细菌启动子识别及应用研究进展

细菌启动子是细菌中基因表达的必需调控元件,决定了细菌基因表达的强度和时机。通过启动子的插入或缺失,可以改变细菌基因的表达,实现对菌体生长发育以及代谢调控的研究。启动子也是构建各种表达系统、实现异源基因表达的基础。启动子的识别和应用研究,对于实现异源基因的可控表达、有效获得目的产物、促进生物催化和代谢工程研究具有重要的意义。以下对细菌启动子进行了简单的介绍,总结了细菌启动子的识别方法,并对细菌启动子的研究进展和具体应用进行了概述。     

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。     

Hybrid Cytomegalovirus-U6 Promoter-based Plasmid Vectors Improve Efficiency of RNA Interference in Zebrafish

Short hairpin RNA (shRNA) directed by RNA polymerase III (Pol III) or Pol II promoter was shown to be capable of silencing gene expression, which should permit analyses of gene functions or as a potential therapeutic tool. However, the inhibitory effect of shRNA remains problematic in fish. We demonstrated that silencing efficiency by shRNA produced from the hybrid construct composed of the CMV enhancer or entire CMV promoter placed immediately upstream of a U6 promoter. When tested the exogenous gene, silencing of an enhanced green fluorescent protein (EGFP) target gene was 89.18 +/- 5.06% for CMVE-U6 promoter group and 88.26 +/- 6.46% for CMV-U6 promoter group. To test the hybrid promoters driving shRNA efficiency against an endogenous gene, we used shRNA against no tail (NTL) gene. When vectorized in the zebrafish, the hybrid constructs strongly repressed NTL gene expression. The NTL phenotype occupied 52.09 +/- 3.06% and 51.56 +/- 3.68% for CMVE-U6 promoter and CMV-U6 promoter groups, respectively. The NTL gene expression reduced 82.17 +/- 2.96% for CMVE-U6 promoter group and 83.06 +/- 2.38% for CMV-U6 promoter group. We concluded that the CMV enhancer or entire CMV promoter locating upstream of the U6-promoter could significantly improve inhibitory effect induced by the shRNA for both exogenous and endogenous genes compared with the CMV promoter or U6 promoter alone. In contrast, the two hybrid promoter constructs had similar effects on driving shRNA.     

A plasmid vector for isolation of strong promoters in Escherichia coli

In order to isolate very strong promoters from bacteria and bacteriophage a plasmid named pProm was constructed. It possesses an origin (ORI) for replication in Gram-negative bacteria, an ORI for replication in Gram-positive bacteria, a promoterless ampicillin resistance gene with a multiple cloning site (MCS) in the position formerly occupied by the ampicillin promoter, a tetracycline resistance gene for selection in Gram-negative bacteria and a chloramphenicol resistance gene for selection in Gram-positive bacteria. Insertion in the MCS of DNA fragments of Staphylococcus aureus bacteriophages resulted in isolation of several clones very resistant to ampicillin. The DNA fragments inserted in these recombinant plasmids were sequenced and all of them contained putative promoter motifs. Direct measurement of the penicillinase activity indicated that one of the isolated promoters could be included within a group of the stronger known prokaryotic promoters. According to these results pProm is a powerful tool to perform studies on promoter strength and for industrial applications.     

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。     

The state of the art of mammalian promoter recognition

The draft sequences of whole genomes are being published at an ever-increasing pace, thus providing access to the human genomic sequence and, more recently, the mouse sequence. Genomes of the invertebrates are also becoming available. Now that the genomic DNA of mammalian species is available, an old problem can be tackled with renewed vigour mammalian promoter prediction. Gene promoters have proved elusive for more than a decade, despite their pivotal role in gene regulation. Recently, however, several new developments have made it possible to make meaningful large-scale predictions. This paper reviews the methods used for the prediction of mammalian, mostly human, promoters.     

高等植物胁迫诱导型启动子的研究进展

逆境胁迫严重影响植物生长发育,降低作物产量。目前在植物抗逆基因工程中,大多使用组成型启动子驱动目的基因表达,组成型启动子的表达虽然能提高转基因植株的抗逆性,但持续过量地表达转化的外源基因有时会阻碍植物的生长且降低其产量。因此,诱导型启动子的研究具有重要的应用价值。该文对近年来植物在逆境胁迫处理下,一些诱导型启动子的种类和功能,可能具有的顺式作用元件,反式作用因子及其研究方法进行了综述。     

Novel and useful properties of a chimeric plant promoter combining CaMV 35S and MAS elements

The CaMV 35S and Ti plasmid mannopine synthetase (mas) promoters are commonly used by plant genetic engineers. To combine their useful properties, we constructed hybrid promoters incorporating elements from both. These promoters were spliced to the beta-glucuronidase reporter gene and introduced into tobacco and tomato plants by Agrobacterium cocultivation. T1 and T2 transgenic plant populations transformed with different constructs were assayed for the marker enzyme. Comparisons were made based on the range of expression levels found for each promoter construct. We found that a hybrid promoter incorporating the mas region from +65 to -301 and the 35S enhancer region from -90 to -941 had new and interesting properties. This promoter, called Mac, expressed gus at a level three to five times that expressed by a double 35S promoter in the leaves, and 10 to 15 times in hypocotyls and roots. The Mac promoter, however, showed only marginal wound inducibility. Five- to seven-fold wound induction required the presence of the region from -301 to -613 of mas. Reiteration of the 35S enhancer region, from -90 to -430, behind the 35S TATA box region or the mas +65 to -301 region had a smaller effect on expression, ranging from equal to twice the level of the single enhancer control.     

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。     

可诱导肿瘤靶向性IFN-α2a重组腺病毒的构建及其表达

腺病毒载体广泛应用于恶性肿瘤的靶向性基因治疗的研究,但这些肿瘤靶向载体缺乏可控性,其疗效和安全性受到很大的影响,因此开发新型可诱导的生物肿瘤靶向载体是当今抗肿瘤靶向药物研究的当务之急。该研究构建了可诱导肿瘤靶向性IFN-α2a重组腺病毒。重组腺病毒能够有效感染人肝癌细胞株HepG2等多种肿瘤细胞株,RT-PCR和Western blot结果表明肿瘤细胞能高效表达IFN-α2a,而其非肿瘤细胞株L02几乎没有表达。诱导试验表明重组腺病毒Ad MT-Ⅱ-hTERT/IFN-α2a能被ZnSO4诱导表达。可诱导肿瘤靶向性重组腺病毒Ad MT-Ⅱ-hTERT/IFN-α2a成功构建为下一步体内外抑癌实验研究打下了基础。     

CMV与T7启动子对仙台病毒微小基因组拯救效率的比较

构建一种以分泌型荧光素酶基因(Gluc)作为报告基因的仙台病毒BB1株微小基因组质粒,比较了CMV启动子与T7启动子对仙台病毒微小基因组的拯救效率。首先设计并合成锤头状核酶序列,仙台病毒trailer、L基因非编码区、N基因非编码区和leader序列以及丁型肝炎病毒核酶序列,插入含有CMV和T7双启动子的质粒pVAX1中,获得仙台微小基因组的通用型载体pVAX-miniSeV。将Gluc基因插入pVAX-miniSeV中,分别获得正向插入的仙台病毒微小基因组载体pVAX-miniSeV-Gluc(+)和反向插入的pVAX-miniSeV-Gluc(-)。用pVAX-miniSeV-Gluc(+)转染BHK21细胞能在上清中检测到高水平的Gluc活性,表明其中的CMV启动子具有正常转录功能。将pVAX-miniSeVGluc(-)和仙台病毒N、P、L蛋白表达质粒共转染BSR T7/5细胞(稳定表达T7RNA聚合酶的BHK-21细胞)检测到Gluc的高效表达,表明pVAX-miniSeV-Gluc(-)能够被有效拯救;但在BHK-21细胞中却未检测到Gluc的有效表达,提示该载体中的CMV启动子对仙台病毒微小基因组的拯救效率可能没有明显作用。为了进一步了解CMV与T7启动子各自对于仙台病毒微小基因组拯救的作用,本研究又构建了单独含有CMV或T7启动子的仙台病毒微小基因组载体pCMV-miniSeV-Gluc(-)和pT7-miniSeV-Gluc(-)。将这两种载体和仙台病毒N、P、L蛋白表达质粒分别共转染BSR T7/5细胞,结果pT7-miniSeV-Gluc(-)共转染组检测到了Gluc的高效表达,而pCMV-miniSeV-Gluc(-)共转染组未检测到,证实了通用型载体pVAX-miniSeV中仅T7启动子对仙台病毒微小基因组的拯救起了关键作用,而CMV启动子作用不明显。本研究成功构建了一种通用型双启动子仙台病毒微小基因组载体pVAX-miniSeV,并证明了T7启动子系统对仙台病毒微小基因组拯救的关键作用。本研究为下一步构建仙台病毒全基因感染性克隆打下了基础。     

肌肉特异性基因启动子的上游转录调控元件研究进展

真核生物启动子位于基因5’端上游转录起始位点附近,是包含核心启动子以及上游转录调控元件的一段DNA序列,这些转录调控元件控制着基因表达的强度和特异性。肌肉特异性启动子的上游调控元件种类、数量和排列顺序决定着基因在肌肉中的特异性表达。深入研究肌肉启动子的上游调控元件,可以进一步了解肌肉基因表达机制,从而为肌肉性状的改良、增殖分化的机理和疾病的基因治疗等研究提供重要依据。该文回顾了近年来肌肉特异性启动子研究领域中的新发现,包括肌肉特异性启动子转录调控元件的分子机制、建立人工合成肌肉启动子的方法及应用,并探讨该领域中急需解决的问题和发展前景。     

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。     

昆虫种群遗传控制技术中启动子的研究

利用昆虫遗传转化技术对害虫进行遗传控制是害虫防治研究的新方向,该技术具有物种特异、防效高且对环境友好的特点。启动子是基因表达调控的重要元件,选择合适的启动子是外源基因高效、准确表达的关键,对获得高效、稳定的遗传修饰昆虫品系至关重要。本文简要介绍了昆虫基因启动子的结构特征,重点描述了昆虫种群遗传防治中组成型启动子、性别和组织特异型启动子、特定发育时期启动子和诱导型启动子的研究和应用概况,并对这几类启动子在害虫遗传控制中的应用前景进行了展望。     

Photosynthesis nuclear genes generally lack TATA-boxes: a tobacco photosystem I gene responds to light through an initiator

A maternal plant exquisitely promotes the success of its offspring by orchestrating embryo development and endowing protection even after the embryos mature. It uses ovule integuments for physical and physiological contact with the developing embryo and for subsequently equipping the seed with a seed coat (testa). The testa is developmentally and metabolically dynamic, but its molecular biology is not well understood. We show here that the inner integument in Brassica napus undergoes organized development and then programmed cell death (PCD), as evident from vacuolation, starch mobilization, DNA fragmentation and eventual compression. We have identified a cysteine proteinase gene (BnCysP1) that is expressed only in the inner integument as it undergoes PCD, well before the embryo begins storage protein synthesis. Two paralogous Cys proteinases have been recruited in rapeseed for the PCD of testa and for leaf senescence, and these differ 25% in their primary structure and post-translational modifications. Despite Arabidopsis being closely related to rapeseed, and an indication of developmental compression of its inner integument, the Arabidopsis genome is suggestive of only one Cys proteinase that shows approximately 72% identity to BnCysP1. It is, however, leaf senescence-associated, and the other Cys proteinases are <52% identical. BnCysP1 also differs from ricinosome-deployed PCD Cys endopeptidases in lacking the hallmark KDEL tail and being glycosylated. BnCysP1, one of the very few plant genes known to function only in the seed coat, will be useful in dissecting post-fertilization development of this important organ in rapeseed.