菊花CmFTL1基因可變剪切的功能機理研究
發(fā)布時間：2018-07-28 20:45
【摘要】：菊花(Chrysanthemum morifolium)是我國十大傳統(tǒng)名花之一,因花色豐富、花型多樣而具有很高的觀賞價值;菊花雖品種繁多,但其短日照品種的花期往往集中在秋季和冬季。目前,為實現(xiàn)周年生產(chǎn)而對短日照菊花隨季節(jié)采取遮光或補光的手段造成大量人力、財力的浪費。因此,從分子機理的層面展開菊花開花機制的研究,將為花期調(diào)控、周年生產(chǎn)及新品種的培育提供重要的理論意義。FT基因作為花發(fā)育多條途徑的匯集點,通過整合不同路徑中的信號參與植物的開花進程。已有研究表明,菊花中的CmFTLs基因?qū)Τ苫ㄅc開花均起重要作用;其中CmFTL1基因會由于發(fā)生可變剪切/選擇性剪切產(chǎn)生多個不同轉(zhuǎn)錄本,但這些轉(zhuǎn)錄本的功能與機制尚不明確。為探明CmFTL1基因不同轉(zhuǎn)錄本在菊花開花進程中的功能,對其可變剪切產(chǎn)物進行一定的功能驗證,意在研究這些轉(zhuǎn)錄本間可能存在的功能差異及其對開花的影響。本文研究的主要內(nèi)容與結(jié)論如下:1.在已發(fā)現(xiàn)的秋菊'神馬'('Jinba')中開花基因CmFTL1存在可變剪切的基礎(chǔ)上,對各轉(zhuǎn)錄本的序列與結(jié)構(gòu)特征進行研究。其中,CmFTL1astE134缺失第二個外顯子;CmFTL1ast In1-1、CmFTL1ast In1-2 分別保留不同長度的內(nèi)含子1;CmFTL1ast In1-3不僅保留內(nèi)含子1,還缺失第三個外顯子。另外為明確CmFTL1發(fā)生可變剪切的位點與剪切類型,在DNA水平上進行其內(nèi)含子1的克隆并分析剪切位點。結(jié)果表明,CmFTL1的第一個內(nèi)含子上存在四個可變剪切位點。氨基酸序列分析表明CmFTL1asts均由于序列重排出現(xiàn)提前終止的現(xiàn)象。因此,CmFTL1不同轉(zhuǎn)錄本對開花的影響需后續(xù)進行功能驗證。2.為確定CmFTL1asts可能的功能,以T-DNA插入導致功能突變的擬南芥ft-10晚花表型突變體為研究材料,利用Gateway技術(shù)構(gòu)建了 pMDC43-CmFTL1astE134、pMDC43-CmFTL1astIn1-1、pMDC43-CmFTL1astIn1-2、pMDC43-CmFTL1astIn1-3 共 4個超表達載體以及既有的pHGW-FTpro::CmFTL1載體進行功能回補實驗。結(jié)果表明,CmFTL1具有部分回補晚花表型的功能,而四個轉(zhuǎn)錄本對ft-10的功能回補程度不盡相同,其中CmFTL1astIn1-1、CmFTL1astIn1-2和CmFTL1astIn1-3能對其晚花表型不同程度的回補,CmFTL1astIn1-1和CmFTL1astIn1-2的回補表型最為明顯;而CmFTL1astE134則幾乎不能回補。經(jīng)過cDNA水平的鑒定發(fā)現(xiàn)轉(zhuǎn)入CmFTL1astIn1-1、CmFTL1astIn1-2和CmFTL1astIn1-3的株系存在剪切前和剪切后的兩種菊花CmFTL1的轉(zhuǎn)錄本存在,說明在擬南芥中發(fā)生菊花CmFTL1基因的可變剪切轉(zhuǎn)錄本的轉(zhuǎn)錄后加工。3.為深入探究CmFTL1asts在菊花中可能的功能及其是否參與菊花的花期調(diào)控,還進行了 '神馬'的遺傳轉(zhuǎn)化。通過農(nóng)桿菌介導法進行菊花'神馬'的葉盤侵染分別轉(zhuǎn)入CmFTL1及CmFTL1基因的四個可變剪切轉(zhuǎn)錄本并獲得超表達株系。自然條件下現(xiàn)蕾及開花統(tǒng)計結(jié)果表明,分別轉(zhuǎn)入CmFTL1、CmFTL1astIn1-1、CmFTL1astIn1-2和CmFTL1astIn1-3的株系現(xiàn)蕾時間均略有提前,而轉(zhuǎn)入CmFTL1astE134無明顯變化,這與轉(zhuǎn)基因擬南芥中的表型一致。
[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.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S682.11;Q943.2
本文編號：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.
【學位授予單位】：南京農(nóng)業(yè)大學
【學位級別】：碩士
【學位授予年份】：2016
【分類號】：S682.11;Q943.2
【參考文獻】
相關(guān)期刊論文 前2條
1 于麗霞;姚有林;武曉璐;湯曉倩;鄢波;;擬南芥成花調(diào)控LFY基因的選擇性剪接(英文)[J];廣西植物;2012年03期
2 潘才博;張啟翔;潘會堂;孫明;;菊花FT類似基因的克隆與表達分析[J];園藝學報;2010年05期
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