基因枪的使用方法介绍

基因枪法是一种新型的遗传转化手段[1-3]。所谓遗传转化是指通过某种途径或技术将外源基因导入受体细胞的基因组中,并使之在受体细胞中表达。人们可利用一个特定的目的基因,如抗虫、抗病基因等进行转化,使转基因植物在保持原有的各种优良农艺性状的同时,又能增加新的目的基因所控制的性状。一般遗传转化主要有两类：一类是农杆菌介导法,一类是DNA直接转化法。DNA直接转化技术包括化学方法和物理方法,如电击、微注射、超声波和基因枪法。基因枪法,又称生物弹法或微粒枪法、微粒轰击法,是依赖高速度的金属微粒将外源基因引入活细胞…     

花生遗传转化的主要方法和研究进展

花生遗传转化研究对于花生品种改良、新品种繁育具有重要意义。农杆菌介导的遗传转化法和基因枪法是花生遗传转化的主要方法。随着对花生研究的深入开展,花生的遗传转化技术也越来越成熟。介绍了农杆菌介导的遗传转化法和基因枪法在花生遗传转化研究中的应用现状及存在的问题。     

植物遗传转化的替代方法及研究进展

农杆菌介导法和基因枪法是两种目前应用最广泛和最可靠的植物遗传转化方法.由于其存在一些不足,因此,需要探索可替代它们的其他植物遗传转化方法以弥补农杆菌介导法和基因枪法的缺点,使其更加适用于特定目标植物的转化事件中,转化效率也随之提高.对这些可替代的遗传转化方法进行概述,并简要介绍各种方法的优缺点及其在农作物转化中的应用情况.     

影响籼稻基因枪法遗传转化效果的几个因素的研究(简报)

基因枪转化技术在水稻的遗传转化上已被广泛地应用并获得显著的成效。与原生质体转化法相比较,基因枪法具有不受或少受基因型限制的优点且提高了转化效率,但对籼稻,仍有不少问题需要解决,转化系统尚须进一步完善。因此有必要对影响转化频率的因素进行深入的研究。我们在以barnase基因对籼稻的遗传转化以诱发工程雄性不育的研究中,特别注     

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。     

月季的植株再生及遗传转化研究进展

本文对近20年月季植株再生和转基因研究进展进行了较为系统的回顾和总结。月季通过器官和体细胞胚发生途径都能再生植株,但遗传转化主要是利用体细胞胚发生途径。通过农杆菌介导法和基因枪法,外源基因如报告基因、抗病基因和改变花色的基因等已转化成功。文章还对今后月季转基因研究的方向进行了讨论。     

基因枪法介导GNA基因遗传转化甘蔗的研究

目的:将含有雪花莲外源凝集素(GNA)基因的植物表达载体用基因枪法分别导入一个果蔗和一个糖蔗品种中,以期获得转基因植株。方法:将GNA基因插入到植物表达载体上,构建出不同选择标记、不同启动子的表达载体,并用基因枪法将之导入甘蔗胚性愈伤组织,分别在G418、PPT和Hyg的选择压力下,筛选抗性植株,并进行分子杂交鉴定。结果:通过斑点杂交和PCR-Southern杂交证明GNA基因已整合到甘蔗基因组中。结论:用基因枪法成功获得了含有GNA基因的甘蔗转化株,为培育抗甘蔗绵蚜(Ceratovacuna lanigeraZehnther)的新品种提供了基础。     

月季组织培养和遗传转化体系的研究进展

月季通过器官和体细胞胚发生途径都可以获得再生植株,在遗传转化中主要是利用体细胞胚作为转化受体。目前,利用农杆菌介导法和基因枪法已成功将外源基因如报告基因、抗病基因和改变花色的基因等导入月季基因组中。本文对近年来月季组织培养和转基因研究进展进行了综述,为建立月季高效遗传转化体系奠定了理论基础。     

农杆菌及基因枪在玉米遗传转化中的应用

目前外源基因导入玉米受体细胞的方法很多 ,最受瞩目的是农杆菌介导法和基因枪法。就农杆菌、基因枪转化玉米的基本原理、影响转化率的因素 ,以及近年来在玉米遗传转化中的最新动态进行了综述     

基因枪法和农杆菌介导法对水稻转基因拷贝数的基因重排概率的影响

基因枪法和农杆菌介导法转化的外源DNA整合到植物梁色上是随机进行的,因此,它们可能会产生不同的转基因拷贝数,得到不同的基因表达盒完整率,这反过来会影响基因的表达,为证实这一假说,作首先将同一质粒pAcPG-CAM分别用基因枪法和农杆菌介导法转化到水稻（Oryza sativa L.cv.TNG67)愈伤组织,为了揭示不同质粒是否也出现类似结果,也用农杆菌介导法,基因枪法分别将pTOK233和pJPM44导入水稻愈伤组织,并获得一批转基因植株,Ｒ０代值转基因表达的分析用ＧＵＳ组织化学染色法,用质粒上的单切点酶酶切基因组DNA后的Southern杂交结果确定转基因拷贝数,总DNA髟双切点酶（分别位于表达盒两侧）酶切后的Southern杂交结果确定了完整转基因表达盒数目,结果表明,农杆菌介导转化法的转基因植株的转基因拷贝数相对少一些（平均为2．1和2．3）,而基因枪法转化产生的转基因植株的转基因撬贝数相对多一些（平均为4．2和5．6）,并且农杆菌介导转化法的转基因植株的基因表达盒DNA重排概率低于由基因枪法转化产生的转基因植株的基因表达盒DNA重排概率－农杆菌介导转化法的DNA重排概率为0．07和0．106,由基因枪法转化的DNA重排概率为0．57和0．66。研究还分析了基因表达情况与转基因的拷贝数或完整表达盒数之间的关系,GUS定量分析结果表明,为了准确揭示转基因的表达情况与转基因之间的关系,用完整基因表达盒数而不是转基因DNA拷贝数更准确,并于用不同转基因方法将同种质粒导入植物体分析基因表达盒DNA重排概率为首次报道。     

促进植物高效遗传转化的发育调节因子及其在玉米中的应用进展*

高效遗传转化技术体系的建立对植物功能基因组学研究和作物新品种的培育均具有促进作用,目前,再生效率低下是限制许多植物高效遗传转化体系建立的主要技术屏障之一。随着对植物分生组织和体细胞胚形成过程研究的深入,鉴定到了一些关键调控基因,统称为发育调节因子。发育调节因子应用于植物遗传转化后,可以有效改善植物分生组织诱导和再生能力,为提高遗传转化效率提供了重要机遇。综述了7类发育调节因子在提高植物遗传转化效率中的研究进展,重点介绍了其中3类在促进玉米遗传转化中的应用,最后展望了建立植物高效遗传转化体系的发展方向。     

Retrofitting YACs for direct DNA transfer into plant cells

The utility of plant YAC libraries prepared in conventional YAC vectors would be dramatically increased if these YACs could be used directly for plant transformation. A pair of vectors that allow clones from YAC libraries to be modified (retrofitted) for plant transformation by direct DNA transfer methods, such as particle bombardment or electroporation, has been developed. Modification of the YAC is achieved in two sequential yeast transformation steps by taking advantage of the homologous recombination system in yeast. Using this approach, two plant-selectable marker genes and DNA sequence elements required for copy number amplification in yeast can be introduced into YACs present in yeast strain AB1380. The utility of these vectors is demonstrated by retrofitting YACs that contain inserts ranging in size from 80 to 700 kb. The 6- to 12-fold increase in copy number of these modified YACs facilitates the isolation of YAC DNA for direct DNA transformation methods. Retrofitted YACs were used for particle bombardment to examine the efficiency with which their large DNA inserts are transferred into plant cells. The availability of these retrofitting vectors should facilitate the transfer of YAC DNA inserts into plant cells and thus help bridge the gap between existing mapping techniques and plant transformation procedures.     

草坪草生物技术研究进展

概述了草坪草植株再生体系和遗传转化体系建立的方法和进展.通过愈伤组织培养、悬浮细胞培养和原生质体培养方法对草坪草的一些种已建立较为完善的植株再生体系.在建立再生体系的基础上,利用原生质体融合、农杆菌介导、基因枪和碳化硅纤维介导等转基因方法在一些草坪草种上建立了遗传转化体系并获得了有一定价值的转基因植株.最后,对草坪草转基因存在的问题和前景作了讨论.     

Floral-Dip Transformation of Flax (Linum usitatissimum) to Generate Transgenic Progenies with a High Transformation Rate

Agrobacterium-mediated plant transformation via floral-dip is a widely used technique in the field of plant transformation and has been reported to be successful for many plant species. However, flax (Linum usitatissimum) transformation by floral-dip has not been reported. The goal of this protocol is to establish that Agrobacterium and the floral-dip method can be used to generate transgenic flax. We show that this technique is simple, inexpensive, efficient, and more importantly, gives a higher transformation rate than the current available methods of flax transformation.In summary, inflorescences of flax were dipped in a solution of Agrobacterium carrying a binary vector plasmid (T-DNA fragment plus the Linum Insertion Sequence, LIS-1) for 1 - 2 min. The plants were laid flat on their side for 24 hr. Then, plants were maintained under normal growth conditions until the next treatment. The process of dipping was repeated 2 - 3 times, with approximately 10 - 14 day intervals between dipping. The T1 seeds were collected and germinated on soil. After approximately two weeks, treated progenies were tested by direct PCR; 2 - 3 leaves were used per plant plus the appropriate T-DNA primers. Positive transformants were selected and grown to maturity. The transformation rate was unexpectedly high, with 50 - 60% of the seeds from treated plants being positive transformants. This is a higher transformation rate than those reported for Arabidopsis thaliana and other plant species, using floral-dip transformation. It is also the highest, which has been reported so far, for flax transformation using other methods for transformation.     

In solium Selection for Arabidopsis Transformants Resistant to Kanamycin

The kanamycin resistance encoded by the neomycin phosphotransferase II gene (nptII) of transposon Tn5 is widely used in higher plant genetic transformation. The general process of plant transformation using nptII as a selectable marker gene, however, requires selecting kanamycin-resistant plants or tissues in culture. Even with the recently developed vacuum infiltration method for Arabidopsis transformation, the plant culture steps are not completely eliminated in selection for kanamycin-resistant transformants. The herbicide resistance genes, such as bar, which provides resistance to bialaphos, allow Arabidopsis transformation to become a true non-culture procedure. In this report, we assessed the feasibility of applying kanamycin as a spray in selecting for kanamycin-resistant Arabidopsis transformants grown in soil. We find that kanamycin-resistant transformants were effectively selected by spraying soil-grown Arabidopsis seedlings.     

Improving plant genetic engineering by manipulating the host

Agrobacterium-mediated transformation is a major technique for the genetic engineering of plants. However, there are many economically important crop and tree species that remain highly recalcitrant to Agrobacterium infection. Although attempts have been made to "improve" transformation by altering the bacterium, future successes might come from manipulation of the plant. Recent studies that identified several plant genes involved in Agrobacterium-mediated transformation, and their over-expression in currently transformable species, suggest that this approach holds great promise for improving the transformation of recalcitrant, but agronomically important, crops.     

Whisker-mediated plant transformation: An alternative technology

A number of different methods, involving direct DNA delivery are now available for plant transformation. Here we review the most recently developed technique which involves the mixing of silicon carbide whiskers with plant cells and plasmid DNA. Fertile transgenic plants have now been produced using whisker-mediated transformation, and this method can now be considered as a simple, inexpensive alternative for plant transformation. A brief review on transformation of animal cells andChlamydomonas using whiskers technology is also included.     

玉米花粉体外萌发方法改进及其对花粉介导转基因的作用

超声波处理花粉介导植物基因转化方法由山西省农业科学院生物技术研究中心发明, 已被国家知识产权局授予发明专利(专利号ZL 99121152.9)。在该专利的基础上, 针对玉米(Zea mays)花粉取样、保存和处理条件等因素对其体外萌发的影响进行深入研究, 提出了改进玉米花粉体外萌发实验的方法。研究结果表明, 在不同时期对开花的玉米进行花粉培养时所需蔗糖溶液的浓度不同; 确定了玉米花粉的保存时间、条件及其对超声波处理后花粉萌发率的影响, 以提高该转化方法中花粉的活力, 并进一步验证了该转基因方法的可靠性; 讨论了玉米花粉体外萌发的操作技巧和各因子的参数, 对提高花粉介导植物基因转化效率有一定的参考价值。     

植物体细胞原生质体遗传转化研究

重点介绍了植物体细胞原生质体遗传转化的方法和当前已经取得的成果,同时提出了目前原生质体遗传转化中存在的问题,展望了今后的工作重点。植物原生质体遗传转化的方法主要有：PEG介导转化法、电击穿孔转化法、脂质体介导转化法、农杆菌共培养转化法等。     

Marker-free transformation: increasing transformation frequency by the use of regeneration-promoting genes

The generation of transgenic plants free of antibiotic resistance markers is a major challenge to plant biologists and plant breeders. Currently, there are two main strategies to achieve this goal: one approach is to excise or segregate marker genes from the host genome after regeneration of transgenic plants, and the second is based on so-called 'marker-free' transformation. Marker-free transformation has been successfully demonstrated by the use of several plant and non-plant genes that are capable of promoting explant regeneration. This approach appears not only to be effective for the generation of marker-free transgenic plants, but also has great potential to improve the transformation frequency of recalcitrant species.