【作者】 邵克强；

【导师】 杨世湖；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2008， 硕士

【摘要】 启动子是位于结构基因5′端上游区的调控序列,在转录水平控制着植物基因表达的时空性。研究植物抗病基因启动子,分析启动区内分子元件的功能对了解植物抗病基因的表达特异性有很重要的意义。不少与启动和诱导活性有关的分子元件在启动区内是多拷贝的。用逐段敲除这些功能性分子元件的启动子缺失体来进行转基因分析是了解这些分子元件的生物学功能的重要方法。Pib基因属于NBS-LRR抗病基因家族,是目前为数不多已克隆的抗稻瘟病主效基因之一。研究报道表明,pib基因表达不受病原菌诱导,而黑暗、温度等环境和一些化学物质却能有效地诱导pib基因的表达。研究结果还显示,将不同长短的pib结构基因序列片段置于35S驱动下,它们各自的转基因植株表现出对稻瘟病生理小种的不同抗性,而pib结构基因上游的启动区与gus基因融合构建的转基因试验也证实了pib启动子具有很强的黑暗诱导活性。由于水稻的根是处于24h黑暗环境,而地上部器官处于光/暗循环交替之下,这就使得pib基因的抗病性表达具有了特殊的器官特异性和昼夜节律性。为了探讨pib启动子这种环境条件诱导性的分子结构和分子机制,笔者用PLACE数据库对pib启动区核酸序列进行了分析,该启动区内有各种定义为启动相关分子元件近百个,除了TATA、CAAT等启动子共有元件外还有数十个不同类型的诱导性元件。其中被定义为光响应相关的顺式作用元件YTCANTYY以6个拷贝形式散布在启动子序列内。为了验证该启动子的暗诱导特性是否与此分子元件有关,这个分子元件又是怎样影响该启动子的暗诱导性的,笔者构建了不同长度、即含有不同数目的YTCANTYY元件的水稻pib基因启动子的5′端缺失体驱动gus基因的双元载体,用对其水稻转基因植株的gus基因表达分析的结果来研究该pib启动子中暗诱导分子元件的生物学功能及其对暗诱导的响应。1.用PCR扩增并测序获得4个不同长度的pib启动子片段,各片段之间的区别仅在于5’端的缺失各不相同。4个片段的3’端均终止于碱基2（ATG信号的G）,分别为:5’缺失体启动子1 PCR产物长222bp,为-219～2bp,无YTCANTYY元件,5’缺失体启动子2PCR产物长569bp,为-566～2bp,含1个YTCANTYY元件（-220～-227bp处）,5’缺失体启动子3 PCR产物长1911bp,为-1908～2bp,含3个YTCANTYY元件（-220～-227,-567～-574,-728～-735bp处）,全长启动子PCR产物长3575bp,为-3572～2bp,含6个YTCANTYY元件（-220～-227,-567～-574,-728～-735,-1909～-1916,-3042～-3049,-3382～-3389 bp处）。801（-219～2bp）、802（-566～2bp）、803（-1908～2bp）和804（-3572～2bp）分别连接进pCAMBIA1301中gus因前,取代原来的CaMV35S启动子,构建成双元载体pNAR801、pNAR802、pNAR803和pNAR804。重组双元质粒经过酶切验证,用农杆菌冻融转化法转化入农杆菌菌株EHA101中。2.利用农杆菌介导的方法,将上述pNAR801、pNAR802、pNAR803、pNAR804重组质粒转化粳稻品种R109,同时以CaMV35S启动子驱动gus基因的pCAMBIA1301作对照。共获得136株再生植株,经PCR筛选确定得到96个阳性的转基因水稻植株,阳性率70.6%。通过Southern blot检测也表明目的基因已经整合到水稻基因组中。3.对上述不同长度5′端缺失的pib启动子驱动gus基因的水稻转基因植株,进行Gus组织化学检测和荧光定量分析,系统研究了pib启动子中分子元件YTCANTYY拷贝数目与该启动子启动活性和暗诱导性的关系。Gus组织化学分析结果表明,含6个、3个、1个拷贝的pib启动子的转基因水稻愈伤在暗处理后均能在X-gluc溶液中显示不同深浅的Gus兰色,而6个拷贝完全缺失的启动子残端的水稻愈伤在X-gluc溶液中不显现Gus兰色。荧光定量分析结果表明,pib启动子序列具有很强的器官特异性,即便是6个暗诱导分子元件都被敲除的3’残端,其转基因植株仍保持根部启动活性高于地上部份的趋势。但其启动活性和暗诱导性随启动子缺失片段的缩短即暗诱导分子元件拷贝数增加而提高。这些结果表明,YTCANTYY在pib启动子序列中是一个暗诱导的功能性分子元件,他能赋予启动子的暗诱导性,起码在6个拷贝以内,pib启动子的暗诱导活性是与其拷贝数目呈正相关,各个YTCANTYY分子元件的启动活性可能是累加的。4.利用pib基因启动区与gus构建的报告基因载体pNAR604得到的T₃转基因水稻,继续对pib基因启动子诱导性进行研究,再次证实pib启动子活性受黑暗和温度影响,无论诱导与否,在根中的Gus活性都远高于叶片具有强烈的器官特异性,pib启动子活性无论是在根还是叶都具有明显的昼夜节律性,白昼低夜晚高,以15:00活性水平为最低,03:00活性水平为最高。这些结果表明,pib基因表达的起始或抗稻瘟病产物的合成地在根系,而不是植株叶、茎等稻瘟病发病部位。也就是说pib基因抗稻瘟病功能性产物的合成起始地与抗病的靶点分离。据此,我们推断,pib基因编码的抗稻瘟病产物是在根部起始合成,抗病基因产物在夜间由根系输送到植株地上的靶组织,实现其抗病的功能。更多还原

【Abstract】 As a important regulatory element of 5’upstream regions of structure gene, promoter controls gene expression characteristic of space and time in transcriptional level. Study on plant promoters of disease resistance and analysis of regulatory element’s function in promoter region are the very significance to expression characteristic of disease resistance gene. The transformation analysis by 5’deletion promoter fragments is the important method to understand the biological function of these molecular elements.Pib gene is the first major blast resistant gene cloned from rice by mapping clone technique and which is belongs to NBS-LRR resistant disease gene family. Previous studies show that the expression of pib gene was induced by darkness and chemistry factors but not pathogen. Our experiment results also indicated that different length fragments of pib structure gene sequence under the drive of 35S promoter, their To transgenic rice plants showed different resistances to rice blast pathogen races. Our results indicated that the pib promoter has the activity of darkness inducing in the rice transgenic experiments with the constructs of pib promoter-gus fusion. In natural environment, only root of rice plant could enjoy full darkness but the above ground organs are in the condition of light or dark cycle alternative, which offer the expression of pib gene the special organ specificity and diurnal rhythm.For studying the molecular structure and mechanism of environmental condition inducing, we analysis this promoter region by using PLACE database. The results show that there are about hundred of molecular regulatory element, for activation in this region, except of TATA、CAAT commonly elements, there are six copies of YTCANTYY element defined for light-response of cis-regluatory element. To verifying whether the dark-inducing property of the pib promoter is relate to the YTCANTYY element and how this molecular element affect the dark-inducing property? By using transgenic rice plants of 5’deletion pib promoter fragment-gus constructions, the relationships between the copy number of molecular element YTCANTYY in pib promoter and activity of pib promoter and its darkness inducing attribute were systematically analyzed.1. Four different regions of pib gene promoter were obtained by PCR, the difference among each fragments only lengths in 5’deletion, the 3’ downstream of four fragments all terminate to base 2（base G of signal ATG）. The PCR products of the first 5’deletion promoter is 222bp which is without YTCANTYY element; the PCR product of the second 5’deletion promoter is 569bp which contains one YTCANTYY element （-220～-227bp）; the PCR products of the third 5’deletion promoter is 1911bp which contain three YTCANTYY element （-220～-227,-567～-574,-728～-735bp）; the PCR products of the full length promoter is 3575bp which contain six YTCANTYY element （-220～-227bp, -567～-574bp, -728～-735, -1909～-1916, -3042～-3049, -3382～-3389bp）, these fragments were ligated into the binary vector pCAMBIA1301 to substitute the CaMV35S promoter which is upstream of gus. The recombined plasmids are pNAR801, pNAR802, pNAR803 and pNAR804. All recombined binary vectors were confirmed by restricted enzyme digestion and transferred into Agrobacterium tumefaciens strain EHA101 by freeze-thaw method.2. By Agrobacterium-mediated method, pNAR801, pNAR802, pNAR803, and pNAR804 were transferred into japonica rice R109. pCAMBIA1301 was also transferred at the same time. Total 136 independent transformed plants were got on regeneration medium. PCR analysis revealed that 96 plants were positive and the transform frequency is 70.6%. PCR and Southern blot tests confirmed the integration of foreign gene.3. By using transgenic rice plants of 5’deletion pib promoter fragment-gus constructions, the relationships between the copy number of molecular element YTCANTYY in pib promoter and activity of pib promoter and its darkness inducing attribute were systematically analyzed. The results from Gus histochemistry detection revealed, after darkness inducing the transgenic rice calli of pib promoter fragments carrying 6, 3 or 1 copy of YTCANTYY motif were appeared different degree Gus blue in X-gluc solution, but the 5’deletion pib promoter fragment with no YTCANTYY element was unable to drive gus gene to show any Gus blue in X-gluc solution. Fluorescence quantitative analysis results showed that pib promoter sequence had strong organ specificity, even though all 6 darkness inducing elements were knocked out, its transgenic rice plants still kept the tendency of promoter activity in root higher than that of above ground parts of plants. But the activity of promotion and darkness inducing were increased with the increase of promoter fragments in length and with increase of darkness inducing element copy. These results indicated that YTCANTYY in pib promoter is a functional motif for darkness inducing and in other words this motif confer the darkness inducing of pib promoter. At least within 6 copies, the activity of darkness inducing was positively correlated with the copy number of YTCANTYY element. And the activity for each individual YTCANTYY element seemed to additive.4. Using transgenic rice progenies of pib promoter-gus recombined plasmid, the inducing activities of pib promoter were analyzed. The fluorescence quantitative analysis of Gus activity in T₃ transgenic rice plants confirmed that 24h darkness treatment and temperature could influence promotion activity of pib promoter. Gus activity in root was always higher than leaf and they have strong organ specificity. Gus activity both in root and leaf of the transgenic rice plants all exhibited a clear diurnal rhythm, the Gus activity in daytime was at low level （15:00 was lowest） and high level was in night （3:00 was highest）. These experimental results of Gus activity in the transgenic rice plants indicated the blast resistance production of pib structure gene was mainly synthesize in root and which was separated from infection position of rice blast. We infer that, the disease resistance product of pib structure gene is synthesized in root, and then transported to the target tissue of aboveground in the night to perform the function of blast resistance.更多还原