韧皮部特异性启动子研究概述

韧皮部特异性启动子可驱动其下游基因在植物的韧皮部特异性表达,通过将几种韧皮部特异性启动子的序列进行比较,发现这类启动子在结构上存在可能与组织特异性有关的保守基序。对这类启动子的结构与功能的研究将有助于提高人们对植物维管组织分化及有机物质运输机制的了解,而且也会为植物抗病虫基因工程的研究提供有应用价值的启动子元件。就韧皮部特异性启动子的结构特点、分类及应用进行了概述。     

高等植物启动子的研究进展

从高等植物启动子的基本结构、启动子克隆方法入手,着重介绍了组成型、组织特异性及诱导型启动子的研究进展及其在植物基因工程方面的应用情况,提出了植物启动子研究中存在的问题与展望。     

种子特异性启动子研究进展

植物的种子,尤其是油料和谷类作物的种子与人类的生产生活关系非常密切,因此,也成为植物基因工程中进行改良的重要目标材料。转化的外源基因在植物受体组织中能否正确、高效并按照人们的意愿特异地表达是人们非常关注的问题。启动子驱使外源基因在受体植株中启动转录是外源基因能够表达的必要条件。目前人们所广泛研究的种子特异性启动子基本上属于Ⅱ类启动子,它可以驱使外源基因在植物的种子中特异表达,按照人们的意愿改进植物代谢途径,提高种子中营养物质含量等。种子特异性启动子的结构符合Ⅱ类启动子的特点,具有基本启动子、起始子和上游元件。它区别于其它类型启动子的一个显著特点是上游存在一些特异的调控元件与调控种子特异性基因的特异表达有关。本文综述了高等植物种子特异性启动子的结构及其在植物基因工程中的最新研究进展。对这类启动子的结构和功能元件的了解,有助于人们更加深入地理解高等植物基因表达调控机制,提高人们对植物种子发育过程及有机物在种子中积累机制的认识,而且可以为植物基因工程中生物反应器的研究提供有应用价值的启动子元件。     

人工合成根特异启动子SRSP的功能分析

根负责吸收水分和养分,是重要的植物组织,但易受生物及非生物胁迫,影响作物的生长发育和产量。设计合成根特异启动子,可为与胁迫相关的抗性基因在作物根部的功能研究及高效表达提供候选启动子。文中将4拷贝的根特异性顺式作用元件(OSE1ROOTNODULE、OSE2ROOTNODULE、SP8BFIBSP8AIB和ROOTMOTIFAPOX1)以串联排列方式设计合成了一个根特异性模块(pro-SRS),并与来自CaMV35S启动子的最小启动子融合,合成一个人工合成启动子SRSP。通过替换CaMV35S启动子将SRSP启动子克隆到pCAMBIA2300.1中以驱动GUS表达。将携带SRSP启动子的构建体通过农杆菌介导的方法转化到烟草中。GUS组织化学染色分析和实时PCR (RT-PCR)分析显示SRSP启动子在转基因烟草中赋予根特异性表达。说明顺式作用元件重复排列可实现启动子预期功能,本研究为理性设计植物组织特异启动子奠定了理论基础。     

种子特异性启动子研究进展

植物的种子,尤其是油料和谷类作物的种子与人类的生产生活关系非常密切,因此,也成为植物基因工程中进行改良的重要目标材料。转化的外源基因在植物受体组织中能否正确、高效并按照人们的意愿特异地表达是人们非常关注的问题。启动子驱使外源基因在受体植株中启动转录是外源基因能够表达的必要条件。目前人们所广泛研究的种子特异性启动子基本上属于II类启动子,它可以驱使外源基因在植物的种子中特异表达,按照人们的意愿改进植物代谢途径,提高种子中营养物质含量等。种子特异性启动子的结构符合II类启动子的特点,具有基本启动子、起始子和上游元件。它区别于其它类型启动子的一个 显著特点是上游存在一些特异的调控元件与调控种子特异性基因的特异表达有关。本文综述了高等植物种子特异性启动子的结构及其在植物基因工程中的最新研究进展。对这类启动子的结构和功能元件的了解,有助于人们更加深入地理解高等植物基因表达调控机制,提高人们对植物种子发育过程及有机物在种子中积累机制的认识,而且可以为植物基因工程中生物反应器的研究提供有应用价值的启动子元件。     

大麦与油菜种皮特异启动子表达模式的比较

启动子位于转录起始位点上游并能特异性地结合RNA聚合酶,其作为调控序列驱动外源基因在异源植物中表达,从而实现转基因的高效性,具有时空表达特异性的启动子对获得有效转基因植物及产物具有重要意义。为了解种皮特异启动子的表达模式,该研究基于前期报道的序列,通过同源克隆的方法分别从大麦和油菜中克隆获得Gerb和Bntt两个种皮特异性启动子,并对其进行生物信息学分析,构建了Gerb::GUS和Bntt::GUS植物表达载体并转化拟南芥,通过组织化学染色观察了GUS的表达情况。结果表明:两种启动子序列中都含有多拷贝种皮特异表达启动子元件以及多种胁迫诱导响应元件;转基因拟南芥幼苗期,大麦Gerb种皮特异启动子驱动GUS全株表达且子叶和下胚轴较真叶和根中表达量高;油菜Bntt种皮特异启动子表达较弱;成株期,Gerb在不同组织(叶片、茎、花序和角果)中均有表达,未显示组织特异性;Bntt仅在叶片及角果维管束中有微弱表达。在各种非生物胁迫下,Gerb表达模式未发生显著变化,而Bntt仅在盐胁迫下显示很强的角果和种子特异性表达,其他胁迫未见明显表达。以上结果显示,大麦种皮特异性启动子Gerb和油菜种皮特异性启动子Bntt在时间和空间表达模式上存在差异,这对今后选择种皮特异启动子具有参考作用,但其具体机制仍需进一步研究验证。     

植物花药花粉特异性基因的调控序列

植物的花药发育和花粉成熟过程中,有大量的基因表达,其中有许多是花药和花粉组织特异性的,本文介绍了几种已知的花药和花粉特异性基因及其上游调控序列,井简单地介绍了花药特异性的启动子在植物基因工程中的应用。     

Calgene获得植物油专利

Calgerie公司已获得一项有关种子-特异性启动子napin的专利,napin是许多公司遗传工程植物油产品的关键成份。此项5,420,034号美国专利涉及3个DNA构建物中的种子一特异性启动子,其中包括napin,还涉及含此构建物的芸苔属宿主植物。 就油料改良而言,种子特异性启动子保证了转移油料基因编码的只是植物贮藏油料,并且对植物本身没有影响。由于长度适宜和定时性,napin启动子是植物油料遗传工程中最常用的启动子。     

人工合成启动子SAR在拟南芥中的表达分析

利用植物防御基因中的病原诱导响应元件和最小35S启动子(-62～+1),人工合成了启动子SAP,并以GUS基因为报告基因,在转基因拟南芥中分析了合成启动子的表达特性.通过对转基因拟南芥GUS组织染色的分析表明:SAR启动子在子叶、毛刺、根茎交接处和根系中优势表达,在老叶中的表达量高于幼叶,说明SAR启动子具有组织和发育表达特异性.     

Comprehensive construction strategy of bidirectional green tissue‐specific synthetic promoters

Bidirectional green tissue‐specific promoters have important application prospects in genetic engineering and crop genetic improvement. However, there is no report on the application of them, mainly due to undiscovered natural bidirectional green tissue‐specific promoters and the lack of a comprehensive approach for the synthesis of these promoters. In order to compensate for this vacancy, the present study reports a novel strategy for the expression regulatory sequence selection and the bidirectional green tissue‐specific synthetic promoter construction. Based on this strategy, seven promoters were synthesized and introduced into rice by agrobacterium‐mediated transformation. The functional identification of these synthetic promoters was performed by the expression pattern of GFP and GUS reporter genes in two reverse directions in transgenic rice. The results indicated that all the synthetic promoters possessed bidirectional expression activities in transgenic rice, and four synthetic promoters (BiGSSP2, BiGSSP3, BiGSSP6, BiGSSP7) showed highly bidirectional expression efficiencies specifically in green tissues (leaf, sheath, panicle, stem), which could be widely applied to agricultural biotechnology. Our study provided a feasible strategy for the construction of synthetic promoters, and we successfully created four bidirectional green tissue‐specific synthetic promoters. It is the first report on bidirectional green tissue‐specific promoters that could be efficiently applied in genetic engineering.     

植物基因启动子研究进展

综述了植物基因启动子的核心结构与功能、植物基因启动子的种类：异源组成型启动子(CaMV35S、MMV)和从植物自身克隆的组成型启动子(PTSB1和PPHYB),以及植物中通过按物理因素(温、光、旱、热等)、化学因素(离子、有机物、激素等)和生物因素(病菌、组织器官、发育阶段等)诱导表达的一些启动子,发育时期特异的启动子、器官特异的启动子(根、茎、叶等)和植物中应用的人工双向启动子的研究进展和应用前景。     

高等植物启动子研究进展

 简述了高等植物来源启动子的多种保守顺式调控元件如TATA盒、转录起始位点、G盒等,以及双向启动子和可变启动子。着重介绍了受环境包括激素、光、创伤、真菌、逆境等因子诱导表达的植物启动子以及显示出植物发育特异性表达的启动子。     

Global functional analyses of rice promoters by genomics approaches

Promoters play key roles in conferring temporal, spatial, chemical, developmental, or environmental regulation of gene expression. Promoters that are subject to specific regulations are useful for manipulating foreign gene expression in plant cells, tissues, or organs with desirable patterns and under controlled conditions, and have been important for both basic research and applications in agriculture biotechnology. Recent advances in genomics technologies have greatly facilitated identification and study of promoters in a genome scale with high efficiency. Previously we have generated a large T-DNA tagged rice mutant library (TRIM), in which the T-DNA was designed with a gene/promoter trap system, by placing a promoter-less GUS gene next to the right border of T-DNA. GUS activity screens of this library offer in situ and in planta identifications and analyses of promoter activities in their native configurations in the rice genome. In the present study, we systematically performed GUS activity screens of the rice mutant library for genes/promoters constitutively, differentially, or specifically active in vegetative and reproductive tissues. More than 8,200 lines have been screened, and 11% and 22% of them displayed GUS staining in vegetative tissues and in flowers, respectively. Among the vegetative tissue active promoters, the ratio of leaf active versus root active is about 1.6. Interestingly, all the flower active promoters are anther active, but with varied activities in different flower tissues. To identify tissue specific ABA/stress up-regulated promoters, we compared microarray data of ABA/stress induced genes with those of tissue-specific expression determined by promoter trap GUS staining. Following this approach, we showed that the peroxidase 1 gene promoter was ABA up-regulated by 4 fold within 1 day of exposure to ABA and its expression is lateral root specific. We suggest that this be an easy bioinformatics approach in identifying tissue/cell type specific promoters that are up-regulated by hormones or other factors. Su-May Yu and Swee-Suak Ko equally contributed to this work.     

Highly interactive nature of flower‐specific enhancers and promoters,and its potential impact on tissue‐specific expression and engineering of multiple genes or agronomic traits

Molecular stacking enables multiple traits to be effectively engineered in crops using a single vector. However, the co‐existence of distinct plant promoters in the same transgenic unit might, like their mammalian counterparts, interfere with one another. In this study, we devised a novel approach to investigate enhancer–promoter and promoter–promoter interactions in transgenic plants and demonstrated that three of four flower‐specific enhancer/promoters were capable of distantly activating a pollen‐ and stigma‐specific Pps promoter (fused to the cytotoxic DT‐A gene) in other tissues, as revealed by novel tissue ablation phenotypes in transgenic plants. The NtAGI1 enhancer exclusively activated stamen‐ and carpel‐specific DT‐A expression, thus resulting in tissue ablation in an orientation‐independent manner; this activation was completely abolished by the insertion of an enhancer‐blocking insulator (EXOB) between the NtAGI1 enhancer and Pps promoter. Similarly, AGL8 and AP1Lb1, but not AP1La, promoters also activated distinct tissue‐specific DT‐A expression and ablation, with the former causing global growth retardation and the latter ablating apical inflorescences. While the tissue specificity of the enhancer/promoters generally defined their activation specificities, the strength of their activity in particular tissues or developmental stages appeared to determine whether activation actually occurred. Our findings provide the first evidence that plant‐derived enhancer/promoters can distantly interact/interfere with one another, which could pose potential problems for the tissue‐specific engineering of multiple traits using a single‐vector stacking approach. Therefore, our work highlights the importance of adopting enhancer‐blocking insulators in transformation vectors to minimize promoter–promoter interactions. The practical and fundamental significance of these findings will be discussed.     

参与水稻光合作用的PsbR3基因启动子的功能分析

本实验旨在研究水稻光合作用蛋白中各基因的表达模式. 采用RT-PCR和定量real-time PCR数据分析水稻不同组织的mRNA表达水平.结果显示,PsaK和PsbR3基因仅在茎、叶等绿色组织表达,而胚、胚乳部分均不表达.通过其启动子克隆、植物表达载体构建,以及农杆菌介导转化后,GUS组织染化分析和GUS荧光定量分析表明,两启动子均为组织特异性优势表达,PsbR3启动报告酶GUS在叶片中的表达活性为Actin启动子的3.29倍,而PsaK启动报告酶GUS在叶片中的表达活性低于Actin启动子的.这些初步结果提示,PsbR3启动子决定水稻绿色组织茎叶的优势表达,PsbR3基因可能参与水稻光合作用.