【作者】 苟吉庆；

【导师】 郭三堆；

【作者基本信息】 中国农业科学院 ， 作物遗传育种， 2001， 博士

【摘要】 目前，启动子的研究是国内外的一个研究热点，现已发现了大量组织特异性启动子和诱导型启动子。在仔细研究了这些特异性启动子的结构特点，以及其中起决定作用的顺式作用调控元件的基础上，本文从中选择了两个研究背景比较清楚、序列结构具有共性的植物顶端新生组织特异性调控元件B1元件和水杨酸诱导型调控元件as-1元件进行了进一步研究。 以35S启动子的基本框架结构（-90至+1区间的序列）为基础，利用人工合成的这两个调控元件进行重新组装、构建了九个分别含有一个、两个、四个B1元件和as-1元件的系列启动子和带有gfp基因的质粒，将它们分别命名为pUC191B1a.GFP、pUC191B2a.GFP、pUC191B4a.GFP、pUC192B1a.GFP、pUC192B2a.GFP、pUC192B4a.GFP、PUC194B1a.GFP、pUC194B2a.GFP和pUC194B4a.GFP。并进一步分别构建出九个不同的植物表达载体，分别命名为pBI1B1a.GFP、pBI1B2a.GFP、pBI1B4a.GFP、pBI2B1a.GFP、pBI2B2a.GFP、pBI2B4a.GFP、PBI4B1a.GFP、PBI4B2a.GFP、pBI4B4a.GFP。利用农杆菌介导的叶盘转化法转化烟草，通过高浓度的卡那霉素连续筛选，最终获得186株转基因烟草植株。经过荧光显微镜初步筛选，获得含有1B1a启动子的植株9株、1B2a启动子的植株12株、1B4a启动子的植株14株、2B1a启动子植株8株、2B2a启动子的植株11株、2B4a启动子的植株9株、4B1a启动子的植株15株、4B2a启动子的植株15株和4B4a启动子的植株19株。经PCR扩增和Southern blot分析表明各种表达载体已分别整合进转基因烟草植株的基因组中。 以每种启动子的转基因烟草植株为一个小群体，分别利用荧光显微镜在蓝色激发光下观察各种转基因烟草植株中各组织部位的GFP表达水平，并利用荧光分光光度计在460nm激发光和509nm发射光下对不同部位叶片中GFP的表达量进行了定量分析。两种不同的分析方法一致证明：（1）含有B1顺式作用元件的启动子具有在植株顶端叶片和茎尖中表达的组织特异性，其在茎中的表达也主要 行 男 二集中在茎的外表皮和韧皮部组织。仅旧 元件具有累加效应，启动子中含有BI 元件越多，该启动子在顶端组织特异性表达就越强，含有四个 田 元件的启动子的顶端组织特异性最显著。在转基因烟草植株的同一部位，含有四个 ＊ 元件启动子的表达水平是含有两个田元件启动子表达水平的2．9倍，是含有单个BI元件启动子表达水平的3．9倍，含有两个BI元件启动子的表达水平是含有单个BI元件启动子表达水平的 1．3倍。o）含有相同 BI元件而 asJ元件数目不同的三种启动子的表达水平没有明显差异，说明正常条件下含有 BI 元件和 as1元件启动子的表达水平主要由 BI 元件的多少决定，而不受asJ元件的影响。 在应用ZmM、20mM和50mM水杨酸溶液喷洒转基因烟草植株叶片后，在不同时间段内选取同一部位的叶片，通过荧光显微镜观察和荧光分光光度计测量两种方法，定性、定量地分析了不同浓度的水杨酸诱导下含有不同启动子的转基因烟草植株中GFP表达水平的变化。两种方法一致证明：（1）as－1 元件能赋予启动子水杨酸诱导特性。（2）水杨酸的诱导效果与所喷洒水杨酸的浓度及 的值密切相关，供试溶液中以50mM PH6．8 的水杨酸溶液诱导效果最佳。（3）水杨酸诱导后基因表达水平的增长幅度与启动子中as－1元件的数目密切相关，含有单个asJ 元件的启动子在诱导后表达水平比诱导前提高2．2～2．8倍，含有两个asJ元件的启动子在诱导后表达水平比诱导前提高3～3．5倍，含有四个asA元件的启动子在诱导后表达水平比诱导前提高 13～17倍。O）增加 BI顺式作用元件数目有协同增强as八元件表达的作用，含有四个BI元件和四个as八元件的4B4a启动子，其不但具有很高的顶端叶片和茎尖组织特异性，而且在水杨酸诱导后 24小时左右，其表达水平比诱导前可提高 17．3倍。如此高效表达目前未见类似报道。 本研究结果有力地证明了来自启动子中的组织特异性BI元件和诱导性as1元件，可被直接用于改造或构建人工融合启动子，并赋予启动子组织特异性表达的特性和水杨酸诱导特性，这一研 MK 3 究结果为今后构建组织特异性或诱导型启动子提供了依据。 将BI和as－1顺式作用调控元件串联叠加起来构建成新型的、 既有顶端组织特异性又有水杨酸诱导性并能高效表达的启动子， 目前尚未见到类似报道。更多还原

【Abstract】 Based on the study of published cis-elements of tissue-specific and induciblepromoters. Salicylic acid (SA) inducible as-I element and the green tissue-specific 81 element, derived from the cauliflower mosaic virus 35S promoter,were selected. Because both of the two promoters have well-known researchbackground and more commonness than other cis-elements.Based on a truncation of?0 355 promoter (-90 to +1), nine different promoterswere constructed with artificially synthesized as-I sequence and BI sequence.Each one of them respectively has one copy, two copies or four copies of as-Isequence and 81 sequence, and were subsequently designated as IBIa promoter,182a promoter, lB4a promoter, 2BIa promoter, 2B2a promoter, 2B4a promoter,4Bta promoter, 4B2a promoter and 4B4a promoter. And all the constructs wereconfirmed by sequencing. Then nine plant expression vectors containing thesecorresponding promoters were constructed, in which the GFP gene wasdownstreamed as a reporter gene. These plant expression vectors weresubsequently designated as p811 B 1 a.GFP, p811 B I a.GFP, p811 B I a.GFP.pBI2Bla.GFP, pBl4BIa.GFP, pBIIB2a.GFP, pBl2B2a.GFP, pBl4B2a.GFP,pBIlB4a.GFP, pBI2B4a.GFP and pBl4B4a.GFP. Tobacco leaf discs weretransformed with Agrobacterium tumefaciences Conn LBA4404 containing theseexpression vectors, and 186 trangenic plants were obtained. Among of them, 106pass muster transgenic plants were firstly screened out by FluorescentMicroscope.Results of testing transgenic plants with PCR, Southern blot methods confirmedthe successful integration of exogenous gene into tobacco plants genome.The GFP expressing level of different tissues located on different parts ofrespective trangenic plants transformed by different promoters were observed bythe Fluorescent Microscopy, and the expressing level of different leaves located5on different parts of transgenic plants were quantitatively analyzed by FluorescentSpectrophotometer. The results of two methods were highly consistent with eachother and confirmed following conclusions:?The promoters containing B! element show a tissue-specific expressionprincipally in new leaves and the stem apex.?The B I element shows a accumulative characteristic when adding the its copynumber in promoter. The expression level of promoters containing fourcopies of B! sequence is 2.9 fold the level of promoters containing twocopies of B1 sequence, is 3.9 fold the level of promoters containing onecopy of 81 sequence. The expression level of promoters containing twocopies of B I sequence is I .3 fold the level of promoters containing one copyof 81 sequence.?No difference has been observed between the different transgenic plants ofthree kinds of promoters that containing same copy number of BI elementand different copy number of as-I element. The results mean that theexpression level of promoter is principally dependent on the number of 81sequence contained in the promoter, and the number of as-I sequence has noobvious influence on it under the general condition.After sprayed by salicylic acid solutions at various concentration, such as 2mM,20mM and 50mM, the transgenic tobacco plants carrying different promoters weretested for their response to the SA treatment by Fluorescent Microscopy andFluorescent Spectrophotometer both qualitatively and quantitatively. The resultsof two methods were highly consistent with each other and confirmed followingconclusions:?The as-I element shows a positive response to the SA signal transduction,and the promoter containing as-I element show a trait of transcriptionactivation by SA inducement.6?Inductive efficiency is closely dependent on concentration and pH value ofSA solution. The results suggest that the inducti更多还原