水稻黄绿叶突变体yellow-green leaf 4的表型鉴定及候选基因定位和功能分析  

Phenotypic identification,candidate gene mapping and functional analysis of yellow-green leaf 4 mutant in rice

作　　者：刘忠学 张渝竣 刘林 刘世家[1] 田云录[1] 周时荣 江玲[1] 万建民 刘玲珑[1] LIU Zhongxue;ZHANG Yujun;LIU Lin;LIU Shijia;TIAN Yunlu;ZHOU Shirong;JIANG Ling;WAN Jianmin;LIU Linglong(State Key Laboratory of Crop Genetics and Germplasm Enhancement/Research Center of Jiangsu Plant Gene Engineering,Nanjing Agricultural University,Nanjing 210095,China;Institute of Crop Sciences,Chinese Academy of Agricultural Sciences,Beijing 100081,China)

机构地区：[1]南京农业大学作物遗传与种质创新国家重点实验室/江苏省植物基因工程技术研究中心,江苏南京210095 [2]中国农业科学院作物科学研究所,北京100081

出　　处：《南京农业大学学报》2022年第4期627-636,共10页Journal of Nanjing Agricultural University

基　　金：江苏省重点研发项目(BE2021360);江苏省农业自主创新资金项目[SCX(19)1079]。

摘　　要：[目的]本研究旨在对水稻黄绿叶突变体yellow-green leaf 4(ygl4)进行表型分析及基因定位和功能分析,探讨水稻叶绿体发育分子机制。[方法]水稻黄绿叶突变体ygl 4来自粳稻品种‘中花11’化学诱变突变体库。ygl 4与籼稻品种‘N22’杂交构建F_(2)分离群体以进行YGL 4基因定位,并利用实时定量PCR和亚细胞定位等技术对基因进行初步功能分析。[结果]相比于野生型,ygl 4在幼苗2~3叶龄出现黄绿叶症状,在6月下旬移栽大田后,黄绿叶症状加剧,甚至开始出现白化,突变性状可以一直保持到全生育期直到种子成熟。温敏性试验表明,ygl 4突变体在20℃表现出更严重黄化表型。透射电镜观察表明,ygl 4的叶片细胞结构中出现了较多的双膜囊泡结构。基因定位显示,ygl 4突变性状由1个隐性核基因LOC_Os 04g42000突变导致。该基因编码一个核黄素合成途径中的关键酶,6,7-二甲基-8-核糖醇基二氧四氢蝶啶合酶(LS),且突变体叶色表型可被外施核黄素恢复为正常表型。亚细胞定位结果显示YGL4定位于叶绿体。实时定量PCR分析表明,在突变体中,叶绿素暗反应阶段参与5-氨基乙酰丙酸(ALA)到原叶绿素酸酯合成过程的基因表达上调。[结论]水稻YGL 4编码LS基因,并通过调控植物体内核黄素合成途径影响叶色和叶绿体发育。[Objectives]The purpose of this study was to analyze the phenotype,gene location and function of rice yellow green leaf mutant yellow-green leaf 4(ygl4),and to explore the molecular mechanism of rice chloroplast development.[Methods]The yellow green leaf mutant ygl 4 of rice comes from the chemical mutation mutant library of japonica rice variety‘Zhonghua 11’.The ygl 4 was hybridized with indica rice variety‘N22’to construct F_(2) segregated population for ygl 4 gene mapping and the preliminary functional analysis was carried out by means of real-time quantitave PCR and subcellular localization techniques.[Results]Compared with the wild-type,ygl 4 had yellow and green leaf symptoms at the age of 2-3 leaves.After transplanting in the field in late June,the yellow and green leaf symptoms worsen and even begin to show albinism.The mutant character could be maintained to the whole growth period until the seed maturity.The temperature sensitivity test showed that the ygl 4 mutant displayed a more severe chlorosis phenotype at 20℃.Transmission electron microscopy showed that there were many double membrane vesicles in the leaf cell structure of ygl 4.Gene mapping revealed that the ygl 4 was caused by a recessive nuclear gene LOC_Os 04g42000 mutation.The gene encoded a key enzyme in the riboflavin synthesis pathway,6,7-dimethyl-8-riboxydioxenidine synthase(LS).The leaf color phenotype of the mutant was restored to normal by external application of riboflavin.Subcellular localization results showed that YGL4 was localized in chloroplasts.Real-time quantitative PCR analysis showed that in the mutant,the gene expression involved in the synthesis of 5-aminolevulinic acid(ALA)to prochlorophyllate was up-regulated in the chlorophyll dark reaction stage.[Conclusions]Rice ygl 4 encodes a LS gene which affected leaf color and chloroplast development by regulating riboflavin content in plants.

关 键 词：水稻 黄绿叶突变体 基因定位 6 7-二甲基-8-核糖醇基二氧四氢蝶啶合酶 表达分析 

分 类 号：S511.032[农业科学—作物学]