控制水稻金黄色颖壳与节间基因OsCAD2的图位克隆

Map-Based Cloning of OsCAD2 Regulating Golden Hull and Internode in Rice

【目的】水稻色素不仅对其自身生长发育有重要生理作用,而且在水稻育种、农副产品改良等方面运用比较广泛。对水稻色素相关基因进行表型分析和基因定位,为进一步研究色素代谢途径及调控机理奠定基础。【方法】利用EMS诱变粳稻长粒粳(CLJ),在突变体库内筛选到一个金黄色颖壳与节间突变体gh881;在成熟期,测定野生型与gh881的主要农艺性状;将gh881与野生型及中恢8015杂交,观察BC1F1及F1植株表型,并对BC1F2及F2表型分离进行卡方检验,对gh881进行遗传分析;利用F2群体和图位克隆的方法对gh881突变基因进行定位;采用q PCR检测颖壳颜色相关基因在gh881与野生型不同发育时期的幼穗、节间以及剑叶叶鞘的相对表达量。【结果】与野生型相比,突变体的颖壳与节间均呈金黄色;除单株有效穗数外,gh881突变体的株高、每穗总粒数及实粒数、结实率和千粒重等性状均极显著降低。遗传分析和基因定位结果表明,gh881的突变表型受1对隐性核基因控制,位于第2染色体短臂,并最终将该基因精细定位于标记FH-13和RH-25之间,物理距离约33.2 kb,该区域中包含4个开放阅读框(ORFs)。【结论】序列分析结果表明,发现其中一个编码肉桂醇脱氢酶(CAD)的基因OsC AD2(Os02g0187800)的3 563 bp处发生了1个单碱基突变(G转换为A),导致该基因编码区的第297位氨基酸由甘氨酸突变为天冬氨酸,由此认为该突变体为OsC AD2基因单碱基突变的新等位基因。q RT-PCR结果表明,突变体的节间中OsCAD2相对表达量极显著下调,而在剑叶叶鞘及穗部则基本都是极显著增加,其他相关基因也发生显著变化,证实OsCAD2是木质素代谢中的重要基因,且可能与其他相关基因存在反馈调节。

【Objective】Rice pigments not only have important physiological effects on their own development,but alsohave been widely used in rice breeding,by-product improvement,and so on.Phenotypic analysis and gene mapping ofpigment-related genes could lay the foundation for the further study of rice pigments metabolism and regulation mechanism.【Method】A golden hull and internode rice mutant,gh881,was isolated from an elite japonica cultivar Changlijing(CLJ)using ethyl methane sulphonate(EMS)mutagenesis strategy;the main agricultural traits of gh881and its wild type(WT)were measured at mature stage;we crossed gh881with WT and ZH8015,respectively,followed by observation of F1andBC1F1plants phenotype,and we made genetic analysis using Chi-square test in BC1F2population and gene mapping usingmap-based cloning in F2population;the related-genes expression of young panicles,internodes and flag leaf sheaths ofgh881and WT at various stages were studied by qPCR.【Result】Compared with wild type,gh881exhibited golden hulland internode at the mature stage.Except for no significant difference in the number of panicles per plant,there weresignificant reduction in plant height,the seed-setting rate,the number of spikelets per panicle,the number of grains perpanicle and1000-grain weight between WT and gh881.Genetic analysis and gene fine-mapping results suggested thatgh881was controlled by a single recessive gene and the mutant gene was mapped to a region of33.2kb in which four openreading flames(ORFs)existed on the short arm of chromosome2between markers FH-13and RH-25.【Conclusion】Sequencing analysis revealed that a single base mutation(G to A)occurred at the site3563bp of OsCAD2(Os02g0187800)encoding a Cinnamyl-Alcohol Dehydrogenase(CAD),which led to a G297D mutation(codon GGC to GAC).This impliedthat gh881might carry a novel allele of OsCAD2.The quantitative real-time PCR analysis showed that the relativeexpression level of OsCAD2decreased significantly in the internode,while it almost increased significantly in the flag leafsheath and panicle,and the related-genes also almost changed significantly.These results demonstrated that OsCAD2is animportant gene involved in lignin metabolic pathway,and may regulate other related-genes expression feedback.