影响根癌农杆菌转化的因素及其在单子叶作物上的应用

在植物转基因方法中,根癌农杆菌介导的遗传转化应用最为广泛,进一步提高其转化频率并扩大其宿主范围到禾谷类作物是人们所关注的问题。有多种因素影响根癌农杆菌的转化频率,包括植物的受伤反应、细菌的吸附、致病基因的诱导、植物细胞ＤＮＡ合成及修复的活力、外植体的状态等。最近的研究结果证明在适宜的条件下,根癌农杆菌还是可以有效地转化禾谷类作物。本文试就这两方面的研究进展作一论述。     

农杆菌介导的单子叶植物遗传转化研究进展

根癌农杆菌(Agrobacterium tumefaciens)Ti质粒转化系统的建立使植物遗传工程进入了一个飞速发展的时期。近年来,发根农杆菌(A.rhizogenes)Ri质粒毛根转化系统的研究十分迅速,展示了美好的前景,农杆菌介导的植物遗传转化已成为目前研究和应用最广泛的系统。但是,农杆菌的宿主范围一般仅限于双子叶植物和一些裸子植物,这就直接防碍着这种比较完善的基因转移技术在单子叶植物,尤其是禾谷类作物转化的应用。本文介绍了农杆菌介导的单子叶植物遗传转化的进展;对扩大农杆菌宿主范围、实现对单子叶植物转化的途径进行了探讨。 (一)农杆菌介导的单子叶植物转化的方法 目前建立的单子叶植物基因转移系统有:(1)农杆菌载体系统;(2)外源DNA     

根癌农杆菌介导转化红曲菌及转化子发酵性能的研究

利用根癌农杆菌介导转化技术成功将潮霉素抗性基因转入发白红曲菌中,优化了抗生素浓度,发白红曲菌孢子浓度,根癌农杆菌浓度,共培养温度及时间,以及乙酰丁香酮浓度等转化条件,最终转化效率可达52个转化子/105个红曲孢子.将转化子在含有潮霉素B的培养基继代培养5代,得到了多株稳定的转化子,对部分转化子进行PCR鉴定,结果进一步...     

根癌农杆菌介导转化番茄的影响因素

综述影响根癌农杆菌介导番茄转化效率的因素,包括根癌农杆菌菌株类型、Vir基因的活化、选择标记基因、植物基因型、外植体类型、培养基中是否附加植物激素和抑菌抗生素、菌液浓度、侵染时间长短,是否预培养和共培养天数等;同时不同的培养方式也是影响番茄转化效率的主要因素,包括液体培养法、农杆菌介导的floral-dip转化法、超声波辅助农杆菌介导法、农杆菌介导与基因枪轰击结合法等.     

根癌农杆菌转化单子叶植物研究概况

单子叶植物过去被人们公认公以为难以用根癌农杆菌转化,但最近有了新的进展。本文对用根癌农杆菌在转比单子叶植物方面的研究历史、现状、所取得的成功以及酚类化合物对根癌农杆菌介导的转化的促进作用做了较为详细的综述。     

基因枪转化技术及其在禾谷类作物遗传转化中的应用

基因枪转化法广泛用于禾谷类作物的遗传转化研究,是目前禾谷类作物遗传转化的有效方法。简要介绍了基困枪转化法的产生与发展、转化的特点以及影响转化频率的主要因素;系统地概述了基因枪法在禾谷类作物遗传转化的应用。     

高等植物在发根农杆菌介导下的遗传转化

本文介绍了发根农杆菌的生物学特性、遗传转化的操作技术、RiT-DNA转化体的形态特征以及发根农杆菌转化的利用。与根癌农杆菌不同,农杆碱型发根农杆菌的RiT-DNA含有生长素合成基因、农杆碱、甘露碱合成基因,不含有细胞分裂素合成基因,它的转化体首先是转化根。发根农杆菌介导的植物遗传转化,在次生代谢产物生产,植物抗逆性育种以及细菌与植物进化关系的研究等方面具有广泛的应用前景。     

影响根癌农杆菌附着禾谷类作物培养细胞的因素

本研究利用水稻、小麦、玉米等主要禾谷类作物悬浮培养细胞,观察农杆菌对禾谷类作物培养细胞的附着,实验结果表明:植物细胞的生理状态和分化发育进程对农杆菌的附着有明显的影响;通过对农杆菌的Vir 区基因进行预活化诱导处理,能显著提高其在禾谷类作物培养细胞上的附着,胭脂碱型农杆菌C 58 C 1(pMP 90+pBI 121)预处理后增加附着的效果较章鱼碱型农杆菌LBA 4404(pAL 4404+pBI121)更显著些。此外,在改善共培养环境条件下(如添加对农杆菌表现强烈趋化性的精氨酸和施行抽气减压处理),农杆菌能大量附着在经过果胶酶预处理的禾谷类作物悬浮培奍细胞上。     

一种适于丝状真菌基因RNA干扰方法的研究

目的：获得一种适于丝状真菌基因研究用的、由根癌农杆菌介导的RNA干扰方法。方法采用基因重组及菌株干扰体系筛选的方法。结果获得适于根癌农杆菌转染的重组载体PCB309?pfgrt,并将其成功用于对孢子丝菌双组份信号组氨酸蛋白激酶DRK1基因的干扰中。结论该方法优化了根癌农杆菌的转化体系,解决了丝状真菌RNA干扰载体构建困难及干扰效率低下的难题,在丝状真菌基因功能及遗传转化研究方面具有广泛的应用前景。     

根癌农杆菌介导里氏木霉转化体系的建立和条件优化

目的:采用根癌农杆菌介导的转化方法实现丝状真菌里氏木霉的遗传转化,并优化转化条件.方法:构建含潮霉素抗性基因(hph)的双元载体pCAM-hph后,转化根癌农杆菌LBA4404获得转化菌株.将根癌农杆菌的转化菌株和里氏木霉的分生孢子共培养后在含100μg/mL潮霉素的抗性平板上筛选里氏木霉转化子,并采用PCR扩增和序列测定对转化子中的插入片段进行了分析.结果:使用根癌农杆菌介导的转化方法转化里氏木霉,每106个分生孢子可获得25.8个转化子.最佳的转化条件为:农杆菌初始浓度为OD660约为0.8,孢子数为106个,共培养时间为48h,pH为5.0～5.5,培养温度为28℃.结论:建立了根癌农杆菌介导的里氏木霉转化方法,并获得了最佳的转化条件.     

Agrobacterium-mediated transformation of cereals: a promising approach crossing barriers

Cereal crops have been the primary targets for improvement by genetic transformation because of their worldwide importance for human consumption. For a long time, many of these important cereals were difficult to genetically engineer, mainly as a result of their inherent limitations associated with the resistance to Agrobacterium infection and their recalcitrance to in vitro regeneration. The delivery of foreign genes to rice plants via Agrobacterium tumefaciens has now become a routine technique. However, there are still serious handicaps with Agrobacterium -mediated transformation of other major cereals. In this paper, we review the pioneering efforts, existing problems and future prospects of Agrobacterium -mediated genetic transformation of major cereal crops, such as rice, maize, wheat, barley, sorghum and sugarcane.     

Genetic transformation of wheat: progress during the 1990s into the Millennium

Wheat transformation technology has progressed rapidly during the past decade. Initially, procedures developed for protoplast isolation and culture, electroporation- and polyethylene glycol (PEG)-induced DNA transfer enabled foreign genes to be introduced into wheat cells. The development of biolistic (microprojectile) bombardment procedures led to a more efficient approach for direct gene transfer. More recently, Agrobacterium-mediated gene delivery procedures, initially developed for the transformation of rice, have also been used to generate transgenic wheat plants. This review summarises the considerable progress in wheat transformation achieved during the last decade. An increase in food production is essential in order to sustain the increasing world population. This could be achieved by the development of higher yielding varieties with improved nutritional quality and tolerance to biotic and abiotic stresses. Although conventional breeding will continue to play a major role in increasing crop yield, laboratory-based techniques, such as genetic transformation to introduce novel genes into crop plants, will be essential in complementing existing breeding technologies. A decade ago, cereals were considered recalcitrant to transformation. Since then, a significant research effort has been focused on cereals because of their agronomic status, leading to improved genetic transformation procedures (Bommineni and Jauhar 1997). Initially, the genetic transformation of cereals relied on the introduction of DNA into protoplasts and the subsequent production of callus from which fertile plants were regenerated. More recently, major advances have been accomplished in the regeneration of fertile plants from a range of source tissues, providing an essential foundation for the generation of transgenic plants. This review summarises procedures, vectors and target tissues used for transformation, high-lights the limitations of current approaches and discusses future trends. The citation of references is limited, where possible, to the most relevant or recent reports.     

Progress and limitations in the culture of cereal protoplasts

A few well-established protoplast systems encompassing morphogenic and non-morphogenic cultures potentially could be used for the somatic hybridization and transformation of cereal plants. However, the present difficulty in obtaining plants from protoplasts makes it important to consider alternative approaches for transferring genes into cereals.     

A set of modular binary vectors for transformation of cereals

Genetic transformation of crop plants offers the possibility of testing hypotheses about the function of individual genes as well as the exploitation of transgenes for targeted trait improvement. However, in most cereals, this option has long been compromised by tedious and low-efficiency transformation protocols, as well as by the lack of versatile vector systems. After having adopted and further improved the protocols for Agrobacterium-mediated stable transformation of barley (Hordeum vulgare) and wheat (Triticum aestivum), we now present a versatile set of binary vectors for transgene overexpression, as well as for gene silencing by double-stranded RNA interference. The vector set is offered with a series of functionally validated promoters and allows for rapid integration of the desired genes or gene fragments by GATEWAY-based recombination. Additional in-built flexibility lies in the choice of plant selectable markers, cassette orientation, and simple integration of further promoters to drive specific expression of genes of interest. Functionality of the cereal vector set has been demonstrated by transient as well as stable transformation experiments for transgene overexpression, as well as for targeted gene silencing in barley.     

三个方便实用的植物表达载体构建与验证

为满足植物功能基因组学研究及转基因安全性需要,本研究根据一些国内外引进或商业化的植物表达载体及其相关元件,构建了3个适合于植物,尤其是单子叶植物转化的表达载体,即p AH006、p WMB022和p WMB025。p AH006载体包含由玉米泛素ubi启动子调控的GUS基因和bar基因的完整T-DNA区域,此区段能够被酶切回收,可用于单子叶植物农杆菌介导转化效率评价及基因枪介导线状DNA转化效果研究;p WMB022载体携带由双35S启动子调控的玉米色素基因Lc和C1,可用作基因枪介导的共转化筛选标记,直观筛选含目标基因转基因材料;p WMB025载体携带由ubi启动子调控的、商业化转基因植物中广泛利用的EPSPS基因,可用于禾谷类作物农杆菌或基因枪介导的遗传转化,载体多克隆位点可通过酶切方式更换目标基因。酶切鉴定结合农杆菌或基因枪介导的小麦幼胚愈伤组织或叶片转化验证此3个载体表明,载体构建正确,其标记基因、可视化基因和报告基因均能正常表达。这3个载体的构建对于小麦等植物转化效率提升、安全型转基因作物获得和植物功能基因组学研究等具有重要意义。     

OBPC Symposium: Maize 2004 &; beyond—Plant regeneration,gene discovery,and genetic engineering of plants for crop improvement

Summary The development of robust plant regeneration technology in cereals, dicots and ornamentals that is in turn coupled to a high-frequency DNA transfer technology is reported. Transgenic cereals that include maize, Tripsacum, sorghum, Festuca and Lolium, in addition to dicots that include soybean, cotton and various ornamentals such as petunia, begonia, and geranium have been produced following either somatic embryogenesis or direct organogenesis independent of genotype. Coupled with these regeneration protocols, we have also identified several interesting genes and promoters for incorporation into various crops and ornamentals. In addition, the phenomenon of direct in vitro flowering from cotyledonary nodes in soybean is described. In in vitro flowering, the formation of a plant body is suppressed and the cells of the cotyledonary node produce complete flowers from which fertile seed is recovered. This in vitro flowering technology serves as a complementary tool to chloroplast transformation for developing a new transgenic pollen containment strategy for crop species. Recently, the center has undertaken to screen the expression response of the 24 000 Arabidopsis genes to nitric oxide. This signaling molecule upregulated 342 genes and downregulated 80 genes. The object here was to identify a population of promoters that can be manipulated by using a signaling molecule. In addition, in keeping with the mission of enhancing greenhouse profitability for North West Ohio growers, we cloned a number of genes responsive for disease resistance from ornamentals that play an important role in disease management and abiotic stress. We have constructed a plant transformation vector with CBF3 gene under the rd29A promoter for engineering cold and freezing tolerance in petunia. Leaf dises of Petunia×hybrida v26 were used for Agrobacterium-mediated transformation, and 44 hygromycin-resistant T0 plants were obtained. The presence of CBF3 gene was confirmed in all the transgenic plants by PCR and Southern analyses.     

Genetic transformation of Sorghum bicolor

Great millet (Sorghum bicolor (L.) Moench) is cultivated across the world for food and fodder. It is typically grown in semiarid regions that are not suitable for cultivation of other major cereals. Sexual incompatibility and shortage of available genes in germplasm to combat biotic and abiotic stresses resulted in marginalized yields of this crop. Genetic modification of sorghum with agronomically useful genes can address this problem. Here, we tried to review and summarize the key aspects of sorghum transformation work being carried out so far by various research groups across the world. The approaches used and the obstacles in generating transgenic sorghum are also pointed out and discussed.     

Recent advances in barley transformation

Barley, an important member of the cereals, has been successfully transformed through various methods such as particle bombardment, Agrobacterium tumefaciens, DNA uptake, and electroporation. Initially, the transformation in barley concentrated on developing protocols using marker genes such as gus, bar, and hpt. Immature embryos and callus derived from immature embryos were targeted for transformation. Subsequently, genes of agronomic and malting importance have been deployed in barley. Particle bombardment appears to be the preferred choice for barley transformation in the majority of the reports, although Agrobacterium-mediated transformation is being used more often. The current review focuses on the challenges encountered in barley transformation such as somaclonal variation, development of transformation systems for commercial cultivars, gene expression, stability and inheritance, and gene flow. Newer markers such as the green fluorescent protein (gfp), firefly luciferase, and phosphomannose isomerase were found to be useful in the selection of transgenic plants. Tissue-specific promoters such as those for B1-hordein and D-hordein genes, and spike-specific promoters, are increasingly used to drive gene expression. The review also describes recent research on gene-tagging through transformation, insertion of disease resistance, and abiotic stress resistance genes, transformation with genes for improved malting quality, nutrient content, feed quality, and the production of feed enzymes and pharmaceutical compounds.     

Pathogenesis-related proteins and their genes in cereals

Pathogenesis-related proteins (PR-proteins) are induced in plants in response to attack by microbial or insect pests. They have been classified into several groups (PR-1 through PR-14 at present) based on their amino acid sequences and biochemical functions. Many of these proteins that have been purified from infected plants or seed extracts possess antifungal or insecticidal activity. Genes and cDNA clones for all classes of PR-proteins have been isolated from a variety of cereals. Some of these genes/cDNAs have been used to transform cereals. This review presents a summary of the PR-proteins and their genes characterized from rice, wheat, barley, sorghum and maize. Efforts to improve disease or insect resistance of these cereal plants by genetic engineering using genes for PR-proteins also are discussed. In many cases, the expression of the PR-proteins either singly or in combination appears to improve resistance to fungi or insects. In addition, chromosomal location of the PR-protein genes indicates that members of the same family of PR-protein genes or sometimes even several families of PR-protein genes often are clustered in the cereal genome, suggesting coordinate regulation. Some of these PR-protein genes map closely to quantitative traits loci. Some concerns regarding the use of genes encoding PR-proteins for genetic modification of cereals also are addressed.