西瓜果实光合产物卸载中碱性α-半乳糖苷酶基因作用的进化分子机制

Phloem unloading of photosynthesis from source to sink is the precondition for sugar accumulation. Only about 5% of the plants such as watermelon and other cucurbitaceae crops, transport raffinose family oligosaccharides (RFOs) as the main sugars in phloem, however, sucrose (disaccharide) is major sugar for fruit storage. This project intends to reveal the unknown RFOs unloading and regulation mechanism. We firstly identified the phenomenon of RFOs "retention" in wild low sugar content watermelon fruit. Then we found that the RFOs are hydrolyzed in fruit vascular. Using genome-wide association analysis (GWAS), we identified the alkaline alpha galactosidase 1 (ClAGA1) is the causal gene for raffinose content. In this project, we will complete transcriptional expression, sub cellular localization and hybridization in situ of ClAGA1 to analyze the spatio-temporal expression pattern. DNA pull down, EMSA (electrophoretic mobility shift assay), LUC (luciferase) reporter and ChIP (chromatin immunoprecipitation) techniques will be performed to analyze the regulating transcription factor of ClAGA1. Haploid, structural variation and domestication of ClAGA1 and its regulator will be studied. We will carry out over expression and CRISPR/Cas9 knock out ClAGA1 in transgenic watermelon to reveal the mechanism of ClAGA1 in photosynthesis distribution. Co-expression network of ClAGA1 will be analyzed by transcriptomics and metabolomics. It will enrich and improve the theory of plant evolution involving fruit photosynthesis distribution.

光合产物从“源”运输并卸载到“库”是糖分积累的前提，约5%的植物如西瓜等葫芦科作物，以棉籽糖系列寡糖（RFOs）作为光合产物运输的主要糖分形式，而果实储存糖分则以蔗糖(二糖)为主，有关RFOs在果实中卸载的分子机制尚不十分清楚。本项目前期研究首次发现：野生低糖西瓜果实存在RFOs“滞留”现象，果实韧皮部是西瓜RFOs分解部位，GWAS与进化选择分析发现碱性α-半乳糖苷酶ClAGA1是控制果实棉籽糖含量主效基因。本项目拟利用转录表达、亚细胞定位、组织原位杂交确定ClAGA1的时空表达；利用DNA pull down、EMSA、LUC报告基因和ChIP等技术明确调控ClAGA1表达的转录因子及调控网络；利用过表达和CRISPR/Cas9敲除ClAGA1转基因验证其在光合产物分配的作用；解析ClAGA1及其调控基因的单倍型、结构变异及进化驯化机制。这将丰富与完善植物果实光合产物分配的进化理论。

葫芦科植物是以棉子糖系列寡糖作为维管束光合产物的主要运输形式，但棉子糖系列寡糖在葫芦科作物怎样被水解和分配利用尚不清楚。野性西瓜和栽培西瓜相互嫁接实验，显示野生不甜西瓜果实中普遍存在棉子糖的“滞留”，而栽培甜西瓜果实中棉子糖被迅速水解含量极低。通过全基因组关联分析我们发现碱性α-半乳糖甘酶ClAGA2(alkaline alpha-galactosidase)是控制西瓜果实棉子糖、水苏糖分解的关键酶。原位杂交显示ClAGA2特异性在果实维管束表达。转基因证明ClAGA2可控制西瓜果实棉子糖水解和糖含量。转录因子ClNF-YC2(nuclear transcription factor Y subunit C)调控ClAGA2表达，ClAGA2启动子区间ClNF-YC2识别位点内的2个关键SNP位点是决定其在栽培和野生材料中表达差异的关键位点。