【作者】 于丽萍；

【导师】 亓宝秀； 李新征；

【作者基本信息】 山东农业大学 ， 细胞生物学， 2010， 硕士

【摘要】 通过基因工程技术,将目的基因片段在特定的组织中以一定的表达量表达,以提高作物对盐碱、干旱等非生物胁迫与病、虫害等生物胁迫的抗性,或改良作物农艺性状或品质等,已成为现在作物品种改良的一种重要手段。而如何控制目的基因片段在特定时期、特定组织中高效表达,是目前限制基因工程技术在作物遗传改良中更有效应用的重要因素。除克隆更有效的目的基因片段外,作为基因表达调控关键元件之一的启动子的获得已成为作物基因工程改良中亟待解决的问题。启动子有组成型启动子(constitutive promoter)、组织特异性启动子(tissue-specific promoter)和诱导型启动子(inducible promoter)三种。迄今为止,植物表达载体中应用最广泛的是以CaMV 35S为代表的组成型启动子,最大优点是活性高,缺点是导致外源基因在转基因植物的几乎所有发育阶段的不同部位都表达,不但会增加细胞内物质和能量的负荷,有时还会引起植物的形态发生改变,影响植物的生长与发育。而采用组织特异性启动子可有效地控制外源基因在特定的组织或器官中表达,减少基因产物对受体生物及环境产生副作用。然而遗憾的是,虽然已相继克隆到许多组织特异启动子,但还没有一种组织特异性启动子能像CaMV 35S一样在作物基因工程改良中得到广泛的应用。2006年,Kriz等(2006)克隆到了玉米Emb5基因启动子emb5的1653bp序列,并将其作为一种玉米胚特异性启动子申请专利(Patent No:US 7,078,234 B2),但是未对emb5启动子进行详细的结构功能分析,至今也未见有这方面研究报导文献。本研究拟在对玉米胚特异性启动子emb5的组织特异性和活性进一步分析鉴定的基础上,对其与胚特异性相关的核心序列结构区域及相关顺式作用元件进行分析鉴定,为通过系统改造获得一个启动活性更强、序列长度更短、易于操控、具有广泛应用价值的胚特异启动子奠定基础。本研究以玉米自交系Qi319为材料,提取基因组DNA,通过PCR技术从中扩增出1653bp的emb5启动子序列。测序结果表明,仅有8个碱基发生突变,与已报道得序列同源性达到99.52%。序列分析表明,emb5的1653bp序列中含有高等植物启动子所特有的基本核心序列TATA-box和CAAT-box,并含有1个真核启动子基本调控元件GC盒,2个种子特异表达调控元件G-box和Skn-1-motif (GTCAT);并多次出现可能增强种子特异表达作用的A-box(CCGTCC)序列和其它如响应激素、光信号等的顺式作用元件等。由于玉米基因转化困难,生长周期长,作为系统分析鉴定emb5启动子功能的研究材料不太现实,所以寻找一种模式植物作为替代研究材料是必需的。由于缺乏易转化、生长周期短、遗传背景相对简单、清楚的单子叶植物模式材料,所以我们想首先验证一下来源于单子叶植物的玉米胚特异性启动子emb5,是否能在双子叶模式植物拟南芥中具有相似的胚特异活性,以便以模式植物拟南芥作为分析emb5启动子结构功能的研究材料。为此,我们分别以emb5和CaMV 35S为启动子,构建了以GUS为报告基因的植物表达载体pBI121,通过Floral-dip法转化拟南芥。对转基因拟南芥GUS活性的组织化学染色分析与GUS活性的组织荧光定量测定分析显示,emb5仅在转基因拟南芥种子胚发育的中后期高效表达,启动GUS表达活性显著高于CaMV 35S,而在根、茎、叶、花中检测不到表达。结果表明emb5在单、双子叶植物中都具有较高的胚特异活性。因此,在生物信息学分析基础上,通过对全长emb5启动子5′-逐步缺失的方法,获得7段不同长度的启动子片段,分别命名为pA(-1143bp~-1bp),pB(-956bp~-1bp),pC(-732bp~-1bp),pD(-523bp~-1bp),pE(-331bp~-1bp),pF(-251bp~-1bp)和pG(-127bp~-1bp),均连接GUS作为报告基因,借助植物表达载体pBI121,转化拟南芥,对emb5启动子的核心序列区域进行分析鉴定,结果如下:1、GUS活性的组织化学染色分析显示,最短的片段pG(-127bp~-1bp)完全丧失了启动子的活性,其余6个片段仅在转基因植株种子胚发育中后期检测到GUS活性。在成苗时期的根、茎、叶和花组织中均没有检测到GUS活性。这些结果清楚地表明emb5启动子中与胚特异活性相关的核心序列区域位于-1653 bp ~-127 bp之间。2、转基因植株种子胚发育后期GUS活性的组织荧光定量测定分析显示:(1)5′端缺失到emb5的约2/3时,片段(即pD)活性与全长启动子活性基本相同,而其余片段的启动子活性随着5′端的逐步缺失而逐渐降低;(2)emb5缺失510bp(-1653bp~-1143bp)区域后(即pA)活性与pD缺失192bp(-523bp~-331bp)区域后(即pE)活性都显著降低,表明-1653bp~-1143bp和-523bp~-331bp区域可能分别含有增强启动子活性的顺式作用元件;(3)pC片段缺失209bp(-732bp~-523bp)区域后(即pD)活性反而显著增强,表明在-732bp~-523bp区域可能存在抑制启动子活性的负调控元件。以上研究分析表明,emb5启动子在单子叶与双子叶植物中都具有较高的胚特异活性,而且emb5启动子5′端缺失约2/3后的片段(即pD)虽然只有500多碱基,但具有与全长启动子几乎相同的胚特异启动子活性。这意味着相对于全长1653bp的emb5启动子,短小的pD,不但更利于基因工程操作,而且由于去掉了许多已知与未知调控元件,减少了在转基因植物中可能被众多调控因子调控的干扰。因此更有可能成为作物遗传改良中理想的单、双子叶植物都适宜的胚高效特异启动子。更多还原

【Abstract】 Plant genetic engineering has become a very important tool in recent years in improving the resistance on plants to abiotic stresses such as salinization, drought, cold and biotic stresses such as diseases and pests. It is also important in improving agronomic traits or crop quality. This is often achieved by expressing a transgene or tansgenes to certain levels at specific tissues and/or organs of the target plants. However, controlling the levels of transgene expression spatially and tissue-specifically has become the current restricting factors in crop genetic improvement by this technology. In addition to employing more effective transgenes, the promoters used to drive the transgene expression are also one of the key regulating factors. Therefore, to obtain such a promoter and/or promoters will no doubt to enhance the further development of crop genetic engineering.There are three types of promoters, i.e., constitutive promoters, inducible promoters and tissue- or organ-specific promoters. To date, the only most widely used promoter in plant genetic engineering is the constitutive cauliflower mosaic virus 35S (CaMV 35S) promoter. The biggest advantage of this promoter is its high activity, leading to the high expression levels of foreign genes in all parts at almost all developmental stages of the transgenic plants. However, this could also become the undesirable property of this promoter since the high levels of gene products would inevitably increase the intracellular burden for resources and energy which are otherwise used for normal metabolism. In addition, it sometimes causes morphological changes of the transgenic plants, affecting plant growth and development. The tissue-specific promoters can overcome these problems by controlling the foreign gene to express in specific tissues or organs, therefore, decrease the side effects caused to the recipient plants. Unfortunately, although many tissue-specific promoters have been cloned in recent years due to this demand, currently there is not a single tissue-specific promoter which has the compatible activity as CaMV 35S promoter to be widely used in crop genetic engineering.Searching through the literature for seed-specific promoters in maize (Zea mays L.), we have found the emb5 promoter of Emb5 gene and emb5 promoter (Kriz et al, 2006). Although it is claimed to be a maize embryo-specific promoter there appears to be no detailed functional analysis for this promoter. The aim of this study is to further analyze the tissue-specific activities of this maize embryo-specific promoter emb5 in Arabidopsis and to dissect its DNA sequence in order to identify the core functional regions that are associated with the embryo-specific characteristics and the related regulatory elements. The ultimate goal would be to isolate an embryo specific promoter that is much smaller, high activity and easy to use therefore it would have extensive application values in transgenic modifications of monocots and dicotsplants. We first isolated the full length of 1653 bp DNA fraMSent by PCR using genomic DNA from maize inbred line Q319 as template according to the known sequence of emb5 promoter (Patent No: US 7,078,234 B2). Sequencing result indicated that there are 8 point mutations and the sequence homology to the published sequence is 99.52%. Detailed analysis of the emb5 sequence shows that it contains the higher plant specific basic core motifs, the TATA-box (TATAATA) and CAAT-box. It also contains: (1) a GC-box, the basic regulation element of the eukaryotic promoters; (2) the seed-specific G-box, Skn-1-box often found in seed-specific promoters; (3) many A-boxes that may enhance the specific expression; (4) many responsive elements of hormone and light signaling.As it is unrealistic to use maize as a research material to systemically analyze and to identify the functional regions of the emb5 promoter due to its difficulties to transform as well as the lengthy growth cycles, it is necessary to look for a model plant as an alternative experimental material. The lack of a monocotyledonous model plant that is easy to transform, has a short growth cycle as well as relatively simple genetic background leads us to choose the most used model plant Arabidopsis thaliana as our experimental material.It would also be interesting to see if the embryo-specific promoter emb5 from maize, a monocot, is still functional in the embryo of Arabidopsis, a dicot. Full length emb5 and 35S promoters were cloned into plant expression vector pBI121 which drive the expression of the reporter gene GUS. These 2 constructs were transformed into Arabidopsis thaliana by floral dipping method. Histochemical and quantitative ?uorometric GUS assays revealed that the full-length emb5 promoter can drive the expression of GUS in high levels from mid to late embryogenesis stages with no detectable GUS activity in the roots, stems, leaves and flowers of the transgenic Arabidopsis thaliana plants. The expression patterns of GUS under 35S promoter are, however, constitutive as expected. Our results indicate that the emb5 promoter has high activities in both monocotyledonous and dicotyledonous plant.Based on bioinformatics analysis, seven promoter fraMSents containing different regions were obtained from 5′-progressively deleted emb5 promoter. They were designated as pA (-1143bp~-1bp), pB (-956bp~-1bp), pC (-732bp~-1bp), pD (-523bp~-1bp), pE (-331bp~-1bp), pF (-251bp~-1bp) and pG (-127bp~-1bp) which were all cloned into the plant expression vector pBI121 and transformed into Arabidopsis in the same fashion as for the full length emb5. Histochemical and activity assays of GUS have been carried from the all the transgenic Arabidopsis lines and the results are as follows:1. GUS activity can only be detected in the late embryogenesis stage for 6 out of the 7 constructs made as pG (-127bp~-1bp) , made from the shortest fraMSent just upstream of the ATG seems to have lost its promoter activities altogether. No GUS activity was detected in the roots, stems, leaves and flowers in all the transgenic lines. Therefore, all six 5′-deletion fraMSents have still maintained the embryo-specific promoter activities and the core sequence regions associated with embryo-specific expression locate between -1653bp and -127 bp.2. Quantitative ?uorometric GUS assays were carried out with seeds of late embryogenesis stage and the results show that:(1). When approximately two-thirds was deleted from its 5′end of the emb5 DNA sequence, the remaining 520 bp DNA fraMSent, i.e. pD, still has similar activity to the full-length emb5 while all other fraMSents only exhibited low promoter activities.(2). When the first 510bp between -1653bp~-1143bp (pA) and the 5th 192bp fraMSent pE between -523bp~-331bp which is within pD are deleted their promoter activities are significantly reduced. This implies that both regions between may contain the positive cis-regulatory elements;(3). The deletion of 209bp of pC (-732bp~-523bp) from pD resulted in high promoter activity of pD, therefore, this region may contain the negative cis-regulatory elements.Our results clearly show that the maize emb5 promoter exhibits higher embryo-specific activity in both monocotyledonous as well as in dicotyledono- us plants. Most importantly we have isolated a much shorter (about 500bp) fraMSent, pD that has compatible promoter activities as the full length emb5 promoter (1653bp). This is achieved by deleting 2/3 of the emb5 sequence from its 5′-end. A shorter version of the emb5 not only offers great flexibility for making plant transformation vectors, especially for making multiple gene vectors for transgenic modifications of plants, but may also reduce the interference from other regulation factors in transgenic plants as it may no longer contain the many known and unknown cis-regulatory elements. Therefore, pD is more likely to become an efficient embryo-specific promoter which could be applied in both monocotyledonous and dicotyledonous plants in crop genetic improvements in the future.更多还原