Tuteja Jigyasa H, Clough Steven J, Chan Wan-Ching, Vodkin Lila O
Program in Physiological and Molecular Plant Biology, University of Illinois, Urbana 61801, USA.
Plant Cell. 2004 Apr;16(4):819-35. doi: 10.1105/tpc.021352.
Chalcone synthase, a key regulatory enzyme in the flavonoid pathway, constitutes an eight-member gene family in Glycine max (soybean). Three of the chalcone synthase (CHS) gene family members are arranged as inverted repeats in a 10-kb region, corresponding to the I locus (inhibitor). Spontaneous mutations of a dominant allele (I or i(i)) to a recessive allele (i) have been shown to delete promoter sequences, paradoxically increasing total CHS transcript levels and resulting in black seed coats. However, it is not known which of the gene family members contribute toward pigmentation and how this locus affects CHS expression in other tissues. We investigated the unusual nature of the I locus using four pairs of isogenic lines differing with respect to alleles of the I locus. RNA gel blots using a generic open reading frame CHS probe detected similar CHS transcript levels in stems, roots, leaves, young pods, and cotyledons of the yellow and black isolines but not in the seed coats, which is consistent with the dominant I and i(i) alleles mediating CHS gene silencing in a tissue-specific manner. Using real-time RT-PCR, a variable pattern of expression of CHS genes in different tissues was demonstrated. However, increase in pigmentation in the black seed coats was associated with release of the silencing effect specifically on CHS7/CHS8, which occurred at all stages of seed coat development. These expression changes were linked to structural changes taking place at the I locus, shown to encompass a much wider region of at least 27 kb, comprising two identical 10.91-kb stretches of CHS gene duplications. The suppressive effect of this 27-kb I locus in a specific tissue of the G. max plant represents a unique endogenous gene silencing mechanism.
查尔酮合酶是类黄酮途径中的关键调控酶，在大豆中构成一个由八个成员组成的基因家族。查尔酮合酶（CHS）基因家族的三个成员在一个10 kb的区域内以反向重复的形式排列，对应于I位点（抑制剂）。已表明显性等位基因（I或i(i)）自发突变为隐性等位基因（i）会删除启动子序列，反常地增加CHS转录本的总水平，并导致黑色种皮。然而，尚不清楚基因家族的哪些成员对色素沉着有贡献，以及该位点如何影响CHS在其他组织中的表达。我们使用四对在I位点等位基因上存在差异的近等基因系，研究了I位点的异常性质。使用通用开放阅读框CHS探针进行的RNA凝胶印迹检测到，黄色和黑色近等基因系的茎、根、叶、幼荚和子叶中CHS转录本水平相似，但种皮中不同，这与显性I和i(i)等位基因以组织特异性方式介导CHS基因沉默一致。使用实时RT-PCR，证明了CHS基因在不同组织中的表达模式可变。然而，黑色种皮中色素沉着的增加与对CHS7/CHS8沉默效应的释放有关，这种释放发生在种皮发育的所有阶段。这些表达变化与I位点发生的结构变化有关，I位点显示包含至少27 kb的更广泛区域，由两个相同的10.91 kb的CHS基因重复片段组成。这个27 kb的I位点在大豆植株特定组织中的抑制作用代表了一种独特的内源基因沉默机制。
