大豆GmCBL基因的eQTL分析及其功能标记开发

Soybean（Glycine max (L.)Merr.）is the important plant protein sources for human diets and livestock feedings. Although soybean protein is rich in most essential amino acids for human, its nutritional quality is limited by a low concentration of sulfur containing amino acids, methionine and cysteine. GmCBL gene codes for the key enzyme involved both in the penultimate step reaction of methionine biosynthesis pathway and in cysteine biosynthesis pathway. Therefore, it plays an important role in sulfur amino acid biosynthesis. Soybean GmCBL gene has been cloned in the previous work of this project.It was found that its overexpression can improve methionine content in transgenic plants. On this basis,this study will detect expression quantitative trait loci (eQTL) for GmCBL gene and quantitative trait loci (QTL) for sulfur containing amino acids using recombinant inbred lines population and explore mechanism for the regulation of GmCBL gene expression. Then, we will analyze correlations between eQTL for GmCBL gene and QTL for sulfur containing amino acids and determine the key eQTL interval. Finally, through the association analysis on the key eQTL interval in natural population, we will detect the optimal haplotype and develop functional markers closely linked with GmCBL gene expression. These results could provide a new train of thought for the breeding of high sulfur containing amino acids soybean varieties by regulating expressing of sulfur containing amino acids genes.

大豆是人类饮食和畜禽饲养重要的植物蛋白来源。尽管大豆蛋白富含大部分人体必需氨基酸，但含硫氨基酸（甲硫氨酸和半胱氨酸）含量低，限制其营养价值。GmCBL基因是甲硫氨酸生物合成路径中参与倒数第二步反应的关键酶基因，也是半胱氨酸代谢途径中的关键酶基因，在含硫氨基酸代谢途径中发挥非常重要的作用。本项目前期工作已克隆了大豆GmCBL基因，发现其过量表达确能提高转基因植物的甲硫氨酸含量。在此基础上，本研究利用大豆重组自交系群体鉴定GmCBL基因的eQTL和含硫氨基酸相关的QTL，探讨GmCBL基因表达调控机理；分析GmCBL基因的eQTL与含硫氨基酸QTL的相关性，确定关键的eQTL区间；并在大豆自然群体中对关键的eQTL区间进行关联分析，确定最优单倍型，开发与GmCBL基因表达水平紧密相关的功能标记。研究结果可为实现大豆高含硫氨基酸新品种的培育提供新的思路。

大豆的含硫氨基酸（甲硫氨酸Met和半胱氨酸Cys）含量低，限制了大豆的营养价值。GmCBL基因在含硫氨基酸代谢途径中发挥重要的作用。本项目利用氨基酸分析仪和近红外仪器测定大豆重组自交系群体NJRIKY（184个家系）种子含硫氨基酸（Met、Cys和总含硫氨基酸SAA）和蛋白（PC）含量，采用实时荧光定量RT-PCR方法测定重组自交系群体和自然群体262分材料GmCBL基因表达量，获得表型数据。统计分析结果表明Met/PC、Cys/PC、SAA/PC、PC和GmCBL基因表达量在两个群体中均存在广泛的变异，GmCBL基因表达量与Met/PC和Cys/PC呈显著和极显著的正相关，和PC呈极显著的负相关，与SAA/PC不相关。利用大豆RIL群体的遗传图谱和表型数据，鉴定到含硫氨基酸相关的QTL28个、蛋白相关的QTL 7个和GmCBL基因表达量的eQTL9个，其中GmCBL基因表达量的eqCBL-3-1、7个含硫氨基酸相关的QTL 和5个蛋白相关的QTL在两个环境条件下可以检测到。通过分析GmCBL基因的eQTL与含硫氨基酸QTL的相关性，确定2个关键的eQTL区间：3号染色体CAPS -satt312标记区间和20号染色体sat_419-satt726标记区间；在大豆自然群体中利用关联作图进行eQTL分析，检测到与GmCBL基因表达水平紧密相关的SNP标记60个，其中3个环境条件下稳定检测到的6个SNP标记AX-93698355、AX-93698360、.AX-93698361、AX-93698362、AX-94274378和AX-94280613均在3号染色体关键区间内；除AX-93698361以外，对其余5个SNP标记进行优异等位变异分析，挖掘出稳定表达的标记位点的优异等位变异及其代表性载体材料为NJAU_C043（2013），NJAU_C246（2014）以及NJAU_C169（2013和2014两年联合分析）。以上研究结果可为实现大豆高含硫氨基酸新品种的培育提供新的基因资源。