调控大豆种子蛋白/油脂含量关键基因的克隆及功能分析

Soybean (Glycine max) seed protein content and oil content, generally negatively correlated, dominants the quality and economic value of soybean. Although pathways for plant protein synthesis and lipid production have already been described, the relative allocation of assimilate to these pathways are unclear. So far, few key genes controlling the content ratio of protein to oil have been reported in soybean. Using genetically recombined inbred lines, we constructed a population of remaining heterozygous lines. With QTL analysis on soybean protein and oil content, we obtained a key regulatory locus PO1 (Protein and Oil 1), which is also a hot spot for protein and oil content studies. We further narrowed the PO1 locus to a 283kb regime. Candidate gene responsible for seed protein and oil content encoding PO1 locus may play an important role in the distribution of protein and oil. Based on our previous work, in this project, we try to clone the candidate gene encoding PO1 locus and reveal the underlying mechanism for its regulation on these seed storage reserve. This research aims to advance our understanding in the regulation network for relative biosynthesis of seed protein and oil in soybean, and provide favorable genetic resources for soybean molecular breeding.

大豆（Glycine max）种子蛋白和油脂含量的是决定大豆品质和经济价值的关键因素，二者成负相关。虽然植物合成蛋白和油脂的途径已有一定阐述；然而，关于途径的整合及蛋白油脂含量比例的分配机制并不清楚，大豆中控制蛋白油脂含量的关键基因还鲜有报道。我们前期工作利用大豆遗传重组自交系构建了剩余杂合系群体，对大豆蛋白含量进行QTL分析，获得一个重要调控位点PO1（Protein and Oil 1），该位点同时也是蛋白油脂含量研究的热门位点，通过进一步的精细定位我们将PO1位点缩小到283kb区间内，该区间内存在编码PO1位点的候选基因，在大豆种子蛋白和油脂含量分配中扮演重要角色。在此基础上，本项目将克隆编码PO1位点的候选基因，并对其调控大豆种子蛋白和油脂含量功能进行分析。本项目的研究将促进对大豆种子蛋白和油脂调控机理的深入认知，同时为大豆分子育种提供有利基因资源。

蛋白与油脂含量是决定大豆品质的关键性状，二者由不同因子通过多种途径协同调控，而这些关键调控因子及其如何调控种子蛋白和油脂含量的具体机制仍不明确，这些重要科学问题严重限制了分子育种的开展和我国大豆品质的全面提高。我们前期工作利用重测序群体和杂交遗传群体，在第20号染色体同时检测到调控蛋白油脂含量的重要位点，在此基础上，本项目主要研究内容为：（1）RHL群体精细定位和后代表型检测，将候选基因PO1（Protein and Oil 1）缩小到146 kb区间内；（2）转基因互补和近等基因系验证PO1基因功能；（3）PO1基因时空表达模式分析；（4）重测序群体PO1自然变异分析与分子标记开发利用。研究结果表明，与野生型大豆亲本相比，栽培大豆TK780基因组中PO1编码区InDel插入导致蛋白含量降低，油脂含量升高；转基因回补和近等基因系分析表明PO1正调控油脂含量，负调控蛋白含量；PO1在种皮和胚乳中特异表达，蛋白定位在细胞核中；该自然变异在栽培大豆中普遍存在，可能是大豆油分含量重要驯化位点；利用突变位点设计分子标记，我们对主栽品种进行了分子遗传改良，BC3F2代材料显著提高了栽培大豆黑河43种子蛋白含量。以上研究克隆并分子验证了大豆蛋白油脂含量重要调控基因PO1，为大豆高蛋白分子育种提供有利基因资源。同时，PO1的基因功能分析将促进对大豆种子蛋白和油脂调控机理的深入认知。