水稻POR基因的分离、定位与功能的初步研究

Preliminary Study on Isolation, Mapping, and Function of NADPH: Protochlorophyllide Oxidoreductase(POR) Gene in Rice(Oryza sativa)

本研究发现水稻ygl3(yellow green leaf3)突变体苗期的叶片呈黄绿色;在营养生长后期,ygl3叶片从叶尖开始严重褪色,形成黄斑或白斑。图位克隆结果表明,YGL3编码一个定位于叶绿体的原叶绿素酸酯氧化还原酶B(OsPORB),转基因互补实验证实了图位克隆的结果。水稻基因组中存在两个POR基因Os PORA和OsPORB。RT-PCR分析表明,Os PORA主要在新生的茎、叶和穗中表达,而OsPORB为组成型表达。亚细胞定位分析发现Os PORA和OsPORB为叶绿体定位蛋白。此外,用35S启动子驱动Os PORA表达能够完全互补ygl3的表型。虽然利用RNAi技术抑制Os PORA表达的转基因水稻叶片与野生型呈现一样的表型,但在ygl3背景下,抑制Os PORA的表达导致转基因水稻黄化致死,说明Os PORA和O s PORB为叶绿素合成所必需的。以上研究结果说明Os PORA和OsPORB在功能上具有一定的冗余性,但OsPORB比Os PORA的作用更重要,揭示了Os POR在叶绿素合成途径上保守性。

In this study, a mutant was identified and named as ygl3（yellow green leaf3） as the leaves of the mutant are yellowish green at seedling stage and turning to yellow/white from the leaf-tip area during the late vegetative stages. The gene YGL3 encoding OsPORB, a chloroplast protein, was isolated through map-based cloning and used to complement the ygl3 mutation successfully. The expression pattern and the relationship between Os PORA and OsPORB, two PORs existed in rice, were then investigated. It was found from RT-PCR that expression of OsPORB was constitutive while the high level expression of Os PORA was occurred only in neonatal stems, leaves and spikes. The analysis of subcellular localization provided evidence that both Os PORA and OsPORB are chloroplast protein. The mutated phenotype of ygl3 could be complemented by Os PORA driven by the 35 S promoter. The inhibition for the expression of Os PORA was then conducted through RNAi for both wild type and ygl3 plants, the same phenotypic characteristics was observed from the transgenic plants of wild type but not the ones of ygl3, illustrating that Os PORA and OsPORB are essential for chlorophyll synthesis. These results indicated that the function of Os PORA and OsPORB are redundant, OsPORB is more important, and the Os PORs are conservative during chlorophyll synthesis.