水稻空育131苗期耐冷候选基因OsrbcS3的克隆和功能分析

发布时间：2018-12-10 11:47

【摘要】：水稻是世界最重要的粮食作物之一,世界上半数以上的人口以稻米为主食。黑龙江省地处中国的最北部,是我国最重要的水稻粮食生产基地,而低温冷害是导致黑龙江省水稻减产的主要障碍之一。其中,随着水稻直播面积的不断扩大,苗期冷害也越来越受到水稻研究者的重视。因此,研究水稻苗期耐冷基因,探明水稻苗期耐冷分子机理,对提高作物抗逆境胁迫具有重要作用。本实验以耐冷水稻品种空育131(K131)和冷敏感品种龙粳11(L11)为研究对象,通过水稻数字表达谱,选取一个与光合作用有关的耐冷响应基因OsrbcS3,利用PCR、实时荧光定量PCR、生物信息学分析、生理指标测定、Gateway克隆技术、农杆菌介导法等方法,从DNA水平、mRNA水平、蛋白水平对OsrbcS3基因进行研究,从而确定OsrbcS3基因为水稻耐冷的相关基因。主要结果如下:1、供试的黑龙江省第一、第二和第三积温区的12个水稻品种苗期耐冷性差异显著,筛选出耐冷性强的水稻品种K131和冷敏感品种L11用于实验。2、通过对12个水稻品种芽期、苗期、孕穗期耐冷相关性进行分析,得出苗期与芽期以及苗期与孕穗期耐冷性呈显著正相关。但也有例外,如龙粳21在苗期耐冷性较弱,但孕穗期耐冷性较强。3、水稻K131和L11中克隆的OsrbcS3基因序列与NCBI上的基因完全吻合,且K131-OsrbcS3与L11-OsrbcS3基因碱基序列完全相同,CDS区均为528bp,编码175个氨基酸。4、以0h为对照,对冷处理2h、5h、12h、24h的OsrbcS3基因进行表达量分析,结果显示,冷胁迫下K131和L11中OsrbcS3基因均上调表达,K131中OsrbcS3基因高表达,L11中OsrbcS3基因表达较低,两个品种耐冷性差异的原因很可能是由于基因表达量的不同导致。5、对低温处理下K131和L11中Rubisco活性等生理指标的测定,进一步说明OsrbcS3基因表达量的差异可能是K131耐冷性强于L11的原因。6、对L11愈伤组织进行遗传转化,获得转OsrbcS3基因植株,为验证OsrbcS3基因的功能准备材料。
[Abstract]:Rice is one of the most important food crops in the world. Heilongjiang Province is located in the northern part of China and is the most important rice grain production base in China. Low temperature chilling injury is one of the main obstacles to yield reduction of rice in Heilongjiang Province. With the continuous expansion of direct seeding area, rice researchers pay more and more attention to chilling injury in seedling stage. Therefore, it is important to study the cold tolerance genes of rice seedlings and to find out the molecular mechanism of cold tolerance in rice seedlings, which plays an important role in improving the stress tolerance of crops. In this experiment, chilling tolerant rice cultivar Gangyu 131 (K131) and cold sensitive cultivar Longjing 11 (L11) were used as research objects. By using digital expression profile of rice, a cold-tolerant response gene OsrbcS3, related to photosynthesis was selected to use PCR, real-time fluorescence quantitative PCR,. Bioinformatics analysis, physiological index determination, Gateway cloning technique and Agrobacterium tumefaciens mediated method were used to study OsrbcS3 gene from DNA level, mRNA level and protein level. The main results are as follows: 1. There are significant differences in cold tolerance of 12 rice varieties in the first, second and third accumulated temperature areas of Heilongjiang Province. K131 and L11, which have strong cold tolerance, were selected for experiment. 2. By analyzing the correlation between cold tolerance of 12 rice varieties at bud stage, seedling stage and booting stage, it was concluded that there was a significant positive correlation between seedling stage and bud stage and between seedling stage and booting stage. But there were some exceptions, such as the cold tolerance of Longjing 21 was weak at seedling stage, but the cold tolerance was stronger at booting stage. The sequence of OsrbcS3 gene cloned in rice K131 and L11 was completely consistent with the gene on NCBI, and the base sequence of K131-OsrbcS3 and L11-OsrbcS3 gene was identical. The CDS region was 528 BP, encoding 175 amino acids. The expression of OsrbcS3 gene was analyzed at 2 h, 5 h, 12 h and 24 h after cold treatment. The results showed that the expression of OsrbcS3 gene in K131 and L11 was up-regulated under cold stress, and the expression of OsrbcS3 gene in K131 was higher than that in K131. The low expression of OsrbcS3 gene in L11 and the difference of cold tolerance between the two cultivars were probably due to the difference in gene expression. The physiological indexes such as Rubisco activity in K131 and L11 were measured under low temperature treatment. It was further explained that the difference of OsrbcS3 gene expression level may be the reason that K131 is stronger than L11 in cold tolerance. 6. The callus of L11 was transformed into transgenic OsrbcS3 gene plants to prepare materials for verifying the function of OsrbcS3 gene.
【学位授予单位】：黑龙江大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S511

【相似文献】