本文選題：水稻 + 著絲粒��； 參考：《揚州大學》2017年碩士論文

【摘要】：著絲粒是真核生物染色體基本的結構與功能元件,每條染色體必須擁有活性著絲粒才能保證染色體有絲分裂與減數(shù)分裂的正常進行、遺傳物質的正常傳遞。長期以來,著絲粒區(qū)域被認為含有大量重復序列,屬于染色質上的轉錄沉默區(qū),并且著絲粒及其側翼區(qū)域的重組是高度受抑制的,所以經(jīng)典遺傳學認為著絲粒區(qū)域不存在功能基因。然而近年來研究表明,著絲粒區(qū)域有活性基因的存在。水稻是世界上重要的糧食作物之一,也是單子葉植物的模式生物。隨著水稻全基因組測序的完成,水稻不同染色體著絲粒區(qū)域活性基因的組成也進行了初步分析。其中不含有大量衛(wèi)星重復序列的水稻第8著絲粒是揭示著絲粒區(qū)域特征的最理想的模型,在水稻第8染色體功能著絲粒區(qū)有16個活性基因,但相關基因的功能很少見報道。水稻葉色的變化是遺傳因素與環(huán)境因素共同作用的結果,其中遺傳因素通常與葉綠素的合成與降解、葉綠體的形成及發(fā)育等相關。水稻葉色變化主要有白化、黃化、淡綠、黃綠、條紋等類型。水稻中有許多與葉色相關的基因,分布在水稻的各條染色體上,然而關于著絲粒區(qū)域與葉色相關基因的報道還很少。本實驗室在前期研究中獲得水稻品種中秈3037著絲粒DNA序列缺失材料,該材料缺失純合體無法在大田條件下正常生長,初步確定該著絲粒區(qū)域存在重要功能基因。本實驗以該材料為基礎,繼續(xù)開展相關研究,明確了該材料在第8染色體著絲粒區(qū)域發(fā)生缺失與表達差異的6個候選基因,并重點對葉色相關基因DCL(defective chloroplasts and leaves)的功能進行初步分析。主要研究結果如下:1.經(jīng)FISH分析明確著絲粒序列缺失片段發(fā)生在第8染色體上,在該區(qū)域沒有水稻著絲粒特異序列CentO的信號。2.進一步通過PCR和RNA-seq分析確定第8染色體的著絲粒缺失片段在LOC_Os08921541至LOC_Os08g217710之間,大小約為171kb,在該區(qū)域包含6個活性基因,其中LOC_Os08921700為葉色相關基因DCL的侯選基因。3.構建6個缺失基因的RNAi載體,并重點對已經(jīng)獲得的著絲粒區(qū)域控制葉色的DCL基因的RNAi材料進行基因表達分析。結果表明RNAi材料中DCL基因的表達均顯著下降。4.進一步對DCL-RNAi材料進行表型觀察。結果顯示DCL-RNAi材料葉片黃化,并在株高、分蘗數(shù)、結實率和千粒重等方面發(fā)生顯著變化。5.通過葉綠素含量的測定及葉綠體電鏡掃描分析表明,DCL-RNAi材料的葉片中總葉綠素含量及葉綠素各組分含量均顯著降低;DCL基因與葉綠體的形成發(fā)育相關。6.對水稻中葉綠體形成發(fā)育相關及光合作用等相關的基因進行表達量分析。結果表明,只有編碼NADPH原葉綠素酸酯氧化還原酶的基因LOC_Os04g58200在DCL-RNAi材料中的表達量較野生型顯著降低,其他基因在野生型和DCL-RNAi材料中的表達量無顯著差異。7.表達分析及GUS染色結果表明,DCL基因在根、莖、葉和穗等組織中均有表達。GFP融合蛋白在水稻原生質體中的瞬時表達表明,DCL蛋白是一個核蛋白。8.構建DCL基因的CRISPR/Cas9載體和過量表達載體,并導入水稻品種鹽稻8號中。目前已獲得dcl突變體及過表達植株,對過表達株系進行基因表達分析,結果表明過表達植株中DCL基因的表達水平顯著提高。
[Abstract]:Centromeric is the basic structural and functional element of eukaryotic chromosomes. Each chromosome must have active centromere to ensure the normal progression of mitosis and meiosis, and the normal transmission of genetic material. The centromere region has long been considered to contain a large number of repeat sequences, which belong to the transcriptional silencing zone on the chromatin. And the recombination of the centromeric and its flanking regions is highly inhibited, so the classical genetics holds that there is no functional gene in the centromere region. However, in recent years, studies have shown that there are active genes in the centromeric region. Rice is one of the most important grain crops in the world and a model organism of mono cotyledon. The composition of the centromere region active genes of different chromosomes of rice was also preliminarily analyzed. The eighth centromere of rice, which did not contain a large number of satellite repeats, was the most ideal model to reveal the characteristics of the centromere region. There were 16 active genes in the fillet region of the eighth chromosome of rice, but the work of the related genes was done. The changes in leaf color of rice are the result of the combination of genetic factors and environmental factors, in which genetic factors are usually related to the synthesis and degradation of chlorophyll, the formation and development of chloroplasts. The changes in leaf color of rice are mainly whitening, yellowing, green, yellow green, stripe and so on. There are many genes related to leaf color in rice. There are few reports on the chromosomes of rice. However, there are few reports on the centromeric region and leaf color related genes. In the previous study, the 3037 centromeric DNA sequences missing from rice varieties were obtained. The missing homozygous material could not grow normally under the condition of field. The important work of the centromeric region was preliminarily determined. On the basis of this material, we continue to carry out the related research, and make clear the 6 candidate genes of the deletion and expression of the material in the centromere region of the eighth chromosome, and a preliminary analysis of the function of the leaf color related gene DCL (defective chloroplasts and leaves). The main results are as follows: 1. by FISH analysis The deletion fragment of the centromere sequence occurred on the eighth chromosome, and there was no signal.2. of the centromere specific sequence CentO of rice in this region. The deletion fragment of the centromere of the eighth chromosome was determined between LOC_Os08921541 and LOC_Os08g217710 by PCR and RNA-seq, and the size was about 171KB, including 6 active genes in the region. LOC_Os08921700, a candidate gene.3. for leaf color related gene DCL, constructed the RNAi vector of 6 missing genes, and focused on the gene expression analysis of the RNAi material of the DCL gene that has been obtained in the centromere region of the centromere. The results showed that the expression of DCL gene in RNAi significantly decreased.4. further to the phenotype of DCL-RNAi materials. The results showed that the leaves of DCL-RNAi were yellow, and there were significant changes in plant height, tiller number, seed setting rate and 1000 grain weight..5. through the determination of chlorophyll content and chloroplast scanning analysis showed that the content of total chlorophyll and the content of chlorophyll in the leaves of the DCL-RNAi material decreased significantly; the DCL gene and the form of the chloroplast were found. The expression of genes related to chloroplast formation and development and photosynthesis in rice was analyzed by development related.6.. The results showed that only the expression of the gene LOC_Os04g58200 encoding the NADPH original chlorophyll-a oxidoreductase decreased significantly in the DCL-RNAi material than the wild type, and the other genes were in the wild type and DCL-RNAi material. .7. expression analysis and GUS staining showed that the DCL gene expressed.GFP fusion protein in the rice protoplast of root, stem and Ye Hesui, indicating that DCL protein was a CRISPR/Cas9 carrier and overexpression vector of a nuclear protein.8. to construct DCL gene, and introduced into rice variety salt rice 8. DCL mutants and overexpressed plants have been obtained, and the expression of overexpressed lines was analyzed. The results showed that the expression level of DCL gene in overexpressed plants was significantly improved.
【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】：Q943.2;S511

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