植物逆境相关启动子及功能

启动子是调控基因表达的重要顺式元件, 在植物基因表达调控过程中起着重要作用。目前植物抗逆基因工程中, 人们大多使用组成型表达启动子驱动目的基因的表达。组成型表达启动子虽然能提高转基因植株的抗逆性, 但是其持续过量地表达转化的外源基因会阻碍植物的生长且减少其产量。因此, 只在胁迫条件下才会驱动外源基因表达的诱导型启动子的研究显得尤其重要, 已成为目前研究的热点。文章综述了受非生物逆境和生物逆境胁迫诱导的植物基因启动子的种类和功能, 并展望了植物逆境诱导启动子的研究方向和前景。     

胁迫诱导型启动子在植物抗逆基因工程中的应用

在抗逆基因工程中,大多采用的是组成型表达启动子,组成型表达启动子驱动外源抗逆基因表达虽然可以提高转基因植物的抗逆性,但会导致转基因植株生长迟缓或不育;而胁迫诱导型启动子则可提高转基因植物的抗逆性,不影响其正常生长发育,所以,胁迫诱导型启动子已逐渐用于植物抗逆基因工程。本文介绍不同胁迫诱导型启动子在植物抗逆基因工程中的应用。     

植物逆境胁迫耐受性启动子的研究进展

逆境胁迫如干旱、极端温度、损伤等非生物胁迫和病虫害等生物胁迫严重影响植物的生长发育及产量。逆境胁迫耐受性启动子能够接受逆境条件下的诱导信号,激活植物体内胁迫应答基因的表达,使植物感知并适应逆境。本文对逆境胁迫耐受性启动子的克隆及功能研究情况进行综合分析,主要包括抗旱、耐盐、耐高温、抗冻、耐损伤、抗病和抗虫基因启动子。     

植物启动子研究进展

植物启动子在转录水平上发挥着重要的调控作用,对其功能进行研究不仅可以反映相应基因的表达模式,还能为利用植物基因工程手段实现基因的高效特异性表达提供有效途径。结合近年来的相关研究,综述了植物启动子的结构特点及其功能研究进展,重点对与生物和非生物胁迫有关的各类诱导型启动子进行了阐述,并展望了植物启动子的未来研究方向。     

植物非生物胁迫诱导启动子顺式作用元件的研究方法

非生物胁迫严重影响植物生长发育,降低作物产量。植物通过各种途径忍受或抵抗非生物胁迫,主要表现是各种抗非生物胁迫基因的表达。基因表达受其上游启动子及转录因子的调控,目前对抗非生物胁迫诱导启动子顺式作用元件及转录因子的研究成为热点。本文综述了植物非生物胁迫诱导启动子顺式作用元件及转录因子的研究方法,并展望了顺式作用元件及转录因子研究的方向及前景。     

胡杨PeNAC121基因启动子的分离鉴定和胁迫应答模式分析

为了探究NAC转录因子家族成员在胡杨（Populus euphratica）逆境胁迫中的响应和调控机制,利用PCR技术从胡杨中克隆了PeNAC121基因的启动子序列,并采用生物信息学工具对该启动子的结构特征进行了分析,最后利用该启动子驱动GUS报告基因在三倍体毛白杨（Populus tomentosa）中表达,并对获得的转基因植株采用不同胁迫处理后进行了GUS染色和酶活性定量分析。结果表明,克隆获得的PeNAC121基因的启动子长度为1 997 bp（起始密码子ATG上游）,启动序列中除了含有大量的光响应元件,还含有多个与非生物逆境胁迫和激素响应相关的元件,如低温响应元件LTR、干旱响应元件MBS、防卫和胁迫响应元件TC-rich repeats、脱落酸（ABA）响应元件、以及赤霉素（GA）响应元件等。基因的组织表达模式检测结果显示,PeNAC121基因主要在茎中表达,在根和叶中的表达较少。GUS组织化学染色和酶活性检测结果表明,胡杨PeNAC121启动子显著受到NaCl、甘露醇、ABA和4 ℃低温的诱导表达。由上述结果推测PeNAC121基因与胡杨的逆境胁迫应答密切相关,表明该基因的启动子是一个能够应答多种逆境胁迫的诱导型启动子。本研究为阐明PeNAC121基因在胡杨逆境响应和调控中的作用机制提供理论参考。     

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。     

玉米钼辅助因子硫酸化酶基因启动子的克隆与功能验证

为克服组成型启动子启动外源基因过量表达引起的诸多问题,同源克隆(Mo-molybdopterin cofactor sulfurase)基因（ABA3）的启动子(ABA3s)序列,并用PlantCARE软件分析其非生物逆境应答元件, 实时定量PCR检测ABA3基因在非生物逆境诱导下的差异表达后。然后,用该启动子构建启动GUS（β-glucuronidase）基因的表达载体, 基因枪法转化玉米愈伤组织。经组织化学染色法检测其表达后, 在高渗、高盐、低温胁迫处理及ABA诱导下检测GUS酶荧光值与荧光素酶(内参)发光值的比值(GUS/LUC), 以此评价ABA3s启动子在非生物逆境胁迫下的启动活性。结果表明, ABA3基因在模拟干旱、低温、高温、高盐胁迫及ABA、乙稀诱导下差异表达, 说明该基因的启动子(ABA3s)具有非生物逆境诱导活性。序列分析表明, ABA3s启动子全长777 bp, 含有ARE、HSE、MBS、TGA、Circadian等多种非生物逆境胁迫应答元件。用ABA3s启动GUS基因构建的表达载体转化的玉米愈伤组织, 响应干旱、低温、高温、高盐胁迫等多种非生物逆境胁迫, 及ABA和乙稀诱导, GUS检测呈阳性。在8%甘露醇高渗条件下, GUS/LUC比值比空白对照高6倍。上述结果表明, ABA3s启动子具有非生物逆境诱导特性, 经进一步验证其功能后, 可用于玉米抗逆转基因研究。     

高等植物启动子的研究进展

启动子是基因表达调控的重要顺式元件,综述了高等植物启动子的构成,包括转录起始位点、TATA框和上游启动子元件。并着重从组成型、组织特异型和诱导型启动子3个方面介绍了其结构特征、功能,以及它们在植物基因工程中的应用和研究进展,简述了双向启动子、可变启动子和串联启动子的研究情况,提出植物启动子研究中存在的问题与展望。     

植物抗病相关启动子及其研究进展

启动子是调控基因表达的重要顺式元件。植物抗病相关启动子的调控特性研究、分离及其应用对于提高植物抗病性极其关键。本文综述了植物基因启动子的基本结构、克隆方法,着重介绍了组成型、组织特异型、天然与人工合成的病原诱导型启动子的研究进展,及其在植物抗病基因工程中的应用现状和存在问题,并展望了植物抗病相关启动子的应用前景。     

Transgenic plants with improved dehydration-stress tolerance: Progress and future prospects

This review summarizes the recent progress made towards the development of transgenic plants with improved tolerance to water stress and salinity. Of the various strategies employed, emphasis has been given to the genes engineered for the biosynthesis of osmoprotectants and osmolytes. This review also briefly discusses the importance of the use of specific stress inducible promoters and the future prospects of transgenic plants with improved agronomic traits.     

Stress-induced synthesis of proline confers tolerance to water deficit in transgenic wheat

Water deficit is one of the main abiotic factors that affect spring wheat planted in subtropical regions. Accumulation of proline appears to be a promising approach to maintain the productivity of plants under stress condition. However, morphological alterations and growth reduction are observed in transgenic plants carrying genes coding for osmoprotectants controlled by constitutive promoters. We report here the effects of water deficit on wheat plants transformed with the Vigna aconitifolia Delta(1)-pyrroline-5-carboxylate synthetase (P5CS) cDNA that encodes the key regulatory enzyme in proline biosynthesis, under the control of a stress-induced promoter complex-AIPC. Transgenic wheat plants submitted to 15 days of water shortage presented a distinct response. We have found that drought resulted in the accumulation of proline. The tolerance to water deficit observed in transgenic plants was mainly due to protection mechanisms against oxidative stress and not caused by osmotic adjustment.     

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。     

Expression of RNA-interference/antisense transgenes by the cognate promoters of target genes is a better gene-silencing strategy to study gene functions in rice

Antisense and RNA interference (RNAi)-mediated gene silencing systems are powerful reverse genetic methods for studying gene function. Most RNAi and antisense experiments used constitutive promoters to drive the expression of RNAi/antisense transgenes; however, several reports showed that constitutive promoters were not expressed in all cell types in cereal plants, suggesting that the constitutive promoter systems are not effective for silencing gene expression in certain tissues/organs. To develop an alternative method that complements the constitutive promoter systems, we constructed RNAi and/or antisense transgenes for four rice genes using a constitutive promoter or a cognate promoter of a selected rice target gene and generated many independent transgenic lines. Genetic, molecular, and phenotypic analyses of these RNAi/antisense transgenic rice plants, in comparison to previously-reported transgenic lines that silenced similar genes, revealed that expression of the cognate promoter-driven RNAi/antisense transgenes resulted in novel growth/developmental defects that were not observed in transgenic lines expressing constitutive promoter-driven gene-silencing transgenes of the same target genes. Our results strongly suggested that expression of RNAi/antisense transgenes by cognate promoters of target genes is a better gene-silencing approach to discovery gene function in rice.     

High-efficiency transformation and selective tolerance against biotic and abiotic stress in mulberry, <Emphasis Type="Italic">Morus indica</Emphasis> cv. K2, by constitutive and inducible expression of tobacco <Emphasis Type="Italic">osmotin</Emphasis>

Osmotin and osmotin-like proteins are stress proteins belonging to the plant PR-5 group of proteins induced in several plant species in response to various types of biotic and abiotic stresses. We report here the overexpression of tobacco osmotin in transgenic mulberry plants under the control of a constitutive promoter (CaMV 35S) as well as a stress-inducible rd29A promoter. Southern analysis of the transgenic plants revealed the stable integration of the introduced genes in the transformants. Real-time PCR analysis provided evidence for the expression of osmotin in the transgenic plants under both the constitutive and stress-inducible promoters. Transgenic plants with the stress-inducible promoter were observed to better tolerate salt and drought stress than those with the constitutive promoter. Transgenic plants when subjected to simulated salinity and drought stress conditions showed better cellular membrane stability (CMS) and photosynthetic yield than non-transgenic plants under conditions of both salinity and drought stress. Proline levels were very high in transgenic plants with the constitutive promoter relative to those with the stress-inducible promoter. Fungal challenge undertaken with three fungal species known to cause serious losses to mulberry cultivation, namely, Fusarium pallidoroseum, Colletotrichum gloeosporioides and Colletotrichum dematium, revealed that transgenic plants with osmotin under control of the constitutive promoter had a better resistance than those with osmotin under the control of the stress-inducible promoter. Evaluation in next generation was undertaken by studying bud break in transgenic and non-transgenic plants under simulated drought (2% polyethylene glycol) and salt stress (200 mM NaCl) conditions. The axillary buds of the selected transgenic lines had a better bud break percentage under stressed conditions than buds from non-transgenic mulberry lines. A biotic assay with Bombyx mori indicated that osmotin protein had no undesirable effect on silkworm rearing and feeding. We therefore conclude that 35S transgenic plants are better suited for both abiotic stress also biotic challenges (fungal), while the rd29A transgenic plants are more responsive to drought.