根癌农杆菌介导的草莓遗传转化研究进展

随着植物离体再生技术的日益成熟和多种外源目的基因的分离与克隆,转基因技术以目的性强、周期短等优点成为草莓品种改良的重要途径。在草莓上采用的遗传转化方法主要是农杆菌介导法。概述了农杆菌介导法的转化机理以及基因型、农杆菌菌株、侵染时间和菌液浓度、共培养时间、酚类物质等对草莓遗传转化的影响,并从草莓果实贮藏保鲜、抗除草剂、抗病毒、抗虫、抗真菌、转基因疫苗等方面对近年来草莓的转基因研究进展进行了总结。     

草莓转基因研究进展

转基因是一项成熟的技术,在草莓中得到广泛应用。本文综述了农杆菌介导的草莓转基因研究进展。     

FaSPS1基因对草莓果实成熟调控的分子机理

该研究以草莓品种‘红颜’(Fragaria×ananassa‘Benihoppe’)为试材,分析了草莓果实发育不同阶段蔗糖磷酸合成酶基因(FaSPS1)的表达量变化,采用PCR方法克隆FaSPS1基因,构建带有报告基因的e-GFP植物表达载体,通过瞬时转基因方法转化草莓果实,采用观察绿色荧光和检测目的基因表达量的方法鉴定转基因植物,并分析FaSPS1基因超表达和反义表达后草莓果实的成熟发育以及与成熟相关的基因表达量变化,探究FaSPS1基因在果实成熟发育中的特殊作用,为深入了解草莓果实发育和成熟调控的分子机理提供思路。结果显示:(1)成功克隆得到FaSPS1基因(GenBank登录号AB267868.1);成功构建带有报告基因e-GFP的FaSPS1基因超表达载体和反义基因表达载体,通过瞬时转基因方法转化并经荧光和目的基因表达量检测的方法鉴定获得转FaSPS1基因草莓植株。(2)与空载对照和非转基因果实相比,FaSPS1基因过表达可促进草莓果实成熟,能够使草莓果实成熟期提前,且果实中蔗糖果糖含量升高;但反义表达后会抑制草莓果实成熟,果实中苹果酸含量升高。(3)基因超表达或者反义表达后,草莓果实成熟相关基因的表达量受到不同程度调控,其中糖代谢基因FaSPS2/3、FaSUT1,果实成软化基因FaEXP1、FaEXP3、FaXYL1以及激素代谢基因FaJAZ1、FaJAZ2、FaJAZ8、FaOPR3、FaPYL1、FaPYL8、FaPYL9、FaNCED1表达量变化最明显。研究推测,FaSPS1基因可能通过影响草莓果实中和成熟相关的糖代谢基因、果实软化基因以及激素代谢基因来调控草莓果实成熟。     

冷诱导转录因子CBF1转化草莓及其抗寒性鉴定

以草莓栽培品种哈尼"为材料,利用根癌农杆菌介导的叶盘法将拟南芥冷诱导转录激活因子CBF1(C-re-peat binding factor)导入草莓中.转化植株经25 mg/L Km筛选后进行PCR和Southern杂交鉴定,结果显示CBF1基因整合进草莓基因组中.采用电解质渗漏法和生长恢复法对转基因株系进行抗寒生理鉴定,结果显示,转基因株系的电导率普遍低于对照;将转基因株系和对照于-2-0℃放置7 d,在转基因株系83中有65%的植株出现了萎蔫,而对照植株有91%出现了萎蔫,经22-25℃下进行恢复生长,转基因株系83有74%完全恢复,而对照株系只有18%恢复.结果表明CBF1基因导入草莓中可以提高草莓对低温胁迫的抵抗力.     

转基因棉花研发及商业化发展态势

正国内外转基因棉花的研发主要集中于抗虫、抗除草剂、抗病、耐盐碱、抗旱和纤维改良等性状研究,但只有转基因抗虫、抗除草剂和纤维改良等性状得到注册应用。转基因棉花的大规模商业化种植,可以有效控制棉花害虫与杂草并改善棉花纤维品质,显著提高棉花产量,降低生产成本。主要描述了棉花转基因的主要技术和规模化转基因技术体系平台,论述了转基因棉花近年来的总体研发状况及在棉花抗虫、抗除草剂及纤维改良方面的研究进展,分析了全球前四大转基因棉花种植国家(印度、美国、中国和巴基斯坦)转基因棉花的商业化发展态势,并归纳总结了转基因棉花的安全性问题及今后主要的发展方向。     

根癌农杆菌介导转录因子CBF1基因对草莓的转化

以草莓(Fragaria ananassa Duch)品种“达斯莱克特“(Darselect)为试材,用根癌农杆菌介导的方法,将转录因子CBF1基因导入草莓叶盘细胞,经多次筛选获得了转基因植株.转化植株经PCR检测,证实了CBF1基因已经整合到草莓的基因组中.以电解质渗透法检测了植株的抗寒性,结果显示转基因草莓的抗寒能力较未转化植株有明显提高,且不同转基因株系之间提高程度有着差异.     

我国转基因鱼研制的历史回顾与展望

中国自从诞生了首例转基因鱼以来,在后续30多年里取得了一系列重要研究进展。全球范围的转基因鱼研究包括多种养殖鱼类,目标性状涉及快速生长、抗病抗逆和品质改良。现在已经初步建成转基因鱼育种技术体系和安全评估体系,为转基因鱼产业化奠定了重要基础。本文以转基因黄河鲤育种研究为主线,简要回顾了转基因鱼研究的发展历程,并对转基因鱼育种面临的问题和发展前景进行了分析和展望。     

转trxS基因大麦发芽种子水解酶活性的变化

利用转基因技术是改良大麦品种品质的有效途径.研究了转trxS基因对大麦种子发芽过程中水解酶活性的影响,结果表明转基因种子中α-淀粉酶、自由态β-淀粉酶和极限糊精酶的活性比未转基因种子高;转基因种子醇溶蛋白和谷蛋白中巯基的含量提高,说明该基因能够表达,为大麦育种和品质改良提供新的途径.     

草莓遗传转化研究进展

草莓是一种重要的小型浆果,近年来育出很多优良品种,种植面积逐渐扩大.然而仍有很多因素限制了草莓生产的发展.基因工程技术为植物育种、种质资源开发利用等领域开辟了新的途径,可以获得常规育种难以或无法得到的新类型.本文综述了近年来国内外在草莓抗病虫、抗逆、抗除草剂、品质特性、耐贮运性等基因工程的研究进展以及基因型、试管苗生理状态、共培养时间、侵染时间和菌液浓度、抗生素等因素对草莓遗传转化的影响.对草莓转基因研究中存在的主要问题和今后的研究方向及应用前景进行了探讨.     

甘蔗转基因育种研究进展

由于复杂的遗传背景、较低的可育性和缺乏优异种质资源等因素给甘蔗品种改良带来了很大的困难,而通过转基因技术为甘蔗品种改良提供了有效的途径。甘蔗通过转基因,不仅在其抗虫性(螟虫和蚜虫)、抗病性(白条病、花叶病和黑穗病)、蔗糖改良(蔗糖产量、蔗糖纯度和色泽)、抗除草剂、抗旱性等方面已经取得了很大的进展,而且转基因甘蔗作为生物反应器生产重组蛋白(GM-CSF等)和生物塑料聚羟基丁酸酯(PHB)也取得了很好的成果。综述甘蔗主要性状遗传改良研究进展、转基因存在的问题以及转基因安全性研究状况,并对今后的甘蔗转基因育种前景和研究方向进行分析。     

Transgenic strawberry: state of the art for improved traits

Strawberry (Fragaria × ananassa Duch.), a member of the Rosaceae family, is one of the most important fruit crops cultivated worldwide. Strawberry is unique within the Rosaceae because it is a rapidly growing herbaceous perennial with a small genome, short reproductive cycle, and facile vegetative and generative propagation for genetic transformation. For these reasons, strawberry has been recognized as excellent germplasm for genetic and molecular studies for the Rosaceae family. Although traditional breeding methods have achieved steady improvement in agronomic traits, the lack of useful economic characters still remains a major challenge. Genetic transformation has opened a new era for greater creativity in strawberry breeding and germplasm by offering an effective method for creating new varieties that selectively targets a specific interested gene or a few heterologous traits. Enormous advances have been made in strawberry genetic transformation since the first transgenic strawberry plant was obtained in 1990. This paper reviews recent progress in genetic transformation of strawberry on increasing resistance to viruses, fungi, insects, herbicides, stress, and achieving better quality. Problems and prospects for future applications of genetic transformation in strawberry are also discussed.     

Characterization of LF9, an octoploid strawberry genotype selected for rapid regeneration and transformation

Cultivated strawberry (Fragaria ×ananassa) is a valuable crop, yet the absence of a rapid, high-throughput transgenic system has precluded meaningful application of biotechnology and translation of information from plant models to this crop. A new octoploid strawberry genetic line Laboratory Festival #9 has been identified, selected solely for its rapid regeneration and efficient transformation. Direct organogenesis has been achieved from all tissues tested, with rapidly-growing shoot initials visible in as few as 13 days. The conditions for optimal shoot regeneration, transformant selection, root generation, and plant acclimatization are presented. The progression from explant to plant in soil can be achieved in about 60 days. The development of transformation protocols in this rapid-cycling genotype allows high-throughput studies of gene function in the octoploid strawberry genetic background.     

Adventitious shoot regeneration from seven commercial strawberry cultivars (<Emphasis Type="Italic">Fragaria </Emphasis>×<Emphasis Type="Italic"> ananassa</Emphasis> Duch.) using a range of explant types

The parameters for optimal regeneration of seven commercial strawberry cultivars were tested using a range of explants and culture conditions. Efficient levels of regeneration--those needed to carry out transformation experiments--with the cultivars Calypso, Pegasus, Bolero, Tango and Emily were achieved with leaf discs, petioles, roots and stipules. Regeneration from cv. Elsanta proved to be difficult from all explant material, although unpollinated ovaries proved to be a promising explant source, with 12% of the explants regenerating shoots. In cv. Eros, regeneration occurred only from root tissue. A comparison of the genetic background suggests that there is a strong genetic component amongst the different cultivars determining their regeneration capacity. The development of these regeneration systems provides a means to use almost the whole stock plant for the efficient genetic transformation of commercial strawberry varieties.     

草莓生物技术研究进展

草莓的生物技术研究始于 6 0年代 ,但进展缓慢 ,进入 2 0世纪 90年代 ,取得了突破性进展 ,草莓的生物技术研究包括花药培养、茎尖培养、叶片和匍匐茎培养和遗传转化。     

草莓生物技术研究进展

草莓的生物技术研究始于60年代,但进展缓慢,进入20世纪90年代,取得了突破性进展,草莓的生物技术研究包括花药培养、茎尖培养、叶片和匍匐茎培养和遗传转化。     

“红颜”草莓原生质体制备及瞬时转化体系的建立

为探索“红颜”草莓悬浮细胞系原生质体提取的最优条件,并建立“红颜”草莓原生质体瞬时转化体系,以“红颜”草莓悬浮细胞为材料,对酶液组成、酶解温度、酶解方式进行研究。用PEG介导的瞬时转化法将标记基因GFP转化到“红颜”草莓原生质体中。结果显示：以“红颜”草莓悬浮细胞系作为分离材料,酶液组合为CPW中含有0.5%PVP+0.1%MES+1%纤维素酶+0.5%离析酶+0.01%半纤维素酶+0.9 mol/L甘露醇,在低速（50 r/min）恒温（31 ℃）震摇下进行酶解反应,酶解10 h时,达到“红颜”草莓原生质体最佳分离效果,每克鲜重产量可得原生质体6×10⁸ 个,活力值可达93.0%。PEG介导法成功将含有绿色荧光蛋白（green fluorescent protein, GFP）的植物表达载体转化“红颜”草莓悬浮细胞原生质体,转化效率达44%。通过实验筛选得到“红颜”草莓悬浮细胞原生质体的最佳制备条件,建立“红颜”草莓悬浮细胞原生质体的瞬时转化体系,为进一步开展“红颜”草莓功能基因及合成生物学研究奠定基础。     

Functional analysis of homologous and heterologous promoters in strawberry fruits using transient expression

The isolation and characterization of fruit-specific promoters are critical for the manipulation of the nutritional value and quality of fruits by genetic engineering. The analysis of regulatory sequences of many ripening-related genes has remained elusive for many species due to their low transformation efficiency and/or lengthy regeneration of a small number of transgenic plants. Strawberry is an important crop and represents one of the most widely studied non-climacteric model systems. However, until recently, its difficult regeneration has limited the functional study of promoters by stable transformation. A protocol based on biolistic transient transformation has been developed in order to study the function of promoters in a fast and efficient manner in strawberry fruits. The protocol has been applied to the study of the GalUR promoter, a gene involved in the biosynthesis of vitamin C in this fruit. The activity of the GalUR promoter is restricted to the fruit, being strictly dependent on light. The analysis of deletion series revealed the presence of a minimum activation region 397 bp upstream of the gene with a putative G-box motif, and a negative regulatory region between -397 and -518 bp, where an I-box was identified. The transient assay has been used to study the activity of the tomato polygalacturonase and the pepper fibrillin promoters in strawberry fruits. Whereas slight activity was observed with the fibrillin promoter, no significant activity was found with the polygalacturonase promoter. The GalUR promoter in transiently transformed ripe tomato fruits showed no activity, indicating the presence of regulatory sequences specific for its function in strawberry fruit.     

Invited review: Transformation of strawberry: The basis for translational genomics in Rosaceae

Summary Translational genomics is defined as the application of molecular-genetic principles derived from model systems to species of experimental or economic interest. The past 20 years of research in plant model systems such as Arabidopsis thaliana have relinquished vast amounts of information regarding gene function, the integration of genetic components into pathways, and the interrelationships between pathways to control form and function in plants and plant-products alike. At present, the challenge is to relate these paradigms to other species of economic or scientific interest. Apart from being an important and valuable crop, strawberry (Fragaria spp.) is a member of the Rosaceae, a plant family containing fruit, nut, ornamental and wood-bearing species. Strawberry is unique within the Rosaceae in that it is a rapidly growing herbaceous perennial with a small genome and the ability to thrive in a laboratory setting. Strawberry species may also be transformed and regenerated in a time scale of weeks or months instead of years. For these reasons, strawberry has been recognized as the translational genomics model for the Rosaceae family. This review summarizes and synthesizes the technical reports of strawberry regeneration and transformation, consolidating the large body of information regarding genetic modification of this important genus.     

Transposon based activation tagging in diploid strawberry and monoploid derivatives of potato

Key message

Diploid strawberry and potato transformed with a transposon tagging construct exhibited either global (strawberry) or local transposition (potato). An activation tagged, compact-sized strawberry mutant overexpressed the gene adjacent to Ds.

Abstract

As major fruit and vegetable crops, respectively, strawberry and potato are among the first horticultural crops with draft genome sequences. To study gene function, we examined transposon-tagged mutant strategies in model populations for both species, Fragaria vesca and Solanum tuberosum Group Phureja, using the same Activation/Dissociation (Ac/Ds) construct. Early somatic transposition during tissue culture occurred at a frequency of 18.5 % in strawberry but not in potato transformants. Green fluorescent protein under a monocot promoter was a more reliable selectable marker in strawberry compared to potato. BASTA (gluphosinate herbicide) resistance served as an effective selectable marker for both species (80 and 85 % reliable in strawberry and potato, respectively), although the effective concentration differed (0.5 % for strawberry and 0.03 % for potato). Transposons preferentially reinserted within genes (exons and introns) in both species. Real-time quantitative PCR revealed enhanced gene expression (670 and 298-fold expression compared to wild type in petiole and leaf tissue, respectively) for an activation tagged strawberry mutant with Ds inserted about 0.6 kb upstream from a gene coding for an epidermis-specific secreted glycoprotein EP1. Our data also suggested that endopolyploid (diploid) cells occurring in leaf explants of monoploid potato were the favored targets of T-DNA integration during transformation. Mutants obtained in these studies provide a useful resource for future genetic studies.     

Genetic transformation of strawberry by Agrobacterium tumefaciens using a leaf disk regeneration system

An efficient genetic transformation protocol has been developed for strawberry cv. Redcoat using Agrobacterium tumefadens. The protocol relies on a high frequency (84%) shoot regeneration system from leaf disks. The leaf disks were inoculated with a non-oncogenic Agrobacterium tumefadens strain MP90 carrying a binary vector plasmid pBI121 which contains a chimeric nopaline synthase (NOS) promoter driven neomycin phosphotransferase (NPT II) gene and a cauliflower mosaic virus 35S (CaMV35S) promoter driven, ß-glucuronidase (GUS) marker gene. The inoculated leaf disks, pre-cultured for 10 days on non-selective shoot regeneration medium, formed light green meristematic regions on selection medium containing 50 g/ml kanamycin. These meristematic regions developed into transformed shoots at a frequency of 6.5% on a second selection medium containing 25 g/ml kanamycin. The selected shoots were multiplied on shoot proliferation medium in the presence of kanamycin. All such shoots were resistant to kanamycin and expressed varying levels of NPT II and GUS enzyme activity. Histochemical assays for GUS activity indicated that the 35S promoter was highly active in meristematic cells of shoot and root apices. Molecular analysis of each transgenic clone confirmed the integration of both marker genes into the strawberry genome. Leaf disks prepared from transformed plants, when put through the second selection cycle on kanamycin, formed callus and exhibited GUS activity. The rooted transformed plants were grown in a greenhouse for further characterization. The protocol may be useful for improvement of strawberry through gene manipulations.NRCC No. 31491During the editorial process, a report has appeared on transformation of strawberry (James et al. 1990 Plant Sci 69:79–94).