水稻黄叶基因YL1的图位克隆和功能研究

发布时间：2018-07-08 07:58

  本文选题：水稻 + 黄叶突变体　； 参考：《扬州大学》2017年硕士论文

【摘要】：水稻是世界上重要的粮食作物之一,其产量大部分来自于叶片的光合作用。叶绿体是光合作用的场所,叶绿体正常的生长发育对于水稻进行正常的光合作用具有重要作用。当叶绿体发育出现异常往往会导致叶色发生变异,影响水稻的光合作用,从而使水稻产量降低。因此利用水稻叶色突变体可以进行基因定位和克隆,鉴定叶绿体发育相关基因。通过基因工程手段提高水稻光合作用,增加水稻产量,既具有理论意义,又有潜在的实际利用价值。本研究以水稻品种蜀恢498经自然突变产生的黄叶突变体(yellow leaf 1,yl1)为材料,对其叶色性状和叶绿体结构进行观察,通过图位克隆的方法鉴定突变的目的基因。主要研究结果如下:1.通过观察发现yl1黄叶突变体在全生育期叶片都表现为黄色,在苗期、分蘖期和成熟期突变体的叶绿素含量相比野生型都明显减少,叶绿体的超显微结构观察发现突变体的叶绿体中基粒片层数和类囊体相比野生型减少很多。2.遗传分析发现该黄叶突变体受单隐性核基因控制的,采用图位克隆的方法将目的基因定位到水稻第1号染色体长臂端29kb的范围内。测序发现在突变体中LOC_Os01g73450的第5外显子有一个单碱基替换,导致精氨酸变为半胱氨酸。互补实验和基因敲除实验证明了该基因就是目的基因。3.qRT-PCR的结果显示,YL1主要在叶片、叶鞘和穗中表达,尤其叶片中表达量最高,而在根和茎中表达量很低。在突变体中YL1的表达量相比野生型也明显降低。从48小时表达量变化来看,YL1的表达受到光的诱导,在光照条件下的表达量明显高于黑暗条件。苗期突变体中与光合作用、叶绿体发育以及叶绿素代谢相关的基因与野生型相比表达量明显降低。4.亚细胞定位观察发现,YL1蛋白定位在叶绿体中。原核表达YL1基因,确认该基因编码的蛋白质大小约37kD。生物信息学分析发现,YL1在水稻中是一个尿苷酸激酶,在细菌和植物中很保守。对43份水稻进行测序发现YL1的序列变异主要位于非编码区。5.通过对蔗糖、可溶性糖以及淀粉含量测定发现,突变体中合成的碳水化合物含量与野生型相比明显减少。通过qRT-PCR分析碳水化合物的合成和转运相关基因发现,突变体中大多数相关基因表达量与野生型相比都降低。
[Abstract]:Rice is one of the most important food crops in the world. Chloroplast is the site of photosynthesis. The normal growth and development of chloroplast play an important role in rice photosynthesis. The abnormal chloroplast development often leads to the variation of leaf color, which affects the photosynthesis of rice and reduces the yield of rice. Therefore, rice leaf color mutants can be used for gene mapping and cloning, and identification of chloroplast development related genes. It is not only of theoretical significance but also of potential practical value to improve photosynthesis and increase rice yield by means of genetic engineering. In this study, the yellow leaf mutant (yellow leaf _ (1), which was produced by natural mutation in rice variety Shuhui 498, was used to observe the leaf color character and chloroplast structure, and the mutant gene was identified by map cloning. The main results are as follows: 1. It was found that the leaves of yl1 yellow leaf mutants were yellow at the whole growth stage, and the chlorophyll content of the mutant at seedling stage, tillering stage and mature stage was significantly lower than that of wild type. Ultrastructural observation of chloroplast revealed that the number of grana lamellae in the chloroplast of mutant was much lower than that of thylakoid. Genetic analysis showed that the yellow leaf mutant was controlled by a single recessive nuclear gene, and the target gene was mapped to the 29kb of the long arm of rice chromosome 1 by the method of map cloning. Sequencing showed that there was a single base substitution at exon 5 of Os01g73450 in the mutant, which resulted in the conversion of arginine to cysteine. The results of complementary experiment and gene knockout showed that the gene was the target gene. 3.qRT-PCR showed that YL1 was mainly expressed in leaves, sheaths and panicles, especially in leaves, but the expression was very low in roots and stems. The expression of YL1 in the mutant was significantly lower than that in the wild type. The expression of YL1 was induced by light, and the expression of YL1 was significantly higher in light than in dark. The expression of genes related to photosynthesis, chloroplast development and chlorophyll metabolism in seedling mutant was significantly lower than that in wild type. Subcellular localization observation showed that YL1 protein was located in chloroplast. Prokaryotic expression of YL1 gene confirmed that the protein size of YL1 gene was about 37 KD. Bioinformatics analysis showed that YL1 is a urolyl kinase in rice and is conserved in bacteria and plants. 43 rice samples were sequenced and found that YL1 sequence variation was mainly located in non-coding region. 5. The contents of sucrose, soluble sugar and starch in the mutant were determined, and the carbohydrate content in the mutant was significantly decreased compared with the wild type. By qRT-PCR analysis of carbohydrate biosynthesis and transportation-related genes, it was found that the expression of most of the related genes in the mutant was lower than that in the wild type.
【学位授予单位】：扬州大学
【学位级别】：硕士
【学位授予年份】：2017
【分类号】：S511
，

本文编号：2106752