Molecular mechanisms of the generation of pigmented seeds from yellow soybean plants

大多数商业大豆品种的种子都是黄色的，因为种皮中的色素缺失。I基因抑制了整个种皮的色素沉着，导致成熟收获的种子呈均匀的黄色。我们先前已经证明，I基因抑制种皮着色是查尔酮合成酶(CHS)基因转录后基因沉默(PTGS)的结果。对I基因的结构及其诱导CHS基因PTGS的机制知之甚少。在本研究中，我们确定了I基因的候选基因GmIRCHS，它由一个含有启动子区域的DNAJ类基因的5‘端部分和一个1.1kb截短的CHS3序列(5’-ΔCHS3和3‘-ΔCHS3)组成。RT-PCRS检测到5‘-ΔCHS3到3’-ΔCHS3的通读产物和ΔCHS3的dsRNA区域的存在，表明ΔCHS3的dsRNA可以从GmIRCHS转录而来，并能诱导CHS基因的PTG。此外，在I改为I的大豆突变体中，GmIRCHS的ΔCHS3的IR结构丢失，支持GmIRCHS是I基因的结论。最后，为了分析GmIRCHS基因的表达模式，我们构建了GmIRCHS启动子和β-葡萄糖苷酶(GUS)基因融合表达的转基因拟南芥。在种皮中没有观察到强烈的染色，这表明GmIRCHS在种皮组织中不是特异表达的。

Most commercial soybean varieties have yellow seeds due to loss of pigmentation in the seed coat. The I gene inhibits pigmentation over the entire seed coat, resulting in a uniform yellow color of mature harvested seeds. We previously demonstrated that the inhibition of seed coat pigmentation by the I gene results from post-transcriptional gene silencing (PTGS) of chalcone synthase (CHS) genes. Little is known about the structure of the I gene and the mechanism by which it induces PTGS of CHS genes. In this study, we identified a candidate of the I gene, GmIRCHS, which consists of a 5'-portion of a DnaJ-like gene containing a promoter region and a perfect inverted repeat (IR) of 1.1-kb truncated CHS3 sequences (5'-ΔCHS3 and 3'-ΔCHS3). RT-PCRs indicated the existence of the read-through product from 5'-ΔCHS3 to 3'-ΔCHS3 and the dsRNA region of ΔCHS3, suggesting that dsRNA of ΔCHS3 could be transcribed from GmIRCHS and could induce PTGS of CHS genes. Moreover, the IR structure of ΔCHS3 in GmIRCHS was lost in the soybean mutants in which I was changed to i, supporting the conclusion that GmIRCHS is the I gene. Finally, to analyze the expression pattern of GmIRCHS, we constructed transgenic Arabidopsis expressing a reporter gene fusion of the GmIRCHS promoter and the β-glucronidase (GUS) gene. Intense staining was not observed in the seed coat, indicating that GmIRCHS is not specifically expressed in the seed coat tissues.