水稻第8染色体着丝粒区域基因-DCL的功能分析

发布时间：2018-06-16 22:10

本文选题：水稻 + 着丝粒　；参考：《扬州大学》2017年硕士论文

【摘要】：着丝粒是真核生物染色体基本的结构与功能元件,每条染色体必须拥有活性着丝粒才能保证染色体有丝分裂与减数分裂的正常进行、遗传物质的正常传递。长期以来,着丝粒区域被认为含有大量重复序列,属于染色质上的转录沉默区,并且着丝粒及其侧翼区域的重组是高度受抑制的,所以经典遗传学认为着丝粒区域不存在功能基因。然而近年来研究表明,着丝粒区域有活性基因的存在。水稻是世界上重要的粮食作物之一,也是单子叶植物的模式生物。随着水稻全基因组测序的完成,水稻不同染色体着丝粒区域活性基因的组成也进行了初步分析。其中不含有大量卫星重复序列的水稻第8着丝粒是揭示着丝粒区域特征的最理想的模型,在水稻第8染色体功能着丝粒区有16个活性基因,但相关基因的功能很少见报道。水稻叶色的变化是遗传因素与环境因素共同作用的结果,其中遗传因素通常与叶绿素的合成与降解、叶绿体的形成及发育等相关。水稻叶色变化主要有白化、黄化、淡绿、黄绿、条纹等类型。水稻中有许多与叶色相关的基因,分布在水稻的各条染色体上,然而关于着丝粒区域与叶色相关基因的报道还很少。本实验室在前期研究中获得水稻品种中籼3037着丝粒DNA序列缺失材料,该材料缺失纯合体无法在大田条件下正常生长,初步确定该着丝粒区域存在重要功能基因。本实验以该材料为基础,继续开展相关研究,明确了该材料在第8染色体着丝粒区域发生缺失与表达差异的6个候选基因,并重点对叶色相关基因DCL(defective chloroplasts and leaves)的功能进行初步分析。主要研究结果如下:1.经FISH分析明确着丝粒序列缺失片段发生在第8染色体上,在该区域没有水稻着丝粒特异序列CentO的信号。2.进一步通过PCR和RNA-seq分析确定第8染色体的着丝粒缺失片段在LOC_Os08921541至LOC_Os08g217710之间,大小约为171kb,在该区域包含6个活性基因,其中LOC_Os08921700为叶色相关基因DCL的侯选基因。3.构建6个缺失基因的RNAi载体,并重点对已经获得的着丝粒区域控制叶色的DCL基因的RNAi材料进行基因表达分析。结果表明RNAi材料中DCL基因的表达均显著下降。4.进一步对DCL-RNAi材料进行表型观察。结果显示DCL-RNAi材料叶片黄化,并在株高、分蘖数、结实率和千粒重等方面发生显著变化。5.通过叶绿素含量的测定及叶绿体电镜扫描分析表明,DCL-RNAi材料的叶片中总叶绿素含量及叶绿素各组分含量均显著降低;DCL基因与叶绿体的形成发育相关。6.对水稻中叶绿体形成发育相关及光合作用等相关的基因进行表达量分析。结果表明,只有编码NADPH原叶绿素酸酯氧化还原酶的基因LOC_Os04g58200在DCL-RNAi材料中的表达量较野生型显著降低,其他基因在野生型和DCL-RNAi材料中的表达量无显著差异。7.表达分析及GUS染色结果表明,DCL基因在根、茎、叶和穗等组织中均有表达。GFP融合蛋白在水稻原生质体中的瞬时表达表明,DCL蛋白是一个核蛋白。8.构建DCL基因的CRISPR/Cas9载体和过量表达载体,并导入水稻品种盐稻8号中。目前已获得dcl突变体及过表达植株,对过表达株系进行基因表达分析,结果表明过表达植株中DCL基因的表达水平显著提高。
[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.
【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】：Q943.2;S511

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