水稻白穗基因WP1的克隆与功能分析

发布时间：2018-03-02 12:10

本文选题：水稻　切入点：叶绿体　出处：《南京农业大学》2016年博士论文　论文类型：学位论文

【摘要】：叶绿体是植物进行光合作用的细胞器,同时还是一些重要物质合成和代谢的场所。因此叶绿体的发育对植物的生长发育及最终的产量形成至关重要。针对叶绿体发育的研究主要集中在双子叶植物拟南芥中,而单子叶植物中研究比较少。叶绿体发育在单子叶植物和双子叶植物中存在较大差异。而水稻作为单子叶植物的模式系统,利用水稻开展叶绿体发育的研究对于全面认识植物的叶绿体发育机制具有重要的意义;同时,研究水稻作为全球最重要的粮食作物之一,研究水稻叶绿体的发育将为培育高光效的超级水稻奠定理论基础。本研究以一个水稻白穗突变体wp1(whitepanicle1)为材料,从细胞学,遗传学和功能基因组学等方面展开研究。主要结果如下:1.水稻白穗突变体wp1在苗期表现为白条纹和白化表型,抽穗后穗子成白色。对苗期叶片和抽穗时的颖壳叶绿素的含量测定发现,与野生型相比wp1突变体上述组织的叶绿素含量显著降低。对上述组织中的叶绿体超微结构观察发现突变体中叶绿体结构严重受损。对早期的叶绿体发育过程进行叶绿素荧光和透射电镜观察,发现wp1突变体的早期叶绿体发育过程严重受损。2.构建定位群体对WP1位点进行了图位克隆。通过对43kb定位区间内的5个ORFs(open reading frames)进行测序发现了可能的突变基因LOC_Os07g06940。通过互补实验进一步证明了该基因的突变导致wp1突变体的一系列的突变表型。3.对WP1的氨基酸序列进行分析,发现其编码一个缬氨酸-tRNA合成酶,命名为OsValRS2。进一步分析发现在水稻基因组中还有一个有功能的缬氨酸-tRNA合成酶(OsValRS1 )。这两个缬氨酸-tRNA合成酶在拟南芥中均有高度同源的直系同源基因存在。对WP1的组织表达分析发现其在各个组织中都有表达,且在绿色组织中表达量相对更高。另一个水稻中的缬氨酸-tRNA合成酶OsValRS1在各个组织中都有表达,表达量很高且分布均匀。对水稻中的两个缬氨酸-tRNA合成酶进行了亚细胞定位发现WP1同时定位在叶绿体和线粒体中,而OsValRS1定位在胞质。WP1和OsValRS1负责了三个亚细胞细胞区域的蛋白质合成,而且这两个蛋白无功能冗余。4.通过RNA-seq的方法检测了wp1突变体中表达量发生变化的基因。在核编码基因中,光系统合成相关的基因(光捕获复合体,光系统Ⅰ,光系统Ⅱ等)的表达量在wp1突变体中均显著下调。我们还发现wp1突变体中缬氨酸合成和降解途径基因的表达量均显著上调,这也间接证明了缬氨酸-tRNA合成酶的突变。通过RNA-seq我们还对质体基因组编码的基因进行了分析,发现质体基因组编码的基因中由NEP负责转录的其表达量都显著上调,而由PEP负责转录的其表达量均显著下调。这一现象是PEP活性受损的一种表现,而PEP活性受损会导致质体早期发育不正常。5.对wp1突变体中质体基因组编码蛋白的含量进行了 Western-Blot分析,发现检测的几类蛋白的含量在wp1突变体中均显著降低。对这些蛋白的mRNA水平进行检测发现其表达量有上调也有下调。因此我们认为wp1突变体中的质体蛋白质合成受阻。对突变体中质体的核糖体进行了分析。发现在突变体幼苗和颖壳中几乎检测不到质体的rRNA和核糖体蛋白。这些结果表明wp1突变体中核糖体生物合成的受损导致质体蛋白质合成的受阻,且我们推测WP1可能是间接地影响到质体核糖体的生物合成的。
[Abstract]:Chloroplasts are organelles of photosynthesis, but also some important material synthesis and metabolism. Therefore the growth and development of plant chloroplast development and yield formation is very important. According to the research of chloroplast development mainly concentrated in dicot Arabidopsis, and monocots. Relatively few studies of chloroplast development exist large differences in monocots and dicots. And rice as a model system of monocotyledonous plants, to carry out the research on chloroplast development has important significance for understanding the plant chloroplast development mechanism using rice; at the same time, the research of rice as one of the most important crops in the world, the research of rice chloroplast development will lay the theoretical foundation for the super rice breeding for high photosynthetic efficiency. In this study, a rice mutant Wp1 (whitepanicle1) as the material, from Study on cytology, genetics and functional genomics. The main results are as follows: 1. white rice panicle mutant Wp1 in seedling white stripe and the albino phenotype after heading, grain white. Determination of chlorophyll in leaves and glume heading when found, compared with the wild type Wp1 mutant of the chlorophyll content of tissue decreased significantly. The chloroplast ultrastructure in the above tissues were found severely damaged the chloroplast structure in the mutant. The early chloroplast development process of chlorophyll fluorescence and TEM observation found that early chloroplast development process Wp1 mutant was severely damaged.2. mapping population of WP1 loci of positional cloning. By 5 ORFs of 43kb the positioning range (open reading frames) were found by sequencing the LOC_Os07g06940. gene mutation may further confirmed by complementary experiments To understand the gene mutation resulted in the amino acid sequence of WP1 mutant phenotypes.3. a series of Wp1 mutants were analyzed, found its encoding a valine -tRNA synthase, named OsValRS2. was analyzed in rice genome and a valine -tRNA synthase function (OsValRS1). The two valine -tRNA synthetase in Arabidopsis thaliana. Have highly homologous orthologous genes. Expression analysis found in various tissues with the expression of WP1 in tissue, and the expression of green tissue was relatively higher. Another rice valine -tRNA synthase OsValRS1 was expressed in all tissues, the expression is very high and uniform distribution of two. A valine -tRNA synthase in rice were also found WP1 subcellular localization localized in the chloroplast and mitochondria, and OsValRS1 localized in the cytoplasm of.WP1 and OsValRS1 Responsible for protein synthesis in three subcellular regions, and these two proteins without functional redundancy detection of.4. expression in the Wp1 mutant gene changes by RNA-seq method. In the nuclear gene encoding, genes associated with the synthesis of optical system (light harvesting complexes of photosystem I, photosystem II expression etc.) the amount was significantly down regulated in the Wp1 mutant. We also found that the amount of gene expression and synthesis of valine degradation pathway of Wp1 mutants were significantly up-regulated, which also indirectly proved that the mutation of valine -tRNA synthase by RNA-seq. We also confront the genome encoding gene was analyzed, found that the expression of transcription by NEP for plastid genome encoding the genes are up-regulated, the expression of PEP is responsible for the transcription were decreased. This phenomenon is a manifestation of the activity of PEP is damaged, and the activity of PEP can lead to impaired By the early development of plastid abnormal content of plastid genome encoding protein Wp1 mutant in.5. was analyzed by Western-Blot, are found in several kinds of protein content decreased significantly in the Wp1 mutant. The protein levels of mRNA were detected that the expression amount increases also lowered. So we think that the plastid protein synthesis Wp1 mutant the blocked. The mutant plastid ribosomes are analyzed. That is almost not detected in plastid ribosomal rRNA and protein in the mutant seedlings and glume. These results suggest that ribosome biogenesis in Wp1 mutants impaired plastid protein synthesis is blocked, and we speculate that WP1 may indirectly affect the biosynthesis of plastid ribosome.

【学位授予单位】：南京农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S511

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