水稻早衰基因PSLS1的精细定位及克隆

发布时间：2018-04-02 14:26

本文选题：水稻　切入点：早衰　出处：《中国农业科学院》2016年硕士论文

【摘要】：叶片是植物重要的源器官,水稻叶片早衰将导致叶绿素、蛋白质等大分子的降解、光合能力降低,并伴随活性氧的积累、衰老相关基因表达发生变化、细胞死亡等现象,严重影响稻米产量及品质。因此深入研究早衰形成的分子机理对控制和延缓衰老的发生具有重要的意义。本研究通过对早衰突变体psls1(premature senescence leaf with spots)形态、生理等多方面分析,并结合图位克隆的方法最终确定候选基因PSLS1:一个编码铁氧还依赖的谷氨酸合酶(Fd-GOGAT)基因的突变导致了早衰表型的发生。主要研究结果如下:1.突变体psls1幼苗期生长正常,发育到7叶期以后,叶片自下而上逐渐黄化褪绿至枯萎(黄化由叶尖开始向内蔓延并伴有黄褐色小斑点);生长后期突变体的衰老明显加快,植株整体呈黄褐色枯死状态。在成熟期,与野生型相比,psls1株高、分蘖数、主穗长、穗粒数和千粒重都显著下降。2.与野生型相比,psls1衰老叶片叶绿素含量显著下降,过氧化氢积累、细胞死亡增加,透射电镜显示psls1叶绿体膜界面不清晰,类囊体基粒片层模糊,嗜饿颗粒明显增多。3.遗传分析表明,psls1受一对隐性基因控制,利用psls1×IRAT129杂交组合F2分离群体中的1690个早衰个体,将目的基因定位在第7染色体长臂末端分子标记ZS-3和ZS-8之间物理距离89Kb的范围内,测序研究发现,区间内一个编码铁氧还依赖的谷氨酸合酶(Fd-GOGAT)基因LOC_Os07g46460的第二外显子末位的G被替换为A,导致转录本的错误剪切,相对于野生型,突变体的cDNA缺失了57bp碱基片段,因此,初步认定LOC_Os07g46460为可能的候选基因,暂命名为PSLS1。4.同前人报道相一致,PSLS1在水稻根、茎、叶、叶鞘、穗部均有表达,且在叶片中表达量最高;PSLS1基因的表达水平在野生型与psls1幼苗中无差异,待分蘖期早衰表型出现后,psls1中PSLS1表达量显著下降,对谷氨酸酶活性测定结果也表明psls1发生早衰后谷氨酸酶活性显著降低。5.psls1抽穗期上部叶片中游离氨基酸的含量与野生型存在显著差异,Glu、Ser、Gly、Ala、Val、Ile的含量显著下降,而Asn、PSer、Thr、Cys、Phe、His、Orn、Lys、Arg的含量却显著增加,表明在psls1突变体叶片中氨基酸代谢紊乱。在水培条件下对野生型和psls1进行不同浓度氮素外施处理,发现psls1对低氮条件响应。表明突变体的早衰可能是由于PSLS1的突变使得谷氨酸合酶失活,突变体氮代谢异常引起的。
[Abstract]:Leaf is an important source organ of plants. Premature senescence of rice leaves will lead to the degradation of chlorophyll, protein and other macromolecules, the decrease of photosynthetic ability, the accumulation of reactive oxygen species, the change of senescence related gene expression and cell death, etc.The yield and quality of rice are seriously affected.Therefore, it is of great significance to study the molecular mechanism of premature senescence to control and delay the occurrence of senescence.In this study, morphology and physiology of psls1(premature senescence leaf with spots were analyzed.The mutation of the candidate gene PSLS1: a glutamate synthase (Fd-GOGATT) gene that encodes ferric oxide dependence resulted in the occurrence of premature senescence phenotype.The main results are as follows: 1.The mutant psls1 grew normally at seedling stage, and after developing to 7 leaf stage, the leaves gradually yellowed and faded to wither from top to top (the yellowish brown blotches spread inwards from the tip of the leaf, and the senescence of the mutant accelerated obviously in the later stage of growth.The whole plant was brown and dead.In the mature stage, the plant height, tiller number, main ear length, grain number per ear and 1000-grain weight of psls 1 decreased significantly compared with wild type.Compared with the wild type, the chlorophyll content, hydrogen peroxide accumulation and cell death in senescent leaves of psls1 decreased significantly. Transmission electron microscopy showed that the interface of chloroplast membrane of psls1 was not clear, the lamellae of thylakoid granulocyte was blurred, and the number of eosinophilic granules increased significantly.Genetic analysis showed that psls1 was controlled by a pair of recessive genes. The target gene was located within the range of the physical distance 89Kb between ZS-3 and ZS-8 in the long arm end of chromosome 7 by using 1690 premature senility individuals in F _ 2 segregated population of psls1 脳 IRAT129 hybrid.Sequencing results showed that G at the end of the second exon of a feroxide-dependent glutamate synthase (Fd-GOGATT) gene in the region was replaced with A, resulting in miscut of the transcripts and the deletion of the 57bp base fragment in the cDNA of the mutant relative to the wild type.Therefore, LOC_Os07g46460 was identified as a possible candidate gene, tentatively named PSLS 1.4.In accordance with previous reports, PSLS1 was expressed in roots, stems, leaves, sheaths and panicles of rice, and the highest expression level of PSLS1 gene was found in wild type and psls1 seedlings.After the phenotype of premature senescence appeared at tillering stage, the expression of PSLS1 in pslS1 decreased significantly.The results showed that amino acid metabolism was disturbed in the leaves of psls1 mutants.Wild type and psls1 were treated with different concentrations of nitrogen under hydroponic conditions, and the response of psls1 to low nitrogen condition was found.It is suggested that the premature senescence of the mutant may be due to the inactivation of glutamate synthase caused by the mutation of PSLS1 and the abnormal nitrogen metabolism of the mutant.
【学位授予单位】：中国农业科学院
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S511

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