水稻Ds插入曲穗突变体及其Ds标记基因的鉴定

发布时间：2018-06-16 02:14

本文选题：水稻 + 穗弯曲　；参考：《苏州大学》2016年硕士论文

【摘要】：稻穗形态是影响水稻产量的重要农艺性状之一,有研究证明,直立穗水稻比弯曲穗水稻有更多高产优势,所以对穗直立与穗弯曲相关基因的研究定位在优化穗型提高水稻产量方面的研究有重要的意义。随着水稻全基因组序列测定的完成,功能基因组学以及利用Ac/Ds系统构建的水稻突变体库在水稻分子遗传育种方面得到广泛应用。经过多年对粳稻品种Dongjin的Ac/Ds插入突变株系的栽培,本实验室从中筛选到一株具有Ds插入的曲穗突变体,分别从形态学、生理学及分子生物学方面初步分析穗弯曲形成的可能原因,为水稻优质高产育种的研究提供参考。本研究主要包括两方面的内容:1.曲穗突变体的形态及生理鉴定。试验田中随机抽取野生型和突变体成熟期植株各50株,测量该突变体与野生型在穗长、千粒重、粒长、粒宽及粒长与宽比值并分析比较其差异;制作突变体与野生型茎秆横切面石蜡切片,从解剖学角度分析比较两者在维管束的数目、形态、大小等方面的差异。结果显示,突变体与野生型在穗长、粒长、粒宽及长与宽比值上无明显差异,但在穗型弯曲度及千粒重上有显著差异,曲穗突变体的穗千粒重低于野生型穗千粒重;解剖学研究发现,突变体与野生型水稻植株在厚壁组织、薄壁组织厚度及维管束数目方面无明显差异,但在内轮维管束的形状上有显著差异,曲穗突变体内轮维管束横切面上呈圆形,而野生型内轮维管束呈椭圆形。由此推测,Ds插入部位基因的表达对水稻茎秆维管束结构的改变有影响,维管束在一定程度上影响籽粒灌浆物质的运转,是营养器官向穗部输送水分、矿物质和有机养分的通道,维管束形状的改变可能与形成曲穗突变体的原因有关。2.Ds插入部位侧翼序列的扩增及相关基因的结构及功能的预测。基于TAIL-PCR技术,通过不同简并引物与特异引物的搭配,扩增并分离到Ds插入部位的侧翼序列,经核苷酸数据库(NCBI-BLAST)在线比对分析,得到Ds插入部位相关基因,并用生物软件分析其可能结构;根据获得的侧翼序列设计引物,采用RACE技术信息学鉴定Ds的插入对相关基因的表达和结构的影响。结果显示,Ds插入在水稻3号染色体(Sequence ID:dbj|AP014959.1|)的基因之间,距离下游类泛素蛋白酶1(Ulp1)基因1554 bp核苷酸。对Ds插入位点下游基因进行启动子预测,Promoter 2.0 Prediction和Web Promoter Scan Service及FGENESH2.6和PLACE软件预测结果显示,Ds插入位点可能位于其下游类泛素蛋白酶1(Ulp1)基因的启动子区域,该预测区域有关于花序形成相关的启动子元件;采用RACE技术对野生型水稻和突变体水稻中获得的Ds插入相关标记基因的cDNA序列进行比较,发现Ds插入前后下游基因的cDNA 3’末端序列无变化,即Ds的插入对下游类泛素蛋白酶1基因(Ulp1)的核苷酸序列无影响。综合上述实验结果,我们初步预测,Ds的插入影响了Ds插入位点下游基因类泛素蛋白酶1(Ulp1)基因的启动子区域,进而影响了该基因的表达,而Ulp1在SUMO/Smt3代谢途径中有重要的调控作用。SUMO/Smt3代谢途径影响植物开花时期,最终在突变体水稻植株表型上就表现为穗型的改变。
[Abstract]:Rice ear morphology is one of the most important agronomic traits affecting rice yield. Studies have proved that erect panicle rice has more advantages than curved panicle rice. Therefore, it is of great significance to study the location of ear erect and panicle bending related genes in optimizing panicle type to improve rice yield. Functional genomics and rice mutant library constructed by Ac/Ds system have been widely used in rice molecular genetics and breeding. After years of cultivation of Ac/Ds inserting mutant lines of Japonica rice variety Dongjin, a panicle mutant with Ds insertion has been screened from the laboratory, from morphology, physiology and molecule, respectively. The possible reasons for the formation of panicle bending are preliminarily analyzed in biology, which can provide reference for the study of rice breeding for high quality and high yield. This study mainly includes two aspects: the morphological and physiological identification of 1. panicle mutants. 50 plants were randomly selected from the wild type and the mature mutant of the mutant in the test field, and the mutant and the wild type were measured in the ear length. The ratio of 1000 grain weight, grain length, grain width and grain length to width was analyzed and compared. The difference in the number, shape and size of vascular bundles between the mutants and the cross section of the wild stem was analyzed and compared. The results showed that the ratio of the mutant and the wild type to the ear length, the grain length, the grain width and the length to width ratio were not clear. There were significant differences between the panicle type and the 1000 grain weight, and the 1000 grain weight per ear of the panicle mutant was lower than the wild type 1000 grain weight. The anatomical study found that there was no difference in the thickness of the parenchyma, the thickness of the parenchyma and the number of vascular bundles in the mutant and the wild type rice plants, but there were significant differences in the shape of the vascular bundle of the inner wheel. The transverse section of the vascular bundle in the panicle mutation was round, but the vascular bundle of the wild type was elliptical. Therefore, the expression of the gene in the Ds insertion site had an influence on the change of the structure of the vascular bundle of the rice stem. The vascular bundle affected the grain filling material to a certain extent, and the nutrient organs conveyed the water, mineral and organic matter to the panicle. The channel of nutrient and the change of the shape of vascular bundle may be related to the cause of the formation of the flanking sequence of the.2.Ds insertion site and the prediction of the structure and function of the related genes. Based on the TAIL-PCR technique, the flanking sequence of the insertion site of the Ds is amplified and separated by the combination of different degenerate primers and specific primers, and the number of nucleotides is obtained by the number of nucleotide sequences. According to the NCBI-BLAST online comparison analysis, the related genes of the Ds insertion site were obtained, and the possible structure was analyzed with the biological software. According to the acquired flanking sequence, the primers were designed and the RACE technique informatics was used to identify the influence of the insertion of Ds on the expression and structure of the related genes. The results showed that Ds was inserted on the 3 chromosome of rice (Sequence ID:dbj|AP01). The gene of 4959.1| is between the downstream ubiquitin protease 1 (Ulp1) gene 1554 BP nucleotides. The promoter of the downstream gene of the Ds insertion site is predicted. The prediction results of Promoter 2 Prediction and Web Promoter Scan Service and FGENESH2.6 and PLACE software suggest that the insertion site may be located at the downstream of its Ubiquitin protease gene. In the promoter region, there are promoter elements related to the formation of inflorescence; RACE technique is used to compare the cDNA sequences of Ds inserting related markers in wild rice and mutant rice. It is found that the cDNA 3 'terminal sequence of the downstream genes before and after Ds insertion does not change, that is, the insertion of Ds to the downstream ubiquitin eggs. The nucleotide sequence of the white enzyme 1 gene (Ulp1) has no effect. Combining the above results, we preliminarily predict that the insertion of Ds affects the promoter region of the ubiquitin protease 1 (Ulp1) gene of the downstream gene of the Ds insertion site, thereby affecting the expression of the gene, and Ulp1 has an important regulatory role in the.SUMO/Smt3 generation pathway in the SUMO/ Smt3 metabolic pathway. The diameter affects the flowering period of plants, and finally changes in the phenotype of the mutant rice plants.
【学位授予单位】：苏州大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S511

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