‘黄花’梨及其绿皮芽变果皮色泽发育相关基因克隆与功能研究

Study on genes involved in russet or green fruit traits and their functions is very significant for molecular breeding of fruit color on sand pear (Pyrus pyrufolia Nakai), and Pyrus pyrufolia Nakai 'Huanghua' and its green skin bud sport (tentatively named as 'Green Huanghua') provide perfect material for carrying out this research. We have found some differentially expressed genes between 'Huanghua' and 'Green Huanghua' fruit peels, including phenylalanine ammonia lyase gene (PAL), cinnamic acid 4-hydroxylase gene (C4H) and cinnamoyl-CoA reductase gene (CCR), which suggest expression level of genes involved in phenylpropane metabolism is changed between the two materials. In order to further study genes related to russet and green fruit traits, in this project we will clone genes related to fruit color development from 'Huanghua' and 'Green Huanghua' pear fruit and study their roles in fruit color formation. On the one hand, comprehensive analysis of differentially expressed genes between 'Huanghua' and 'Green Huanghua' fruit peel at the key developmental stage of fruit color would be performed by transcriptome sequencing; On the other hand, GC-MS is used to analyze and identify the different metabolites between peel epidermises of the two pear fruits, which are used to select the key genes involving in fruit color development from the above differentially expressed genes. Based on the candidate genes which might identify fruit color, their full length cDNAs would be cloned and used to construct sense or antisense plant expressing vector, and then the roles of these genes in fruit color development would be studied by agro-injection technique. In addition, the selected important genes would be transferred into tomato to further reveal their functions. Exploring genes controlling russet or green skin development and their functions to provide important functional genes for molecular breeding of pear fruit color.

砂梨果实褐皮和绿皮性状相关基因克隆及其功能研究对果皮色泽分子育种具有重要意义，'黄花'梨及其绿皮芽变'绿黄花'为该研究提供了理想的试材。我们已找出了该对材料一些差异表达基因，如PAL、C4H、CCR，至少是苯丙烷代谢相关基因表达发生了改变。为了进一步研究褐皮和绿皮性状相关基因及其功能，本项目以克隆果皮色泽发育相关基因及其在色泽发育过程中的功能为主要研究内容，一方面通过转录组测序对果皮色泽发育关键期差异表达基因进行更全面的分析；另一方面利用GC-MS技术，分析果皮表皮差异代谢产物并与差异表达的基因进行比照分析确定出候选基因，克隆基因全长序列，构建其正义/反义表达载体，利用农杆菌微注射基因瞬时表达技术研究这些基因在果皮色泽发育过程中的作用，同时筛选重要的基因转化番茄深入研究其功能。预期结果是探明控制褐皮/绿皮发育的关键基因及其作用，为果皮色泽分子育种提供重要的功能基因。

砂梨果实褐皮和绿皮性状差异基因的鉴定、相关基因的克隆及其功能研究，对果皮色泽分子育种具有重要意义。本研究主要对‘黄花’梨及其绿皮芽变‘绿黄花’果皮形态学、生理生化及其代谢组差异、转色关键期差异基因的筛选及其表达分析、砂梨果皮褐色性状形成的相关基因功能验证分析、梨SSR和SNP分子标记开发等五方面进行了研究，主要研究结果如下：.1、通过对‘黄花’梨及其绿皮芽变‘绿黄花’果实的果点发育、不同发育时期的果皮表面结构和表皮膜特点、成熟梨果皮结构的比较研究，发现‘黄花’梨果点形成早而集中，果点发育快，花后10周后果面蜡质减少，果点周缘出现裂口，角质膜逐渐破裂，直至完全龟裂，黄褐色表皮膜为果皮颜色的决定者；而‘绿黄花’梨果点形成晚，果点发育慢，花后10周蜡质一直存在，角质膜从幼果到果实成熟都保持完整，表皮膜无色透明，不决定果皮色泽。.2、‘黄花’高于‘绿黄花’木质素和酚类物质的含量，果皮高于果肉；两种材料果皮代谢产物差异成分主要为蜡质、角质组分及苯丙烷类代谢产物，说明苯丙烷代谢途径在‘黄花’果皮褐色性状形成中发挥作用；‘黄花’梨果皮蜡质和木栓质成分中单体含量高，表明脂肪酸同样参与木栓质形成；‘黄花’梨果实套袋和未套袋果皮代谢物差异物质分析表明一些脂肪酸类、酚类和苯丙烷类等代谢产物可能参与了‘黄花’梨果皮褐色物质的形成。.3、通过DDRT-PCR法找到了‘黄花’与‘绿黄花’差异表达基因PpyDFN1、PpyPAL1、PpyC4H和PpyCCR，通过转录组分析法，从注释到GO数据库中的41,137个Unigene中找到6,408个差异显著基因，从KEGG代谢通路中的29,961条Unigene中筛选出1,441条差异表达基因，其中678个参与96个代谢或信号通路，分别有55个和33个参与到“苯丙烷类生物合成”和“角质、木栓质和蜡质生物合成”代谢途径中。.4、克隆了砂梨防御素基因PpyDFN1（登录号HM044853）、苯丙氨酸解氨酶基因PpyPAL1、促进组织木栓化的ω-羟基棕榈酸O-阿魏酰转移酶基因PpyHHT。.5、利用‘黄花’果实发育期果皮转录组数据开发了4,300对SSR分子标记，获得162,048个SNP位点，利用梨杂交群体的转录组数据开发的了332对SSR分子标记。