基于启动子序列变异和DNA甲基化分析的红梨花青苷合成相关基因转录调控机制研究

Red pears belonging to different species with different genetic backgrounds and coloration patterns may have the distinct transcriptional regulation for related anthocyanin biosynthesis genes compared with apples and grape. However, more attention was payed on the isolation, expression and function analysis of related genes in the study on molecular mechanism of coloration in red pears, few have carried out on the mechanism of transcriptional regulation. What's more, the limited and single material in the previous studies related to red pears would not be favorable to the comprehensive understanding of molecular regulation mechanism of different kinds of red pears. Therefore, in this project, typical red pears including Oriental pear, Occidental pear and some special types with different coloration patterns will be used to study transcriptional regulation for related anthocyanin biosynthesis genes. The promoter region will be isolated for analyzing the correlation between the sequence variations in the promoter region and peel phenotypes of red pears. The dual luciferase assay, promoter deletion analysis, electrophoretic mobility shift assay, yeast two-hybrid assay, transgenic technology will be further used for the analysis of their function. Methylation levels of these genes will also be investigated to understand molecular mechanism for coloration unrelated with the sequence variations in the promoter region. The research will be of help to clarify the regulation mechanism of anthocyanin biosynthesis in red pears, and also to enrich the theory of molecular mechanism of fruit anthocyanin biosynthesis.

红梨着色模式多样，现有红梨着色机制的研究多集中在花青苷合成相关基因克隆，表达分析和功能鉴定的传统思路上，对这些基因转录调控机制研究极为薄弱，所涉及的研究材料也相对单一，难以从全局上认识不同着色类型红梨着色的分子调控机制。本研究从启动子序列变异和DNA甲基化分析两个角度系统研究红梨花青苷合成相关基因转录调控机制，拟以包括东方梨、西方梨和特殊类型等具有不同着色模式的典型红与非红梨为试材，分离其花青苷合成相关基因编码区和启动子，并对这些基因序列变异与色泽变异进行关联分析，找出与色泽变异相关的序列变异，再利用双荧光素酶检测、启动子删截、酵母双杂交、凝胶阻滞分析和转基因等实验手段对该序列进行功能鉴定分析，对于无法从基因序列变异角度解释的色泽变异现象，则对其进行DNA甲基化水平检测。研究成果有助于阐明红梨花青苷合成调控机制，同时可进一步丰富果实花青苷生物合成的分子机制的理论。

本项目以绿皮梨‘早酥’及其红色芽变‘早酥红’，红色砂梨品种‘美人酥’和‘满天红’和红色西洋梨品种‘凯斯凯德’为主要试材，探讨了DNA甲基化和miRNA对红梨着色调控的相关机制及光和茉莉酸甲酯（MJ）对红梨着色的影响，结果表明：‘早酥红’各组织的花青苷含量显著高于‘早酥’，且‘早酥红’条纹着色成熟果中红条纹的花青苷含量高于绿条纹。基因表达结果显示，PpUFGT2 和PyMYB10的表达则与花青苷的积累呈现显著的正相关，但是不同材料上PpUFGT2 和PyMYB10基因编码区和启动子区域均未产生序列变异。对不同材料上PyMYB10基因启动子区域的甲基化水平进行分析发现，红色材料上的甲基化水平要显著低于绿色材料。去袋过程中，‘美人酥’果皮花青苷进行积累，花青苷合成相关结构基因和转录因子表达上调，通过sRNA和降解组测序，发现在梨果皮中表达的27个miR156家族成员，并发现4个可以被miR156降解的SPL基因。通过梨全基因组SPL家族成员鉴定，发现19个PpSPL成员，其中11个成员拥有miR156的剪切位点。在‘美人酥’果皮着色过程中，miR156表达上调，而PpSPL基因中，PpSPL2、PpSPL5、PpSPL7、PpSPL9、PpSPL10、PpSPL13、PpSPL16、PpSPL17和PpSPL18 的表达下调。酵母双杂结果显示，梨上调控花青苷合成的3类转录因子可以形成MYB/bHLH/WD40转录因子复合体，并且PpSPL10和PpSPL13与PyMYB10存在互作。红色砂梨品种‘满天红’和红色西洋梨品种‘凯斯凯德’对采前套袋和采后紫外光/可见光诱导处理同样具有不同的响应模式，PpCHS3、PpCHI3、PpUFGT1和PyMYB10等4个基因的转录与花青苷的积累呈显著正相关。MJ处理显著提高了红色砂梨‘满天红’果皮中PpCHS2、PpCHI3、PpF3H、PpANS、PpDFR1、PpUFGT1、PpUFGT2、PyMYB10、PpbHLH和PpWD40的表达水平，从而促进其果皮中花青苷的积累。研究结果丰富了梨果实花青苷合成的分子机制，对于指导优质红梨生产的着色调控提供了理论基础。