本文選題：水稻 + 黃葉突變體�。� 參考：《揚州大學》2017年碩士論文

【摘要】：水稻是世界上重要的糧食作物之一,其產量大部分來自于葉片的光合作用。葉綠體是光合作用的場所,葉綠體正常的生長發(fā)育對于水稻進行正常的光合作用具有重要作用。當葉綠體發(fā)育出現(xiàn)異常往往會導致葉色發(fā)生變異,影響水稻的光合作用,從而使水稻產量降低。因此利用水稻葉色突變體可以進行基因定位和克隆,鑒定葉綠體發(fā)育相關基因。通過基因工程手段提高水稻光合作用,增加水稻產量,既具有理論意義,又有潛在的實際利用價值。本研究以水稻品種蜀恢498經自然突變產生的黃葉突變體(yellow leaf 1,yl1)為材料,對其葉色性狀和葉綠體結構進行觀察,通過圖位克隆的方法鑒定突變的目的基因。主要研究結果如下:1.通過觀察發(fā)現(xiàn)yl1黃葉突變體在全生育期葉片都表現(xiàn)為黃色,在苗期、分蘗期和成熟期突變體的葉綠素含量相比野生型都明顯減少,葉綠體的超顯微結構觀察發(fā)現(xiàn)突變體的葉綠體中基粒片層數(shù)和類囊體相比野生型減少很多。2.遺傳分析發(fā)現(xiàn)該黃葉突變體受單隱性核基因控制的,采用圖位克隆的方法將目的基因定位到水稻第1號染色體長臂端29kb的范圍內。測序發(fā)現(xiàn)在突變體中LOC_Os01g73450的第5外顯子有一個單堿基替換,導致精氨酸變?yōu)榘腚装彼�。互補實驗和基因敲除實驗證明了該基因就是目的基因。3.qRT-PCR的結果顯示,YL1主要在葉片、葉鞘和穗中表達,尤其葉片中表達量最高,而在根和莖中表達量很低。在突變體中YL1的表達量相比野生型也明顯降低。從48小時表達量變化來看,YL1的表達受到光的誘導,在光照條件下的表達量明顯高于黑暗條件。苗期突變體中與光合作用、葉綠體發(fā)育以及葉綠素代謝相關的基因與野生型相比表達量明顯降低。4.亞細胞定位觀察發(fā)現(xiàn),YL1蛋白定位在葉綠體中。原核表達YL1基因,確認該基因編碼的蛋白質大小約37kD。生物信息學分析發(fā)現(xiàn),YL1在水稻中是一個尿苷酸激酶,在細菌和植物中很保守。對43份水稻進行測序發(fā)現(xiàn)YL1的序列變異主要位于非編碼區(qū)。5.通過對蔗糖、可溶性糖以及淀粉含量測定發(fā)現(xiàn),突變體中合成的碳水化合物含量與野生型相比明顯減少。通過qRT-PCR分析碳水化合物的合成和轉運相關基因發(fā)現(xiàn),突變體中大多數(shù)相關基因表達量與野生型相比都降低。
[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.
【學位授予單位】：揚州大學
【學位級別】：碩士
【學位授予年份】：2017
【分類號】：S511
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本文編號：2106752