Analysis and application of regulatory genes controlling anthocyanin accumulation in rice

水稻（Oryza sativa L.）影响各种植物组织中花青素苷生物合成的调节。花色苷色素沉着的组织特异性模式以及同线性关系表明，水稻Pl基因座可能在花色苷途径中发挥类似于玉米R/B基因座的作用。我们分离了两个cDNA OSB 1和OSB 2，它们与玉米R基因家族中发现的碱性螺旋-环-螺旋（bHLH）蛋白具有显著的同一性。对相应基因组区域的表征表明，与OSB 2的5 '部分相同的序列存在于OSB 1编码区下游约10-kb处。OSB 2缺乏保守的C-末端结构域。利用F_2群体的RFLP分析表明，这两个基因与紫色叶表型共分离。为了检查这些cDNA是否真的促进了体内花青素苷的积累，我们开发了一种瞬时互补测定法，通过该测定法，我们表明花青素苷途径是由OSB 1或OSB 2诱导的。这些 ...更多信息 结果表明，Pl^w等位基因可能是复杂的，由至少两个编码bHLH蛋白的基因组成。由此产生的转基因植物在营养组织中表现出花青素积累。进一步的研究表明，花色素苷也存在于种子组织中，但仅限于种皮和果皮，而不积累在胚和胚乳中。推测胚和胚乳中的花青素途径可能需要其他调控成分的参与，除了紫叶外，还有其他基因位点控制着水稻的花青素色素形成。一个例子是基于水稻和玉米基因组之间的同线性，已被建议对应于DFR基因的Activator（A）。为了检验这一点，我们采用了我们的瞬时测定，并表明，花青素色素是诱导时，DFR与OSB 1和OSB 2一起引入。结果表明，A编码DFR。少

The Purple leaf (Pl) locus of rice (Oryza sativa L.) affects regulation of anthocyanin biosynthesis in various plant tissues. The tissue-specific patterns of anthocyanin pigmentation, together with the syntenic relationship, indicate that the rice Pl locus may play a role in the anthocyanin pathway similar to the maize R/B loci. We isolated two cDNAs OSB1 and OSB2 showing significant identity to the basic helix-loop-helix (bHLH) proteins found in the maize R gene family. Characterization of the corresponding genomic region revealed that the sequence identical to a 5'-portion of OSB2 existed 〜10-kb downstream of the OSB1 coding region. OSB2 lacks a conserved C-terminal domain. RFLP analyses using an F_2 population indicate that both genes co-segregate with the purple leaf phenotype. To examine if these cDNAs actually promote anthocyanin accumulation in vivo, we developed a transient complementation assay by which we showed that the anthocyanin pathway is inducible by OSB1 or OSB2. These … More results suggest that the Pl^w allele may be complex and composed of at least two genes encoding bHLH proteins.We introduced OSB1 and OSB2 in rice plants under the control of a constitutive CaMV 35S promoter. The resulting trangenic plants showed anthocyanin accumulation in vegetative tissues. Further examination revealed that anthocyanin is also present in seed tissues, but it is limited to seed coat and pericarp and not accumulated in embryo and endosperm. It is postulated that other regulatory components may be required for inducing anthocyanin pathway in embryo and endosperm.In addition to Purple leaf, there are other loci genetically known to control anthocyanin pigmentation in rice. An example is Activator (A) that has been suggested to correspond to DFR gene, based on the synteny between rice and maize genomes. To examine this, we employed our transient assay and showed that anthocyanin pigments were inducible when DFR was introduced together with OSB1 and OSB2. The result indicated that A encodes DFR. Less