinidia eriantha Benth）富含抗坏血酸、叶酸、多酚等营养物质，且具有较强的抗病性和耐热性，是我国特有的极具开发潜力的野生浆果资源。植物多酚为植物体内次生代谢产物，具有抗氧化、抗自由基等特性，更能直接影响果品的色泽、口感、硬度、风味以及贮藏加工等方面的特性。本项目以毛花猕猴桃‘赣猕6号’为试材，通过响应面分析法对毛花猕猴桃果实酚类物质提取的主要因素进行了优化，确定最佳的提取工艺条件为：丙酮浓度为51%、提取温度为47℃、提取时间为43 min、液料比为7:1。在此条件下毛花猕猴桃果实总酚提取量达到519.24 mg/100 g。对果实生长发育过程中的总酚、类黄酮含量和抗氧化活性能力的动态变化测定发现，‘赣猕6号’果实生长发育期间总酚含量极显著（p<0.01）高于其对照品种‘红阳’与‘金魁’，最高含量高达1112.26 mg/100 g，最低也有466.06 mg/100 g，成熟期为568.20 mg/100 g，其对照品种‘红阳’与‘金魁’最高时分别为214.33 mg/100 g、245.76 mg/100 g。果实总酚、类黄酮与抗氧化活性之间存在显著的相关性，说明总酚与类黄酮物质对毛花猕猴桃抗氧化活性起到至关重要的作用。通过HPLC测定分析了猕猴桃果实生长发育过程中酚类物质相关组分（没食子酸、儿茶素、对羟基苯甲酸、表儿茶素、咖啡酸、香草酸、芦丁、阿魏酸、槲皮素）含量，‘赣猕6号’果实在各阶段发育期间，酚类物质组分均极显著（p<0.01）高于对照‘金魁’与‘红阳’；‘赣猕6号’果实酚类物质以没食子酸为主，最高达620.55 mg/100 g，最低为109.13 mg/100 g；没食子酸、芦丁、表儿茶素、咖啡酸、槲皮素这五种组分是毛花猕猴桃果实各发育期间含量较高的酚类物质。经代谢组学检测分析，毛花猕猴桃果实生长发育期间大量的差异代谢物存在于类黄酮、异黄酮、花青苷、黄酮和黄酮醇等代谢合成路径中。本项目结果为揭示毛花猕猴桃果实酚类物质积聚的分子机制研究奠定良好基础，亦为毛花猕猴桃种质创新和新品种选育提供新的思路和理论依据。