【作者】 张欢欢；

【导师】 吴平；

【作者基本信息】 浙江大学 ， 生物化学与分子生物学， 2011， 硕士

【摘要】 水稻根系发育具有很强的可塑性,外界环境的变化能够影响水稻根系的发育。本研究通过筛选EMS诱变的水稻突变体库,获得一个在缺氮(-N)营养液中根伸长受抑制的突变体Osgatb。该突变体在正常营养液中早期主根发育迟缓,10天苗龄主根长是野生型的60%左右。在缺氮营养液中,Osgatb根长受到抑制,为野生型根长的40%左右,野生型的根长为正常营养液中野生型根长的125%左右,说明N饥饿抑制该突变体根伸长。突变体后期生长发育受到影响,植株矮小,分蘖数和每穗种子数减少。利用图位克隆的方法,克隆到该突变基因为OsGatB,该基因位于第11号染色体(LOC_Os11g34210),是tRNA依赖性转氨基酶GatCAB复合体的一个组成亚基,该复合体在线粒体中催化Glu-tRNAGln转变为Gln-tRNAGln,在线粒体蛋白翻译中起重要作用。测序结果表明突变体的OsGatB基因在第四个外显子发生突变,致使GatB_N结构域上第342位氨基酸由脯氨酸变成亮氨酸。转基因回复实验证明了OsGatB的单碱基突变是造成突变体短根表型的原因。qRT-PCR分析结果显示,该基因的点突变并没有显著改变其转录水平的表达。缺氮条件下,OsGatB的表达量下调但并不明显,说明该基因的突变虽然改变了突变体的N响应,但并不是通过自身表达量的改变实现的。生物信息学分析显示,OsGatB在水稻中是单拷贝,有八个外显子和七个内含子。该蛋白有两个重要结构域Gat_N和Gat_Yqey,两个结构域对GatCAB酶的GatB亚基活性都很重要。进化树分析发现GatB非常保守,从古菌,到低等植物藻类、酵母再到高等植物水稻、拟南芥、蓖麻都有一定的同源关系,氨基酸序列比对显示,在GatB的两个结构域氨基酸序列有很强的保守性,OsGatB突变体的突变位点是很保守的一个氨基酸位点。之前对GatCAB的报道都集中在结晶蛋白的生化研究和亚细胞定位分析,本研究首次发现了GatCAB和植物根系发育的关系,这对该蛋白的生理生化功能提供了新的知识。更多还原

【Abstract】 Plants roots development is of plasticity in response to environmental conditions. Nitrogen (N) is one of the most important nutrients for plant growth. In this study, a mutant with reduced root length was isolated in rice from an Ethyl-methyl sulfonate (EMS) mutant library. At 10-day-old seedling stage, the mutant exhibits about 40% shorter primary root length than the wild type (WT) plants. The mutant root length reduces 70% compared to WT under N-starvation condition, indicating that the mutant is sensitive to N-starvation. The plants of the mutant showed less tiller number, panicle number and seeds per panicle compared to WT plants.Genetic analysis indicated that the mutant phenotype is controlled by a single recessive gene. The mutated gene was detected on chromosome 11 within 73kb region between STSllgl9271k and STSllg19344k by map-based cloning. Sequencing analysis revealed that a point mutation occurred in the forth exon of the gene OsGatB (LOC_Osllg34210). The point mutation results in proline-to-leucine change in the GatB_N domain of the gene product. OsGatB is a subunit of tRNA-dependent amidotransferase GatCAB. Glu-tRNAGln is converted into Gln-tRNAGln by the GatCAB in plant mitochondria, which is important to translation of protein in mitochondria.Overexpression of OsGatB in the Osgatb mutant can rescue the mutation phenotype which confirm that the mutation phenotype is caused by the point mutation of OsGatB. quantitative PCR analysis showed that OsGatB transcriptional level is not change in the mutant remarkable. The root of mutant decreased compared to induced of WT root is not changed transcription of the gene under N-starvation by quantitative PCR analysis.Alignment of the cDNA with the genomic DNA sequences revealed that OsGatB contains 7 introns and 8 exons. Analysis for conserved domains of OsGatB indicated that the OsGatB contains two conserved domains, Gat_N and GatB_Yqey, both of them are important to the function of OsGatB. Alignment of amino acids showed that the protein is highly conserved from archaea to plants, and the mutant proline is conserved point in rice GatB.更多还原