本文選題：神農(nóng)香菊　切入點(diǎn)：DiaMYB　出處：《東北林業(yè)大學(xué)》2016年碩士論文　論文類型：學(xué)位論文

【摘要】：神農(nóng)香菊(Dendranthema indicum var.aromaticum)是菊科菊屬中一種新資源植物,本文以神農(nóng)香菊中成功克隆出的DiaMYB基因?yàn)檠芯繉ο?以構(gòu)建成功的含有DiaMYB基因的PCAMBIA1301-MYB表達(dá)載體,通過農(nóng)桿菌蘸花法轉(zhuǎn)化擬南芥,篩選培養(yǎng)獲得成活率均為100%的三個陽性植株,繼續(xù)篩選并培養(yǎng)至T3代。研究轉(zhuǎn)基因擬南芥在鹽和干旱條件下的酶活性、葉綠素含量、光合作用生理指標(biāo),構(gòu)建含有綠色熒光蛋白pBI121-MYB-GFP表達(dá)載體,觀察亞細(xì)胞定位。實(shí)時熒光定量分析在逆境脅迫下轉(zhuǎn)DiaMYB基因植株中脅迫相關(guān)基因表達(dá)量。本文主要研究結(jié)果如下：1.利用農(nóng)桿菌蘸花法將神農(nóng)香菊DiaMYB基因轉(zhuǎn)入擬南芥中,獲得三個陽性株系,命名為MYB-1、MYB-2、MYB-3,經(jīng)過PCR和GUS染色鑒定,證實(shí)DiaMYB基因已成功轉(zhuǎn)入擬南芥基因組中。實(shí)時熒光定量方法分析過表達(dá)DiaMYB基因擬南芥不同器官的表達(dá)量,結(jié)果表明：在莖、葉和花中的表達(dá)量稍高,其次是果,在根中的表達(dá)量最低。利用DiaMYB基因構(gòu)建含有綠色熒光蛋白融合表達(dá)載體pBI121-MYB-GFP,通過基因槍法轉(zhuǎn)入洋蔥表皮細(xì)胞,顯示DiaMYB基因定位于細(xì)胞核上。2.取T3代轉(zhuǎn)DiaMYB基因擬南芥3個株系和野生型擬南芥進(jìn)行高鹽脅迫,結(jié)果顯示：隨著鹽溶液濃度的增加, 轉(zhuǎn)DiaMYB基因擬南芥各株系種子的萌發(fā)率及成苗期成活率、超氧化物歧化酶(SOD)和過氧化物酶(POD)活性、葉綠素￡L/葉綠素b、類胡蘿卜素/葉綠素、凈光合速率(Pn)、氣孔導(dǎo)度(Gs)以及胞間CO2濃度(Ci)的數(shù)值結(jié)果均得出轉(zhuǎn)DiaMYB基因擬南芥各株系表現(xiàn)出了更強(qiáng)于野生型的耐鹽性。高鹽脅迫下的實(shí)時熒光定量PCR結(jié)果顯示,轉(zhuǎn)DiaMYB基因擬南芥中RD22、 RD29A、RAB18、COR47、ABA1的表達(dá)量明顯增加,與此同時,ABI1、HAB1基因的表達(dá)量都稍有下降且都低于野生型植株,DiaMYB基因在鹽脅迫下的表達(dá)量逐漸升高。3.取T3代轉(zhuǎn)DiaMYB基因擬南芥3個株系和野生型擬南芥進(jìn)行干旱脅迫,結(jié)果顯示：隨著干旱脅迫的遞增,轉(zhuǎn)DiaMYB基因擬南芥各株系種子的萌發(fā)率及成苗期成活率、超氧化物歧化酶(SOD)和過氧化物酶(POD)活性、葉綠素a/葉綠素b、類胡蘿卜素／葉綠素、凈光合速率(Pn)、氣孔導(dǎo)度(Gs)以及胞間C02濃度(Ci)的數(shù)值結(jié)果均得出轉(zhuǎn)DiaMYB基因擬南芥各株系表現(xiàn)出了更強(qiáng)于野生型的耐旱性。干旱脅迫下的實(shí)時熒光定量PCR結(jié)果顯示,轉(zhuǎn)DiaMYB基因擬南芥中RD22、 RD29A、RAB18、COR47、ABA1的表達(dá)量明顯增加,與此同時,ABI1、HAB1基因的表達(dá)量都稍有下降且都低于野生型植株,DiaMYB基因在干旱脅迫下的表達(dá)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.
【學(xué)位授予單位】：東北林業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2016
【分類號】：S682.11

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