神农香菊DiaMYB基因的表达特性及抗逆功能研究

发布时间：2018-03-19 14:04

本文选题：神农香菊　切入点：DiaMYB　出处：《东北林业大学》2016年硕士论文　论文类型：学位论文

【摘要】：神农香菊(Dendranthema indicum var.aromaticum)是菊科菊属中一种新资源植物,本文以神农香菊中成功克隆出的DiaMYB基因为研究对象,以构建成功的含有DiaMYB基因的PCAMBIA1301-MYB表达载体,通过农杆菌蘸花法转化拟南芥,筛选培养获得成活率均为100%的三个阳性植株,继续筛选并培养至T3代。研究转基因拟南芥在盐和干旱条件下的酶活性、叶绿素含量、光合作用生理指标,构建含有绿色荧光蛋白pBI121-MYB-GFP表达载体,观察亚细胞定位。实时荧光定量分析在逆境胁迫下转DiaMYB基因植株中胁迫相关基因表达量。本文主要研究结果如下：1.利用农杆菌蘸花法将神农香菊DiaMYB基因转入拟南芥中,获得三个阳性株系,命名为MYB-1、MYB-2、MYB-3,经过PCR和GUS染色鉴定,证实DiaMYB基因已成功转入拟南芥基因组中。实时荧光定量方法分析过表达DiaMYB基因拟南芥不同器官的表达量,结果表明：在茎、叶和花中的表达量稍高,其次是果,在根中的表达量最低。利用DiaMYB基因构建含有绿色荧光蛋白融合表达载体pBI121-MYB-GFP,通过基因枪法转入洋葱表皮细胞,显示DiaMYB基因定位于细胞核上。2.取T3代转DiaMYB基因拟南芥3个株系和野生型拟南芥进行高盐胁迫,结果显示：随着盐溶液浓度的增加, 转DiaMYB基因拟南芥各株系种子的萌发率及成苗期成活率、超氧化物歧化酶(SOD)和过氧化物酶(POD)活性、叶绿素￡L/叶绿素b、类胡萝卜素/叶绿素、净光合速率(Pn)、气孔导度(Gs)以及胞间CO2浓度(Ci)的数值结果均得出转DiaMYB基因拟南芥各株系表现出了更强于野生型的耐盐性。高盐胁迫下的实时荧光定量PCR结果显示,转DiaMYB基因拟南芥中RD22、 RD29A、RAB18、COR47、ABA1的表达量明显增加,与此同时,ABI1、HAB1基因的表达量都稍有下降且都低于野生型植株,DiaMYB基因在盐胁迫下的表达量逐渐升高。3.取T3代转DiaMYB基因拟南芥3个株系和野生型拟南芥进行干旱胁迫,结果显示：随着干旱胁迫的递增,转DiaMYB基因拟南芥各株系种子的萌发率及成苗期成活率、超氧化物歧化酶(SOD)和过氧化物酶(POD)活性、叶绿素a/叶绿素b、类胡萝卜素／叶绿素、净光合速率(Pn)、气孔导度(Gs)以及胞间C02浓度(Ci)的数值结果均得出转DiaMYB基因拟南芥各株系表现出了更强于野生型的耐旱性。干旱胁迫下的实时荧光定量PCR结果显示,转DiaMYB基因拟南芥中RD22、 RD29A、RAB18、COR47、ABA1的表达量明显增加,与此同时,ABI1、HAB1基因的表达量都稍有下降且都低于野生型植株,DiaMYB基因在干旱胁迫下的表达量先升高后下降。
[Abstract]:Dendranthema indicum var. aromaticum is a new resource plant of Compositae. In this paper, the DiaMYB gene which was successfully cloned from Shennong Chrysanthemum was used as the research object, and the PCAMBIA1301-MYB expression vector containing DiaMYB gene was constructed to transform Arabidopsis thaliana into Arabidopsis by Agrobacterium tumefaciens dipping flower. Three positive plants with survival rate of 100% were obtained by screening and culturing to T3 generation. The enzyme activity, chlorophyll content, photosynthesis physiological index of transgenic Arabidopsis thaliana under salt and drought conditions were studied. Construction of green fluorescent protein pBI121-MYB-GFP expression vector, The expression of stress related genes in transgenic DiaMYB plants under stress stress was analyzed by real-time fluorescence quantitative analysis. The main results were as follows: 1.The DiaMYB gene was transferred into Arabidopsis thaliana by Agrobacterium tumefaciens dipping. Three positive strains, named MYB-1, MYB-2 and MYB-3, were obtained. The results of PCR and GUS staining confirmed that the DiaMYB gene had been successfully transferred into Arabidopsis thaliana genome. The expression of DiaMYB gene in different organs of Arabidopsis thaliana was analyzed by real-time fluorescence quantitative method. The DiaMYB gene was used to construct the fusion expression vector pBI121-MYB-GFP, which was transferred into onion epidermis cells by gene gunshot. The results showed that the DiaMYB gene was located in the nucleus. T3 generation transgenic Arabidopsis thaliana lines and wild type Arabidopsis thaliana were selected for high salt stress. The results showed that with the increase of salt solution concentration, the germination rate, survival rate, superoxide dismutase (SOD) and peroxidase (POD) activity, chlorophyll L / chlorophyll b, carotenoid / chlorophyll, and carotenoid / chlorophyll of Arabidopsis thaliana transgenic with DiaMYB gene were increased. The numerical results of net photosynthetic rate (PN), stomatal conductance (GS) and intercellular CO2 concentration (CO2) showed that the transgenic Arabidopsis thaliana lines showed stronger salt tolerance than wild-type Arabidopsis plants under high salt stress, and the results of real-time fluorescence quantitative PCR under high salt stress showed that the transgenic Arabidopsis strains showed higher salt tolerance than wild ones. The expression of RD22, RD29, RAB18, Cor47 and ABA1 in Arabidopsis thaliana (Arabidopsis thaliana) transfected with DiaMYB gene was significantly increased. At the same time, the expression of ABI1HAB1 gene decreased slightly and was lower than that of wild type plants. The expression of DiaMYB gene increased gradually under salt stress. 3. The T3 generation transgenic Arabidopsis thaliana and wild type Arabidopsis thaliana were used for drought stress. The results showed that with the increasing of drought stress, the germination rate and survival rate of Arabidopsis thaliana lines transformed with DiaMYB gene, the activities of superoxide dismutase (SOD) and peroxidase (POD), chlorophyll a / chlorophyll b, carotenoid / chlorophyll, and the activity of superoxide dismutase (SOD) and peroxidase (POD), chlorophyll a / chlorophyll b, carotenoid / chlorophyll were increased. The numerical results of net photosynthetic rate (PN), stomatal conductance (GS) and intercellular C02 concentration (C02) showed that the transgenic Arabidopsis strains showed stronger drought tolerance than wild-type Arabidopsis strains, and the real-time fluorescence quantitative PCR results showed that the transgenic Arabidopsis strains were more resistant to drought than wild-type Arabidopsis plants under drought stress. In Arabidopsis thaliana with DiaMYB gene, the expression of RD22, RD29, RAB18, Cor47 and ABA1 increased significantly, while the expression of ABI1HAB1 gene in Arabidopsis thaliana was slightly decreased and lower than that of wild type plant, DiaMYB, which increased at first and then decreased under drought stress.
【学位授予单位】：东北林业大学
【学位级别】：硕士
【学位授予年份】：2016
【分类号】：S682.11

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