《植物生理学报》 2015, 51(6): 860-868

水稻POR基因的分离、定位与功能的初步研究

高小丽^1,2, 李素娟², 邵健丰², 刘洪家^2,*, 陶跃之²

¹杭州师范大学生命与环境科学学院, 浙江杭州310036; ²浙江省农业科学院作物与核技术利用研究所, 浙江杭州310021

通信作者：刘洪家；E-mail: lhjzju@aliyun.com；Tel: 15857135706

摘　要：

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OsPORA和OsPORB。RT-PCR分析表明, OsPORA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OsPORA和OsPORB为叶绿体定位蛋白。此外, 用35S启动子驱动OsPORA表达能够完全互补ygl3的表型。虽然利用RNAi技术抑制OsPORA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OsPORA的表达导致转基因水稻黄化致死, 说明OsPORA和OsPORB为叶绿素合成所必需的。以上研究结果说明OsPORA和OsPORB在功能上具有一定的冗余性, 但OsPORB比OsPORA的作用更重要, 揭示了OsPOR在叶绿素合成途径上保守性。

关键词：水稻; 叶绿素合成; OsPOR; 图位克隆; 叶绿体

收稿：2015-03-06   修定：2015-05-14

资助：国家自然科学基金(31171530)。

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

GAO Xiao-Li^1,2, LI Su-Juan², SHAO Jian-Feng², LIU Hong-Jia^2,*, TAO Yue-Zhi²

¹College of Life and Environmental Sciences, Hangzhou Normal University, Hangzhou, Zhejiang 310036, China; ²Institute of Crops and Utilization of Nuclear Technology, Zhejiang Academy of Agricultural Sciences, Hangzhou, Zhejiang 310021, China

Corresponding author: LIU Hong-Jia; E-mail: lhjzju@aliyun.com; Tel: 15857135706

Abstract:

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 OsPORA 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 OsPORA was occurred only in neonatal stems, leaves and spikes. The analysis of subcellular localization provided evidence that both OsPORA and OsPORB are chloroplast protein. The mutated phenotype of ygl3 could be complemented by OsPORA driven by the 35S promoter. The inhibition for the expression of OsPORA 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 OsPORA and OsPORB are essential for chlorophyll synthesis. These results indicated that the function of OsPORA and OsPORB are redundant, OsPORB is more important, and the OsPORs are conservative during chlorophyll synthesis.

Key words: rice (Oryza sativa); chlorophyll synthesis; OsPOR; map-based cloning; chloroplast