樟树LFY、UFO和FUL基因克隆及功能分析
发布时间：2018-10-26 13:03
【摘要】：香樟(Cinnamomum camphora)是樟目樟科樟属的代表树木,是一种常绿大乔木。成年樟树枝叶繁茂,气味芬香,是中国长江中下游地区常见园林树种,广泛用于园林绿化和市政道路绿化中。但是,樟树的核果成熟后,会从树上掉落下来,砸在行人、车辆以及地面上,给人们日常生活带来不便,同时还造成一定的污染。针对这个问题,本课题从植物花发育关键基因LFY、UFO、FUL出发,分离克隆樟树中这三个基因的同源基因,分别命名为Cc LFY、Cc UFO、Cc FUL,进行基因功能分析,为以后获得少花少果乃至无花无果的樟树提供一定指导信息。本课题获得Cc LFY、Cc UFO、Cc FUL序列后,构建系统进化树,进行基因序列分析和构建系统进化树,然后进行半定量表达分析,确定这三个基因功能。最后,构建含目的基因的超表载体,进行拟南芥的遗传转化,观察分析转基因拟南芥表型,进行目的基因的功能鉴定。此外,本课题还进行了酵母双杂交实验,分析Cc LFY、Cc UFO之间是否存在互作关系。本课题主要研究结果如下:(1)通过同源克隆技术、侧翼扩增技术和常规PCR,扩增得到了Cc FUL基因全长序列;根据转录组数据、侧翼扩增技术和常规PCR,扩增得到了Cc UFO基因全长序列;利用转录组数据和常规PCR,扩增得到了Cc LFY基因全长序列。然后根据扩增得到的序列,构建系统进化树,进行同源分析,结果表明这三个目的基因与双子叶植物亲缘关系较近。(2)采集樟树不同部位和时期的组织,利用CTAB法提取RNA,进行半定量表达分析,结果表明Cc LFY、Cc UFO、Cc FUL与樟树花发育和果实形成有关。(3)利用p CAMBIA2300表达载体,通过传统质粒重组的方法,成功构建了35S-Cc LFY、35S-Cc UFO、35S-Cc FUL等三个超表载体。然后将其电转进农杆菌菌株GV1303中,最后通过农杆菌介导的方法,进行拟南芥的遗传转化,获得了8株Cc LFY阳性转基因拟南芥,11株Cc UFO阳性转基因拟南芥以及6株Cc FUL阳性转基因拟南芥。观察阳性转基因拟南芥的表型,发现Cc LFY、Cc UFO阳性转基因拟南芥均出现了早花、侧枝转变为花的表型。同时Cc LFY阳性转基因拟南芥还出现分支少,植株底端侧枝直接转化成花的表型,Cc UFO阳性转基因拟南芥还呈现出植株矮小的表型。此外,Cc FUL阳性转基因拟南芥呈现晚花、果荚变短弯曲的表型。这些转基因拟南芥表型有力证明了这三个基因与花发育和果实形成有关。(4)酵母双杂交实验表明Cc LFY、Cc UFO之间的不存在互作关系。
[Abstract]:Cinnamomum camphora (Cinnamomum camphora) is the representative tree of Camphor family, and is a kind of large evergreen tree. The adult camphor tree is a common garden tree in the middle and lower reaches of the Yangtze River. It is widely used in landscape and municipal road greening. However, when the stone fruit of camphor tree ripens, it will fall down from the tree and crash on pedestrians, vehicles and the ground, which will bring inconvenience to people's daily life and cause certain pollution at the same time. In order to solve this problem, we isolated and cloned the homologous genes of the three genes in camphor tree from the key gene LFY,UFO,FUL of plant flower development, and named Cc LFY,Cc UFO,Cc FUL, for gene function analysis. It provides some guiding information for obtaining camphor trees with few flowers and few fruits or no flowers and no fruit. After the Cc LFY,Cc UFO,Cc FUL sequence was obtained, the phylogenetic tree was constructed, gene sequence analysis and phylogenetic tree were constructed, and semi-quantitative expression analysis was carried out to determine the function of the three genes. Finally, the supersurface vector containing the target gene was constructed, the genetic transformation of Arabidopsis thaliana was carried out, the phenotype of transgenic Arabidopsis was observed and analyzed, and the function of the target gene was identified. In addition, yeast two-hybrid experiments were carried out to analyze the interaction between Cc LFY,Cc UFO. The main results are as follows: (1) the full-length sequence of Cc FUL gene was obtained by homologous cloning, flanking amplification and conventional PCR, amplification. According to the transcriptional data, flanking amplification technique and conventional PCR, amplification, the full-length sequence of Cc UFO gene was obtained, and the full-length sequence of Cc LFY gene was obtained by using transcriptome data and conventional PCR, amplification. Then the phylogenetic tree was constructed according to the amplified sequence and the homology analysis was carried out. The results showed that the relationship between the three target genes and dicotyledonous plants was close. (2) the tissues of different parts and periods of camphor tree were collected and RNA, was extracted by CTAB method. The results of semi-quantitative expression analysis showed that Cc LFY,Cc UFO,Cc FUL was related to flower development and fruit formation of camphor tree. (3) 35S-Cc LFY,35S-Cc UFO, was successfully constructed by using p CAMBIA2300 expression vector and traditional plasmid recombination method. 35S-Cc FUL and other three supermeter carriers. At last, through Agrobacterium tumefaciens mediated transformation, 8 Cc LFY positive transgenic Arabidopsis thaliana strains were obtained. 11 Cc UFO positive transgenic Arabidopsis and 6 Cc FUL positive transgenic Arabidopsis thaliana. The phenotype of positive transgenic Arabidopsis thaliana was observed. It was found that all transgenic Arabidopsis thaliana with Cc LFY,Cc UFO positive appeared early flower and the lateral branch changed into flower phenotype. At the same time, Cc LFY positive transgenic Arabidopsis had fewer branches, and, Cc UFO positive transgenic Arabidopsis showed short phenotype. In addition, Cc FUL positive transgenic Arabidopsis thaliana showed the phenotype of late flower and short curving fruit pod. The phenotypes of these transgenic Arabidopsis thaliana showed that the three genes were related to flower development and fruit formation. (4) yeast two-hybrid experiments showed that there was no interaction between Cc LFY,Cc UFO.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S792.23
本文编号：2295805
[Abstract]:Cinnamomum camphora (Cinnamomum camphora) is the representative tree of Camphor family, and is a kind of large evergreen tree. The adult camphor tree is a common garden tree in the middle and lower reaches of the Yangtze River. It is widely used in landscape and municipal road greening. However, when the stone fruit of camphor tree ripens, it will fall down from the tree and crash on pedestrians, vehicles and the ground, which will bring inconvenience to people's daily life and cause certain pollution at the same time. In order to solve this problem, we isolated and cloned the homologous genes of the three genes in camphor tree from the key gene LFY,UFO,FUL of plant flower development, and named Cc LFY,Cc UFO,Cc FUL, for gene function analysis. It provides some guiding information for obtaining camphor trees with few flowers and few fruits or no flowers and no fruit. After the Cc LFY,Cc UFO,Cc FUL sequence was obtained, the phylogenetic tree was constructed, gene sequence analysis and phylogenetic tree were constructed, and semi-quantitative expression analysis was carried out to determine the function of the three genes. Finally, the supersurface vector containing the target gene was constructed, the genetic transformation of Arabidopsis thaliana was carried out, the phenotype of transgenic Arabidopsis was observed and analyzed, and the function of the target gene was identified. In addition, yeast two-hybrid experiments were carried out to analyze the interaction between Cc LFY,Cc UFO. The main results are as follows: (1) the full-length sequence of Cc FUL gene was obtained by homologous cloning, flanking amplification and conventional PCR, amplification. According to the transcriptional data, flanking amplification technique and conventional PCR, amplification, the full-length sequence of Cc UFO gene was obtained, and the full-length sequence of Cc LFY gene was obtained by using transcriptome data and conventional PCR, amplification. Then the phylogenetic tree was constructed according to the amplified sequence and the homology analysis was carried out. The results showed that the relationship between the three target genes and dicotyledonous plants was close. (2) the tissues of different parts and periods of camphor tree were collected and RNA, was extracted by CTAB method. The results of semi-quantitative expression analysis showed that Cc LFY,Cc UFO,Cc FUL was related to flower development and fruit formation of camphor tree. (3) 35S-Cc LFY,35S-Cc UFO, was successfully constructed by using p CAMBIA2300 expression vector and traditional plasmid recombination method. 35S-Cc FUL and other three supermeter carriers. At last, through Agrobacterium tumefaciens mediated transformation, 8 Cc LFY positive transgenic Arabidopsis thaliana strains were obtained. 11 Cc UFO positive transgenic Arabidopsis and 6 Cc FUL positive transgenic Arabidopsis thaliana. The phenotype of positive transgenic Arabidopsis thaliana was observed. It was found that all transgenic Arabidopsis thaliana with Cc LFY,Cc UFO positive appeared early flower and the lateral branch changed into flower phenotype. At the same time, Cc LFY positive transgenic Arabidopsis had fewer branches, and, Cc UFO positive transgenic Arabidopsis showed short phenotype. In addition, Cc FUL positive transgenic Arabidopsis thaliana showed the phenotype of late flower and short curving fruit pod. The phenotypes of these transgenic Arabidopsis thaliana showed that the three genes were related to flower development and fruit formation. (4) yeast two-hybrid experiments showed that there was no interaction between Cc LFY,Cc UFO.
【学位授予单位】：华中农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S792.23
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1 周琴;樟树LFY、UFO和FUL基因克隆及功能分析[D];华中农业大学;2016年
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