番茄DR8基因启动子的克隆及其调控初探
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摘要
番茄DR8(Development Regulation 8)基因是一个与果实成熟相关的新基因,与拟南芥的生长素反应基因家族(Aux/IAA)具有很高的同源性。DR8基因作为一个生长素诱导表达的基因,其表达水平却受到乙烯的负调控和生长素的正调控,预示着该基因不仅对果实成熟过程具有重要的调控作用,并且可能和生长素与乙烯的相互作用密切相关。目前,DR8全长基因已构建到植物表达载体pGA643,通过农杆菌介导的基因转化方法将其转入番茄基因组中,获得了DR8超表达和抑制表达的转基因植株。为了深入阐明该基因的表达调控机制及其对乙烯、生长素的反应,十分必要分离DR8基因的启动子,分析并确定其激素调控区域及精确调控位点,为该基因的功能研究及启动子的应用奠定基础。本研究利用DNA步移PCR法进行启动子扩增,并从中设计构建了3个5’端缺失片段的pGreen-GFP荧光表达载体,转化烟草原生质体,经2,4-D处理后的荧光表达分析发现在250 bp区域内存在1个生长素的正调控位点。另外,在本研究中还构建了7个pBI121植物表达载体,为DR8基因启动子的番茄转化研究奠定了很好的基础
主要研究结果如下:
①利用DNA步移PCR技术从番茄基因组DNA中分离了DR8基因的部分启动子,1031 bp。
②启动子序列分析表明,启动子序列中含有大量AT丰富区域,TATA-box,CAAT-box,G-box,此区域为启动子的基本功能区,在该启动子序列中还存在其它调控区域,如ERE是乙烯响应元件,HSE是热激响应元件,3-AF1 binding site是光响应元件,TCA-element是水杨酸响应元件。
③构建了3个5’端缺失片段的pGreen-GFP荧光表达载体,其缺失片段大小分别为250 bp、500 bp、1031 bp。
④通过PEG法将荧光表达载体转化烟草原生质体,经2,4-D处理后发现启动子中存在生长素的正调控区域,位于3’端250 bp范围内。
⑤构建了7个pBI121植物表达载体。
DR8(Development Regulation 8)-a new gene about fruit ripening,has higher homology with Aux/IAA in the Arabidopsis. DR8 gene as an auxin-induce gene,is also negatively regulated by ethylene and positively regulated by auxin,it suggests DR8 gene has important regulation effect on fruit ripening and the relation between auxin and ethylene probably. At present,DR8 gene’s full long sequence was inserted into vector-pGA643,and was transformed into tomato genome by Agrobactera transform technology,DR8 over-expression and under-expression transgenic plants were obtained. In order to clarify the expression and regulation mechanism about DR8 gene and the response of ethylene and auxin,in the research intending to amplify DR8 gene’s promoter,according to analysis the function of DR8 gene promoter,confirming the regulated region and exact regulated site,establishing the base for researching DR8 gene’s function and promoter’s application. Using genome walking PCR technology amplified promoter,designed and constructed three 5’delete pGreen-GFP fluorescent expression vectors,and transformed into tobacco protoplast,after treated with 2,4-D and fluorescent detecting,the results showed that there was one auxin positively regulated site in 250 bp of 3’end . Meanwhile,the seven pBI121 plant expression vectors were construced,preparing for the transformation work of DR8 promoter in tomato.
The main results of research in this thesis are followed:
①The 1031 bp promoter region of DR8 gene from tomato was amplified by genome walking PCR technology.
②Promoter sequence analysis showed that this cloned fragment has lots of AT rich-regions,TATA-box,CAAT-box,G-box,they are the base function region in all promoters. Meanwhile,there are some other regulation regions in this promoter sequence,such as ERE is ethylene response element,HSE is hot response element,3-AF1 binding site is light response element, TCA-element is salicylic response element.
③Constructing three fluorescence vectors,the constructed fragment were 250 bp、500 bp、1031 bp.
④The expression vectors were transformed into tobacco protoplast by PEG technology and were treated with 2,4-D,fluorescence analysis showed that the auxin positive regulation region likely in the 250 bp of 3’end.
⑤Constructing seven pBI121 plant expression vectors.
主要研究结果如下:
①利用DNA步移PCR技术从番茄基因组DNA中分离了DR8基因的部分启动子,1031 bp。
②启动子序列分析表明,启动子序列中含有大量AT丰富区域,TATA-box,CAAT-box,G-box,此区域为启动子的基本功能区,在该启动子序列中还存在其它调控区域,如ERE是乙烯响应元件,HSE是热激响应元件,3-AF1 binding site是光响应元件,TCA-element是水杨酸响应元件。
③构建了3个5’端缺失片段的pGreen-GFP荧光表达载体,其缺失片段大小分别为250 bp、500 bp、1031 bp。
④通过PEG法将荧光表达载体转化烟草原生质体,经2,4-D处理后发现启动子中存在生长素的正调控区域,位于3’端250 bp范围内。
⑤构建了7个pBI121植物表达载体。
DR8(Development Regulation 8)-a new gene about fruit ripening,has higher homology with Aux/IAA in the Arabidopsis. DR8 gene as an auxin-induce gene,is also negatively regulated by ethylene and positively regulated by auxin,it suggests DR8 gene has important regulation effect on fruit ripening and the relation between auxin and ethylene probably. At present,DR8 gene’s full long sequence was inserted into vector-pGA643,and was transformed into tomato genome by Agrobactera transform technology,DR8 over-expression and under-expression transgenic plants were obtained. In order to clarify the expression and regulation mechanism about DR8 gene and the response of ethylene and auxin,in the research intending to amplify DR8 gene’s promoter,according to analysis the function of DR8 gene promoter,confirming the regulated region and exact regulated site,establishing the base for researching DR8 gene’s function and promoter’s application. Using genome walking PCR technology amplified promoter,designed and constructed three 5’delete pGreen-GFP fluorescent expression vectors,and transformed into tobacco protoplast,after treated with 2,4-D and fluorescent detecting,the results showed that there was one auxin positively regulated site in 250 bp of 3’end . Meanwhile,the seven pBI121 plant expression vectors were construced,preparing for the transformation work of DR8 promoter in tomato.
The main results of research in this thesis are followed:
①The 1031 bp promoter region of DR8 gene from tomato was amplified by genome walking PCR technology.
②Promoter sequence analysis showed that this cloned fragment has lots of AT rich-regions,TATA-box,CAAT-box,G-box,they are the base function region in all promoters. Meanwhile,there are some other regulation regions in this promoter sequence,such as ERE is ethylene response element,HSE is hot response element,3-AF1 binding site is light response element, TCA-element is salicylic response element.
③Constructing three fluorescence vectors,the constructed fragment were 250 bp、500 bp、1031 bp.
④The expression vectors were transformed into tobacco protoplast by PEG technology and were treated with 2,4-D,fluorescence analysis showed that the auxin positive regulation region likely in the 250 bp of 3’end.
⑤Constructing seven pBI121 plant expression vectors.
引文
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