甘藍BolSDG8基因克隆與功能的初步研究
發(fā)布時間：2019-01-04 11:03
【摘要】：開花是植物由營養(yǎng)生長向生殖生長轉(zhuǎn)變的重要過程,對生殖發(fā)育特別是種子的發(fā)育有重要影響。已有研究證明：擬南芥SDG8通過調(diào)控開花關鍵基因FLC位點上組蛋白H3中第36位賴氨酸的二、三甲基化水平促進其轉(zhuǎn)錄表達,進而抑制植株早花。甘藍型油菜是甘藍與白菜型油菜雜交形成的異源四倍體,在長江中下游地區(qū)有廣泛的栽培。深入了解甘藍型油菜的開花機制對油菜生產(chǎn)有重要作用。而了解甘藍的開花調(diào)控機制對深入理解甘藍型油菜的開花有重要的奠基作用。我們在甘藍的全基因組序列中,以擬南芥SDG8基因序列作為比對,找到一段與SDG8相似度很高的片段,命名為BolSDG8基因。利用生物信息學、基因工程和分子生物學的技術和方法對甘藍BolSDG8基因進行了初步研究,獲得了如下研究結(jié)果：1生物信息學分析的結(jié)果顯示：BolSDG8具有一個AWS結(jié)構(gòu)域、一個SET結(jié)構(gòu)域和一個post SET結(jié)構(gòu)域,在結(jié)構(gòu)上和擬南芥的AtSDG8相似。AWS結(jié)構(gòu)域與SET結(jié)構(gòu)域相關聯(lián),其具體功能尚不清楚。Post SET結(jié)構(gòu)域在組蛋白賴氨酸甲基轉(zhuǎn)移酶中大量存在,可以與組蛋白C末端殘基作用提供一個芳香基團形成疏水通道,并參與構(gòu)成部分酶活位點。SET結(jié)構(gòu)域則由130個氨基酸組成在進化上保守的結(jié)構(gòu)域,幾乎存在于所有的甲基轉(zhuǎn)移酶中。2通過對BolSDG8和AtSDG8進行比對,找出一段約為359bp的保守序列,將其命名為BolSDG8-RRAi。構(gòu)建了的RNA干擾載體,并將其轉(zhuǎn)化至哥倫比亞野生型擬南芥中,并獲得了穩(wěn)定遺傳的轉(zhuǎn)基因植株。3構(gòu)建了BolSDG8表達載體并將將其轉(zhuǎn)入擬南芥sdg8突變體中,獲得可穩(wěn)定遺傳的植株。4通過對干擾載體T3代和表達載體T3代轉(zhuǎn)基因植物的表型觀察和統(tǒng)計分析,我們發(fā)現(xiàn)：BolSDG8 RNA干擾轉(zhuǎn)基因植株表現(xiàn)出了與擬南芥sdg8突變體相似的生物學表型,即植株相對變小,開花時間較野生型早5天,表現(xiàn)出明顯早花的表型。同時轉(zhuǎn)入擬南芥sdg8的pFGC5941-BolSDG8-1載體,使原本早花的sdg8,在開花時間、蓮座葉數(shù)目等表型上出現(xiàn)了與野生型相似的表型,說明BolSDG8能恢復擬南芥SDG8的功能。上述結(jié)果暗示BolSDG8與擬南芥SDG8在調(diào)控植物開花上具有相似的生物學功能。
[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.
【學位授予單位】：湖南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S635;Q943.2
本文編號：2400199
[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.
【學位授予單位】：湖南農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S635;Q943.2
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