甘蓝BolSDG8基因克隆与功能的初步研究

发布时间：2019-01-04 11:03

【摘要】：开花是植物由营养生长向生殖生长转变的重要过程,对生殖发育特别是种子的发育有重要影响。已有研究证明：拟南芥SDG8通过调控开花关键基因FLC位点上组蛋白H3中第36位赖氨酸的二、三甲基化水平促进其转录表达,进而抑制植株早花。甘蓝型油菜是甘蓝与白菜型油菜杂交形成的异源四倍体,在长江中下游地区有广泛的栽培。深入了解甘蓝型油菜的开花机制对油菜生产有重要作用。而了解甘蓝的开花调控机制对深入理解甘蓝型油菜的开花有重要的奠基作用。我们在甘蓝的全基因组序列中,以拟南芥SDG8基因序列作为比对,找到一段与SDG8相似度很高的片段,命名为BolSDG8基因。利用生物信息学、基因工程和分子生物学的技术和方法对甘蓝BolSDG8基因进行了初步研究,获得了如下研究结果：1生物信息学分析的结果显示：BolSDG8具有一个AWS结构域、一个SET结构域和一个post SET结构域,在结构上和拟南芥的AtSDG8相似。AWS结构域与SET结构域相关联,其具体功能尚不清楚。Post SET结构域在组蛋白赖氨酸甲基转移酶中大量存在,可以与组蛋白C末端残基作用提供一个芳香基团形成疏水通道,并参与构成部分酶活位点。SET结构域则由130个氨基酸组成在进化上保守的结构域,几乎存在于所有的甲基转移酶中。2通过对BolSDG8和AtSDG8进行比对,找出一段约为359bp的保守序列,将其命名为BolSDG8-RRAi。构建了的RNA干扰载体,并将其转化至哥伦比亚野生型拟南芥中,并获得了稳定遗传的转基因植株。3构建了BolSDG8表达载体并将将其转入拟南芥sdg8突变体中,获得可稳定遗传的植株。4通过对干扰载体T3代和表达载体T3代转基因植物的表型观察和统计分析,我们发现：BolSDG8 RNA干扰转基因植株表现出了与拟南芥sdg8突变体相似的生物学表型,即植株相对变小,开花时间较野生型早5天,表现出明显早花的表型。同时转入拟南芥sdg8的pFGC5941-BolSDG8-1载体,使原本早花的sdg8,在开花时间、莲座叶数目等表型上出现了与野生型相似的表型,说明BolSDG8能恢复拟南芥SDG8的功能。上述结果暗示BolSDG8与拟南芥SDG8在调控植物开花上具有相似的生物学功能。
[Abstract]:Flowering is an important process from vegetative growth to reproductive growth, which has an important effect on reproductive development, especially seed development. It has been shown that Arabidopsis thaliana SDG8 promotes its transcription expression by regulating the dimethylation level of lysine at the 36th position of histone H3 on the FLC site of the key flowering gene and thus inhibits the early flowering of plants. Brassica napus (Brassica napus L.) is an allotetraploid formed by crossing Brassica napus with Brassica campestris. It is widely cultivated in the middle and lower reaches of the Yangtze River. Understanding the flowering mechanism of Brassica napus has an important role in rape production. Understanding the flowering regulation mechanism of Brassica napus has an important role in understanding the flowering of Brassica napus. Using Arabidopsis thaliana SDG8 gene as alignment, we found a fragment with high similarity to SDG8 in the whole genome sequence of Brassica oleracea. We named it BolSDG8 gene. Using bioinformatics, genetic engineering and molecular biology techniques and methods, the BolSDG8 gene of cabbage was preliminarily studied. The following results were obtained: 1 the results of bioinformatics analysis showed that BolSDG8 had a AWS domain. A SET domain and a post SET domain are similar to AtSDG8 in Arabidopsis thaliana. AWS domain is associated with SET domain. The specific function of. Post SET domain in histone lysine methyltransferase is not clear. It can provide a hydrophobic channel to form an aromatic group and participate in part of the enzyme activity site. The SET domain is composed of 130 amino acids and is an evolutionarily conserved domain. In almost all methyltransferases. 2 by comparing BolSDG8 and AtSDG8, a conserved sequence about 359bp is found and named BolSDG8-RRAi. The RNA interference vector was constructed and transformed into wild-type Arabidopsis thaliana, and a stable genetic transgenic plant was obtained. 3 BolSDG8 expression vector was constructed and transferred into Arabidopsis thaliana sdg8 mutant. Stable and hereditary plants were obtained. 4 phenotypic observation and statistical analysis of transgenic plants with interference vector T3 and expression vector T3, We found that the transgenic plants with BolSDG8 RNA interference showed the same biological phenotype as Arabidopsis sdg8 mutants, that is, the plants were relatively small, the flowering time was 5 days earlier than that of the wild type, and the phenotype was obviously earlier than that of the wild type. At the same time, pFGC5941-BolSDG8-1 vector of Arabidopsis thaliana sdg8 was transferred to make sdg8, with early flowering appear similar phenotypes to wild type in flowering time and number of rosette leaves, indicating that BolSDG8 can restore the function of SDG8 in Arabidopsis thaliana. These results suggest that BolSDG8 and Arabidopsis SDG8 have similar biological functions in regulating the flowering of plants.
【学位授予单位】：湖南农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S635;Q943.2

【参考文献】