菊花CmFTL1基因可变剪切的功能机理研究
发布时间：2018-07-28 20:45
【摘要】：菊花(Chrysanthemum morifolium)是我国十大传统名花之一,因花色丰富、花型多样而具有很高的观赏价值;菊花虽品种繁多,但其短日照品种的花期往往集中在秋季和冬季。目前,为实现周年生产而对短日照菊花随季节采取遮光或补光的手段造成大量人力、财力的浪费。因此,从分子机理的层面展开菊花开花机制的研究,将为花期调控、周年生产及新品种的培育提供重要的理论意义。FT基因作为花发育多条途径的汇集点,通过整合不同路径中的信号参与植物的开花进程。已有研究表明,菊花中的CmFTLs基因对成花与开花均起重要作用;其中CmFTL1基因会由于发生可变剪切/选择性剪切产生多个不同转录本,但这些转录本的功能与机制尚不明确。为探明CmFTL1基因不同转录本在菊花开花进程中的功能,对其可变剪切产物进行一定的功能验证,意在研究这些转录本间可能存在的功能差异及其对开花的影响。本文研究的主要内容与结论如下:1.在已发现的秋菊'神马'('Jinba')中开花基因CmFTL1存在可变剪切的基础上,对各转录本的序列与结构特征进行研究。其中,CmFTL1astE134缺失第二个外显子;CmFTL1ast In1-1、CmFTL1ast In1-2 分别保留不同长度的内含子1;CmFTL1ast In1-3不仅保留内含子1,还缺失第三个外显子。另外为明确CmFTL1发生可变剪切的位点与剪切类型,在DNA水平上进行其内含子1的克隆并分析剪切位点。结果表明,CmFTL1的第一个内含子上存在四个可变剪切位点。氨基酸序列分析表明CmFTL1asts均由于序列重排出现提前终止的现象。因此,CmFTL1不同转录本对开花的影响需后续进行功能验证。2.为确定CmFTL1asts可能的功能,以T-DNA插入导致功能突变的拟南芥ft-10晚花表型突变体为研究材料,利用Gateway技术构建了 pMDC43-CmFTL1astE134、pMDC43-CmFTL1astIn1-1、pMDC43-CmFTL1astIn1-2、pMDC43-CmFTL1astIn1-3 共 4个超表达载体以及既有的pHGW-FTpro::CmFTL1载体进行功能回补实验。结果表明,CmFTL1具有部分回补晚花表型的功能,而四个转录本对ft-10的功能回补程度不尽相同,其中CmFTL1astIn1-1、CmFTL1astIn1-2和CmFTL1astIn1-3能对其晚花表型不同程度的回补,CmFTL1astIn1-1和CmFTL1astIn1-2的回补表型最为明显;而CmFTL1astE134则几乎不能回补。经过cDNA水平的鉴定发现转入CmFTL1astIn1-1、CmFTL1astIn1-2和CmFTL1astIn1-3的株系存在剪切前和剪切后的两种菊花CmFTL1的转录本存在,说明在拟南芥中发生菊花CmFTL1基因的可变剪切转录本的转录后加工。3.为深入探究CmFTL1asts在菊花中可能的功能及其是否参与菊花的花期调控,还进行了 '神马'的遗传转化。通过农杆菌介导法进行菊花'神马'的叶盘侵染分别转入CmFTL1及CmFTL1基因的四个可变剪切转录本并获得超表达株系。自然条件下现蕾及开花统计结果表明,分别转入CmFTL1、CmFTL1astIn1-1、CmFTL1astIn1-2和CmFTL1astIn1-3的株系现蕾时间均略有提前,而转入CmFTL1astE134无明显变化,这与转基因拟南芥中的表型一致。
[Abstract]:Chrysanthemum (Chrysanthemum morifolium) is one of the ten traditional flowers in China, which has high ornamental value because of its rich color and various flower forms. Although there are many varieties of chrysanthemum, the flowering period of chrysanthemum varieties with short sunshine is usually concentrated in autumn and winter. At present, in order to realize annual production, it is a waste of manpower and financial resources to take the means of shading or replenishing light on the short sunshine chrysanthemum with the season. Therefore, the study on flowering mechanism of chrysanthemum from the molecular mechanism will provide important theoretical significance for the regulation of flowering, annual production and breeding of new varieties. FT gene can be used as a collection point for multiple pathways of flower development. The flowering process of plants is involved by integrating the signals in different paths. It has been shown that CmFTLs gene in chrysanthemum plays an important role in flower formation and flowering, and that CmFTL1 gene can produce many different transcripts due to variable shear / selective shearing, but the function and mechanism of these transcripts are not clear. In order to study the function of different transcripts of CmFTL1 gene in flowering process of chrysanthemum, the function of its variable shearing products was verified in order to study the possible functional differences among these transcripts and their effects on flowering. The main contents and conclusions of this paper are as follows: 1. The sequence and structural characteristics of the transcripts were studied on the basis of the variable splicing of the flowering gene CmFTL1 found in Akiba 'Shenma'. The second exon of CmFTL1ast In1-1ast CmFTL1ast In1-2 contains intron 1 and CmFTL1ast In1-3 of different lengths, not only in intron 1, but also in the third exon of CmFTL1astE134. In addition, in order to identify the site and type of variable shear in CmFTL1, the intron 1 was cloned at DNA level and the shear site was analyzed. The results showed that there were four variable shear sites in the first intron of CmFTL1. Amino acid sequence analysis showed that CmFTL1asts was terminated early due to sequence rearrangement. Therefore, the effect of different transcripts of CmFTL1 on flowering needs further functional verification. In order to determine the possible function of CmFTL1asts, four pMDC43-CmFTL1astIn1-1pMDC43-CmFTL1astIn1-3 (pMDC43-CmFTL1astIn1-3) pMDC43-CmFTL1astIn1-3 (pMDC43-CmFTL1astIn1-3) superexpression vectors were constructed using Gateway technique. The results showed that CmFTL1 had the function of partially complementing late flowering phenotypes, and the four transcripts had different degree of functional recharge to ft-10, and CmFTL1astIn1astIn1-2 and CmFTL1astIn1-3 could complement the phenotypes of CmFTL1astIn1-1 and CmFTL1astIn1-2 with different degrees of late blooming phenotypes, and the phenotypes of CmFTL1astIn1-1 and CmFTL1astIn1-2 were the most obvious. CmFTL1astE134, on the other hand, can hardly make up for it. The identification of cDNA level showed that there were two kinds of CmFTL1 transcripts of chrysanthemum before and after shearing in transgenic lines CmFTL1astIn1-2 and CmFTL1astIn1-2, indicating that the post-transcriptional processing of variable shearing transcripts of CmFTL1 gene of chrysanthemum occurred in Arabidopsis thaliana. In order to explore the possible function of CmFTL1asts in chrysanthemum and whether it is involved in flowering regulation of chrysanthemum, the genetic transformation of 'Shenma' was carried out. Agrobacterium tumefaciens was used to infect the leaves of chrysanthemum 'Shenma' and transferred into four variable shear transcripts of CmFTL1 and CmFTL1 genes, respectively, and superexpressed lines were obtained. The results of budding and flowering statistics showed that the budding time of the lines transferred to CmFTL1astIn1-1 and CmFTL1astIn1-2 and CmFTL1astIn1-3 were slightly earlier than that of CmFTL1astE134, which was consistent with the phenotype of transgenic Arabidopsis thaliana.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S682.11;Q943.2
本文编号：2151526
[Abstract]:Chrysanthemum (Chrysanthemum morifolium) is one of the ten traditional flowers in China, which has high ornamental value because of its rich color and various flower forms. Although there are many varieties of chrysanthemum, the flowering period of chrysanthemum varieties with short sunshine is usually concentrated in autumn and winter. At present, in order to realize annual production, it is a waste of manpower and financial resources to take the means of shading or replenishing light on the short sunshine chrysanthemum with the season. Therefore, the study on flowering mechanism of chrysanthemum from the molecular mechanism will provide important theoretical significance for the regulation of flowering, annual production and breeding of new varieties. FT gene can be used as a collection point for multiple pathways of flower development. The flowering process of plants is involved by integrating the signals in different paths. It has been shown that CmFTLs gene in chrysanthemum plays an important role in flower formation and flowering, and that CmFTL1 gene can produce many different transcripts due to variable shear / selective shearing, but the function and mechanism of these transcripts are not clear. In order to study the function of different transcripts of CmFTL1 gene in flowering process of chrysanthemum, the function of its variable shearing products was verified in order to study the possible functional differences among these transcripts and their effects on flowering. The main contents and conclusions of this paper are as follows: 1. The sequence and structural characteristics of the transcripts were studied on the basis of the variable splicing of the flowering gene CmFTL1 found in Akiba 'Shenma'. The second exon of CmFTL1ast In1-1ast CmFTL1ast In1-2 contains intron 1 and CmFTL1ast In1-3 of different lengths, not only in intron 1, but also in the third exon of CmFTL1astE134. In addition, in order to identify the site and type of variable shear in CmFTL1, the intron 1 was cloned at DNA level and the shear site was analyzed. The results showed that there were four variable shear sites in the first intron of CmFTL1. Amino acid sequence analysis showed that CmFTL1asts was terminated early due to sequence rearrangement. Therefore, the effect of different transcripts of CmFTL1 on flowering needs further functional verification. In order to determine the possible function of CmFTL1asts, four pMDC43-CmFTL1astIn1-1pMDC43-CmFTL1astIn1-3 (pMDC43-CmFTL1astIn1-3) pMDC43-CmFTL1astIn1-3 (pMDC43-CmFTL1astIn1-3) superexpression vectors were constructed using Gateway technique. The results showed that CmFTL1 had the function of partially complementing late flowering phenotypes, and the four transcripts had different degree of functional recharge to ft-10, and CmFTL1astIn1astIn1-2 and CmFTL1astIn1-3 could complement the phenotypes of CmFTL1astIn1-1 and CmFTL1astIn1-2 with different degrees of late blooming phenotypes, and the phenotypes of CmFTL1astIn1-1 and CmFTL1astIn1-2 were the most obvious. CmFTL1astE134, on the other hand, can hardly make up for it. The identification of cDNA level showed that there were two kinds of CmFTL1 transcripts of chrysanthemum before and after shearing in transgenic lines CmFTL1astIn1-2 and CmFTL1astIn1-2, indicating that the post-transcriptional processing of variable shearing transcripts of CmFTL1 gene of chrysanthemum occurred in Arabidopsis thaliana. In order to explore the possible function of CmFTL1asts in chrysanthemum and whether it is involved in flowering regulation of chrysanthemum, the genetic transformation of 'Shenma' was carried out. Agrobacterium tumefaciens was used to infect the leaves of chrysanthemum 'Shenma' and transferred into four variable shear transcripts of CmFTL1 and CmFTL1 genes, respectively, and superexpressed lines were obtained. The results of budding and flowering statistics showed that the budding time of the lines transferred to CmFTL1astIn1-1 and CmFTL1astIn1-2 and CmFTL1astIn1-3 were slightly earlier than that of CmFTL1astE134, which was consistent with the phenotype of transgenic Arabidopsis thaliana.
【学位授予单位】：南京农业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S682.11;Q943.2
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相关期刊论文 前2条
1 于丽霞;姚有林;武晓璐;汤晓倩;鄢波;;拟南芥成花调控LFY基因的选择性剪接(英文)[J];广西植物;2012年03期
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