一个含LoxP-FRT重组酶位点及易于操作的植物表达双元载体pYBA100

在农杆菌(Agrobacterium tumefaciens)介导的植物遗传转化中,常常需要用到植物表达双元载体。为便于基因工程操作并能在获得转基因植物后方便将标记基因删除,我们构建了植物表达双元载体pYBA100,以满足用于生产安全转基因植物的需要。本研究通过融合PCR方法,尽可能去除所有非必需的序列,将pYBA载体最小化。pYBA载体在大肠杆菌中为多拷贝,骨架为3.69kb。我们构建的含NptⅡ植物筛选标记基因的载体pYBA100仅为5.37kb,多克隆位点为22个,方便基因工程操作。载体pYBA100的T-DNA植物筛选基因表达框两侧预留有LoxP-FRT融合位点,方便在获得转基因植物后通过Cre或FLP重组酶将植物筛选标记基因删除。pYBA100载体能于大肠杆菌和农杆菌中自我复制,并成功转化了拟南芥,符合农杆菌介导的植物转基因要求。离体删除实验结果证明,载体pYBA100能经Cre重组酶删除植物标记基因表达框。     

含双边界序列植物双价表达载体的构建

转基因植物的安全性是基因工程改良作物的一个重要问题.构建含双边界序列（双T-DNA区）载体,将选择标记基因和目标外源基因分开在不同的T-DNA区,通过转基因植株有性杂交及后代分离,可在转基因后代中获得无选择标记的转基因安全植株.将2个外源基因置于同一载体上,可以提高其共转化的效率.本研究构建了1个中间载体pAHC17-PTA和1个含双边界序列的植物双价表达载体pDB13PS.pAHC17-PTA含有由Ubiquitin启动子引导的具有抗虫效果的半夏凝集素基因（PTA）.pDB13PS含2个独立的T-DNA区,在其中一个T-DNA区,含两个目的基因的完整的表达盒,一个是由Ubiquitin启动子引导的半夏凝集素基因（PTA）,另一个是由水稻胚乳特异表达启动子（Glutelin-B1 promoter）驱动的马铃薯高赖氨酸蛋白基因的cDNA（SB401）.pDB13PS的成功构建,为提高PTA和SB401的共转化效率和获得无选择标记的转基因后代植株奠定了基础.     

The biotechnology of crop legumes

Summary The absence of variety-independent gene transfer methods for major agronomic species has, until now, limited the usefulness of recombinant DNA techniques to crop improvement programs. Until recently, only Solanaceous crops could be used to study fundamental and applied problems in plant sciences. During the past five years rapid advances in cell biology, in combination with the development of novel gene transfer methodology allowed utilization of the tools of plant molecular biology in conventional breeding programs. Cereal and leguminous species were considered to be recalcitrant to genetic manipulation. As a result of the development of direct DNA transfer methodology into organized tissue, we are now in a position to introduce any foreign gene into almost all of the major cereals and legumes. This can be achieved efficiently, often in a variety-independent fashion. The object of this review is to provide a comprehensive account of the state of the art in gene transfer for the cultivated leguminous crops. Important oilseed and feed species primarily in industrialized countries, as well as minor but equally important species for sustaining growth populations in developing countries will be examined. Advantages of the various gene transfer methods that were shown to be useful for specific crops, as well as limitations and problems associated with each crop and gene transfer method will be discussed. Data from field trials of transgenic legumes, where available, will be presented.     

Production of Transgenic Plants： More, Better, and Faster

Over the last two decades, transgenic plants have moved from being solely laboratory vehicles for basic research work to providing new varieties grown on large areas throughout the world. A number of plant     

用核基质结合区(SAR)序列提高小麦最小表达框转基因表达的稳定性

采用最小表达框技术转化植物可以规避由骨架序列引起的安全风险。核基质结合区序列SAR (scaffold attachment region)可作为边界元件与核基质结合阻挡转基因片段邻近染色质区的作用与影响, 提高外源基因稳定性。本研究在最小表达框序列两端添加SAR序列, 提高小麦最小表达框转基因表达的稳定性, 提高转化基因的表达效率。首先, 以GUS为目的基因构建带有SAR序列的最小表达框, 以科农199为受体进行基因枪转化, 同时以不加SAR序列的最小表达框片段为对照。带有SAR序列的最小表达框片段共轰击857个幼胚, T₀代获得40株再生植株, PCR检测到16株阳性植株, 转化效率为1.87%; 对这16个阳性单株进行GUS染色, 15株显色; 从来自4个T₀阳性植株的18个T₁代植株中随机选取18株进行PCR和GUS染色检测, 有15株表现为阳性。不带SAR序列的对照片段轰击1012个幼胚, 获得31株再生植株, 其中5株PCR阳性, 转化效率0.49%, 这5个阳性植株中仅2株为GUS染色阳性; 来自于5个T₀代PCR阳性株系的10个T₁代单株中没有发现PCR和GUS染色阳性株。表明SAR序列可以提高基因枪转基因效率和目的基因表达稳定性。为了创制抗旱转基因小麦, 以来自大豆的抗旱相关转录因子基因GmDREB3为目的基因, Bar基因为筛选标记基因, 转化受体小麦济麦22, 共轰击6045个幼胚, 获得再生植株130株, PCR检测阳性植株30株, 转化效率为0.50%; 随机选取6株PCR阳性植株进行RT-PCR分析, 其中5株可检测到外源基因的转录。进一步对这5株RT-PCR阳性植株插入片段完整性进行分析, 其中4株插入片段基本完整。通过real-time PCR分析, 发现T₀代6个RT-PCR阳性植株的外源GmDREB3的拷贝数为1~3个。以上结果证明, 在最小表达框两端加上SAR序列后可以提高小麦最小表达框转基因表达的稳定性。     

根癌农杆菌介导获得稗草Ecppc转基因小麦的研究

采用携带pUbi-Ecppc质粒的3个根癌农杆菌菌株（LBA4404、EHA105和C58c1）,对经过预培养10~12 d的春小麦品种扬麦158、Bobwhite和扬麦12的幼胚愈伤组织进行了遗传转化。对筛选中的抗生素浓度、菌液浓度、共培养温度和时间、受体基因型、菌株-质粒组合等影响转化的重要因素进行了研究。首次将单子叶野生C₄植物稗草的磷酸烯醇式丙酮酸羧化酶基因（Ecppc）导入小麦受体基因型,并得到具有潮霉素（Hyg）抗性的转化植株。从816块共培养愈伤组织中转化得到34株抗性植株,其中14株PCR检测为阳性。扬麦158的转化效率达3.03%。Southern和RT-PCR分析表明外源基因已整合到小麦基因组并得到正确的转录。生理学检测显示,转基因小麦植株的光合速率和PEPC活性都有所提高。说明Ubiqintin基因启动子控制的稗草PEPC cDNA基因在小麦中可以正确表达和起到一定的生理作用。这些工作为进一步探讨PEPC对小麦光合作用及其他生理过程的影响奠定了基础。     

The Experimental and Commercial Release of Transgenic Crop Plants

With advances in recombinant DNA methods and transformation procedures, it is possible to transfer genes into crop plants from unrelated plants, microbes and animals. Many of the modifications being carried out, or envisaged, are for disease and pest resistance, product quality and tolerance to environmental stress, but there are additional opportunities to modify crops to give specialized products for industrial or pharmaceutical use. Some of the characteristics of transgenic plants are considered, including: transgene copy number, position, expression, stability, pleiotropy, selectable marker genes and somaclonal variation. There have been several hundreds of field trials with transgenic plants, and the first transgenic varieties are likely to be approved for commercial production in 1993. Before releasing transgenic plants, it is necessary to carry out a risk assessment to determine whether the transgenic variety will behave differently from a conventionally bred variety. Assessment procedures are being harmonized internationally by various organizations. There is a growing commitment to apply these genetic modification methods to crops in developing countries, as genes relevant to their crops and environments become available.     

海藻糖酶基因RNA干扰载体对花烟草的转化

为了能够使花烟草更好地应用于园林美化,需要进一步提高其抵抗非生物胁迫的能力。海藻糖在植物抗逆性方面具有重要作用。利用RNA干扰技术抑制植物海藻糖酶基因的表达,可以阻断海藻糖降解过程,从而使植物体内海藻糖含量增加,有望以此提高植物抵抗非生物胁迫的能力。本实验利用叶盘侵染法将带有拟南芥海藻糖酶基因干扰载体(iTre-1285)的农杆菌导入花烟草中,经过抗生素筛选初步获得阳性植株后,对阳性植株提取DNA,完成了目的基因的PCR鉴定,初步证明目的基因已转入花烟草植株中。通过本试验,在建立起花烟草基因转化方法的同时,成功地将海藻糖酶干扰基因转入花烟草中。转基因花烟草植株的获得为后期能够进一步筛选出具有抵抗非生物胁迫的转基因植株奠定了基础。     

花生抗黄曲霉的植物双价表达载体的构建

通过常规育种培育的抗黄曲霉品种皆表现抗性不稳定,鉴此开展通过基因工程手段以培育高抗以及无筛选标记的转基因花生品种。几丁质酶基因CHI和葡聚糖酶基因GLU皆为广谱性的抗病基因且具有协同增效作用。分别以pSC1300-8-CHI和pSC1300-13-GLU载体为基础,通过PCR技术在CHI基因的特异启动子8#前端和T-nos末端分别加入NcoⅠ酶切位点和AflⅡ酶切位点,在GLU基因的特异启动子13#前端和T-nos末端分别加入ApaⅠ酶切位点和SpeⅠ酶切位点,通过酶切、连接将上述2个基因连接在抗生素自我删除载体pLoxp上,获得了具有抗生素自我删除选择标记的双价果种皮特异表达载体pLoxp-8-CHI-13-GLU。经过限制性内切酶鉴定,该植物表达载体构建成功。     

Tomato: a crop species amenable to improvement by cellular and molecular methods

Summary Tomato is a crop plant with a relatively small DNA content per haploid genome and a well developed genetics. Plant regeneration from explants and protoplasts is feasable which led to the development of efficient transformation procedures.In view of the current data, the isolation of useful mutants at the cellular level probably will be of limited value in the genetic improvement of tomato. Protoplast fusion may lead to novel combinations of organelle and nuclear DNA (cybrids), whereas this technique also provides a means of introducing genetic information from alien species into tomato. Important developments have come from molecular approaches. Following the construction of an RFLP map, these RFLP markers can be used in tomato to tag quantitative traits bred in from related species. Both RFLP's and transposons are in the process of being used to clone desired genes for which no gene products are known. Cloned genes can be introduced and potentially improve specific properties of tomato especially those controlled by single genes. Recent results suggest that, in principle, phenotypic mutants can be created for cloned and characterized genes and will prove their value in further improving the cultivated tomato.     

菊花DgLsL基因RNAi表达载体的构建及遗传转化

培育少侧枝的标准切花菊品种具有重要的实际意义。本研究利用RNAi的方法抑制菊花侧枝发育相关基因DgLsL基因的表达,以期得到侧枝生长受抑制的转基因植株。从切花菊‘神马’基因组DNA中克隆侧枝发育相关基因DgLsL基因,经过测序分析,将一个413bp长的保守片段连接到植物表达载体pBI121和pFGC5941,构建植物RNAi载体pBI121-R2,并运用农杆菌介导法将其导入切花菊‘神马’。结果:PCR得到的DgLsL基因片段测序后与GenBank上的DgLsL基因比对,DNA序列一致性为99.4%;构建的RNAi表达载体经过PCR和酶切证实外源基因的正确插入;转化后得到了18株抗性植株,PCR结果显示,12株抗性植株为阳性。本试验通过将DgLsL基因干扰片段导入切花菊,获得了转基因植株,以此为基础可进一步选育少侧枝的切花菊品种。     

Wheat transformation – an update of recent progress

Summary Genetic transformation is vital to the transfer of novel genes into crop plants as well as to the emerging area of functional genomics. However, the successful genetic transformation of wheat still remains time consuming and genotype dependent. This paper updates the progress made in last 3 years towards developing a robust genetic transformation system for wheat. Agrobacterium-mediated wheat transformation offers advantages such as single-copy gene insertion, minimal rearrangement of DNA, low cost and comparatively high efficiency. The reported recent developments in wheat transformation will lead to increased efficiency of wheat breeding programs. The most promising recent progress is in the development of drought-tolerant wheat, since water stress continues to be a major limiting factor hindering world wheat productivity under adverse hot and dry weather conditions.     

Genetic engineering of cereal crop plants: a review

Summary Many aspects of basic and applied problems in plant biology can be investigated by transformation techniques. In dicotyledonous species, the ability to generate transgenic plants provides the tools for an understanding of plant gene function and regulation as well as for the directed transfer of genes of agronomic interest.For many dicotyledonous plants Agrobacterium tumefaciens can be routinely used to introduce foreign DNA into their genome. However, cereals seem to be recalcitrant to Agrobacterium-mediated transformation.In cereals, many efforts have been made in recent years to establish reliable transformation techniques. Several transformation techniques have been developed but to date only three methods have been found to be suitable for obtaining transgenic cereals: transformation of totipotent protoplasts, particle bombardment of regenerable tissues and, more recently, tissue electroporation. The current state of transformation methods used for cereals will be reviewed.     

农杆菌介导轮状病毒抗原蛋白VP4基因遗传转化花生的研究

选用鲁花14与花育23花生品种, 通过农杆菌介导开展了轮状病毒抗原蛋白VP4基因遗传转化研究, 从转化植株中随机选取26株表现Kan抗性植株进行npt II基因的PCR检测, 结果有22株能扩增出620 bp左右的npt II基因条带, 阳性率约为84.62%。提取npt II基因显示为阳性的植株叶片DNA作模板, 用VP4基因特异引物进行PCR扩增, 经琼脂糖凝胶电泳分析, 所有npt II基因阳性的植株均扩增出了约2 350 bp的特异性条带, 而野生型没有。对部分转基因植株进一步进行PCR-Southern杂交和Southern杂交分析, 发现转基因植株中出现了阳性杂交信号, 表明VP4基因的确已经整合到花生的基因组中, 并且是1~2个拷贝。用RT-PCR分析了11株转G1VP4基因的植株, 证明插入鲁花14中的VP4基因已经正常转录, 利用Western blot方法检测筛选到的4株转基因花生, 分别提取其蛋白, 在30 kD处出现特异性蛋白条带, 这为获得转基因创新种质材料奠定了基础。     

Coat protein-mediated protection to cucumber mosaic virus infections in cultivated tomato

Summary Cucumber mosaic virus (CMV) infections rank among the most devastating diseases in the commercial culture of tomato (Lycopersicon esculentum Mill.), for which suitable sources of natural resistance are not available. The concept of pathogen-derived resistance, however, offers an alternate approach to combat plant viral diseases by transformation of crops with nucleotide sequences derived from the viral genome. This report demonstrates the successful application of such a pathogen-derived resistance gene comprising the CMV coat protein (CP) gene, to generate protection to CMV infections in cultivated tomato. Transformation of an inbred tomato line with the CMV CP gene isolated from a subgroup I strain, engendered high levels of protection to various CMV strains, including a virulent strain causing lethal necrosis and a typical subgroup II strain. Moreover, when challenged by natural infection through aphid vectors in open field, levels of protection were largely maintained in hemizygous hybrids. In all, these results demonstrate that synthetic resistance genes based on the CMV CP gene make excellent sources of broad spectrum resistance to CMV infections for introgression into tomato breeding programs.     

转甜菜碱醛脱氢酶基因马铃薯的抗旱耐盐性

通过根癌农杆菌介导法将甜菜碱醛脱氢酶(BADH)基因导入马铃薯栽培品种甘农薯2号, 经PCR、Southern杂交和Northern杂交证明BADH基因已整合到马铃薯基因组中并在转基因植株中转录和表达。测定表明对照植株没有BADH酶活性, 各转化株系在胁迫前后BADH酶活性近似, 在2~11 U之间。BADH酶活性与叶片的相对电导率呈一定的负相关(y= –3.7738x+57.083, r=0.989^**)。在NaCl和PEG胁迫下, 转基因植株生长正常, 株高比对照提高0.41~1.00 cm, 单株重量比对照增加10%~35%, 说明外源BADH基因的导入提高了马铃薯植株对干旱和盐碱的抗性。     

农杆菌介导轮状病毒抗原蛋白VP4基因遗传转化花生的研究

选用鲁花14与花育23花生品种, 通过农杆菌介导开展了轮状病毒抗原蛋白VP4基因遗传转化研究, 从转化植株中随机选取26株表现Kan抗性植株进行npt II基因的PCR检测, 结果有22株能扩增出620 bp左右的npt II基因条带, 阳性率约为84.62%。提取npt II基因显示为阳性的植株叶片DNA作模板, 用VP4基因特异引物进行PCR扩增, 经琼脂糖凝胶电泳分析, 所有npt II基因阳性的植株均扩增出了约2 350 bp的特异性条带, 而野生型没有。对部分转基因植株进一步进行PCR-Southern杂交和Southern杂交分析, 发现转基因植株中出现了阳性杂交信号, 表明VP4基因的确已经整合到花生的基因组中, 并且是1~2个拷贝。用RT-PCR分析了11株转G1VP4基因的植株, 证明插入鲁花14中的VP4基因已经正常转录, 利用Western blot方法检测筛选到的4株转基因花生, 分别提取其蛋白, 在30 kD处出现特异性蛋白条带, 这为获得转基因创新种质材料奠定了基础。     

转甜菜碱醛脱氢酶基因马铃薯的抗旱耐盐性

通过根癌农杆菌介导法将甜菜碱醛脱氢酶(BADH)基因导入马铃薯栽培品种甘农薯2号, 经PCR、Southern杂交和Northern杂交证明BADH基因已整合到马铃薯基因组中并在转基因植株中转录和表达。测定表明对照植株没有BADH酶活性, 各转化株系在胁迫前后BADH酶活性近似, 在2~11 U之间。BADH酶活性与叶片的相对电导率呈一定的负相关(y= –3.7738x+57.083, r=0.989^**)。在NaCl和PEG胁迫下, 转基因植株生长正常, 株高比对照提高0.41~1.00 cm, 单株重量比对照增加10%~35%, 说明外源BADH基因的导入提高了马铃薯植株对干旱和盐碱的抗性。     

果树基因转化技术的选用和转基因植株的鉴定与性状分析

植物基因工程为果树育种开辟了一条新途径,在果树重要性状遗传改良方面具有重要意义。果树转基因是一个复杂而长期的工作,包括从对外源基因功能的全面认识、高效表达载体的构建、基因转化、以及转基因植株的鉴定与性状观测等多个重要环节。应该说,每一个环节甚至每一实验步骤都需严格把握。迄今,鉴于已经有大量果树再生体系成功建立的报道,笔者在总结实际研究经验的基础上,结合文献的报道,有针对性地对果树基因转化技术和转基因植株的鉴定与性状分析进行简要介绍,为果树基因转化技术的选用,以及进行高效转基因植株的鉴定与性状分析提供一定的参考。     

叶用莴苣无缝克隆构建LsE3基因过表达载体和新RNAi载体及遗传转化体系优化

研究旨在应用新技术和新载体构建过表达和沉默载体,优化遗传转化体系,验证生菜LsE3基因功能,探究高温抽薹机制。以pCAMBIA1304和pFGC5941载体为材料,通过无缝克隆构建叶用莴苣转基因载体,并通过筛选培养基配方优化遗传转化体系。试验成功地以pCAMBIA1304为基础,插入源自pFGC5941的dsRNA表达框序列,获得了新RNAi空载pCAMBIA1304- FGC5941,构建了LsE3沉默载体,并成功以双元质粒载体pCAMBIA1304构建了LsE3过表达载体。试验对易抽薹叶用莴苣品种‘GB-31’进行潮霉素浓度、直接芽诱导培养基配方和生根培养基配方的筛选,确立了潮霉素适宜筛选浓度为5 mg/L,并确立了最优培养芽诱导和生根培养基配方。试验成功获得新RNAi空载并构建了转基因载体,优化了遗传转化体系,为解决载体构建繁琐复杂的问题提供新思路,并奠定了生菜转基因技术的基础。