普通小麦品种Alondra＇s遗传转化体系的建立

小麦（Triticum aestivum）幼胚愈伤组织的诱导和分化再生有高度依赖基因型特征。为了建立和优化Alondra’s的高效再生及遗传转化体系,为小麦遗传转化提供更多的受体基因型,以Alondra’s的幼胚为外植体,研究了培养基种类、不同激素配比等对其幼胚愈伤组织诱导及再生的影响。结果表明,在使用N6培养基时,添加3mg·L^-1的2,4-D并附加1000mg·L^-1的CH对愈伤组织的诱导效果较好;添加4mg·L^-1的ZT、不附加IAA对愈伤组织的分化效果最好。通过构建植物表达载体pCAMBIA1301-220.6,利用基因枪法将HYG基因导入Alondra’s幼胚愈伤组织中,以建立Alondra’s的高效遗传转化体系。结果在含100mg·L^-1潮霉素的选择培养基上进行筛选、分化,获得了30棵抗性植株。经PCR检测,其中5株为阳性转基因植株,转化率为0.5%。Alondra＇s遗传转化体系的建立丰富了小麦遗传转化的基因型,为小麦品种的转基因改良和在不同背景下研究基因的功能奠定了良好的基础。     

普通小麦品种Alondra's遗传转化体系的建立

小麦(Triticum aestivum)幼胚愈伤组织的诱导和分化再生有高度依赖基因型特征。为了建立和优化Alondra’s的高效再生及遗传转化体系, 为小麦遗传转化提供更多的受体基因型, 以Alondra’s的幼胚为外植体, 研究了培养基种类、不同激素配比等对其幼胚愈伤组织诱导及再生的影响。结果表明, 在使用N₆培养基时, 添加3 mg·L^–1的2,4-D并附加1 000 mg·L^–1的CH对愈伤组织的诱导效果较好; 添加4 mg·L^–1的ZT、不附加IAA对愈伤组织的分化效果最好。通过构建植物表达载体pCAMBIA1301-220.6, 利用基因枪法将HYG基因导入Alondra’s幼胚愈伤组织中, 以建立Alondra’s的高效遗传转化体系。结果在含100 mg·L^–1潮霉素的选择培养基上进行筛选、分化, 获得了30棵抗性植株。经PCR检测, 其中5株为阳性转基因植株, 转化率为0.5%。Alondra's遗传转化体系的建立丰富了小麦遗传转化的基因型, 为小麦品种的转基因改良和在不同背景下研究基因的功能奠定了良好的基础。     

农杆菌介导的半夏凝集素基因(Pinellia Ternate Agglutinin Gene,pta)对小麦的遗传转化及鉴定

以甘肃主要推广春小麦品种陇春22幼胚为转基因受体材料,建立了农杆菌介导的小麦遗传转化体系。以预培养4天的幼胚愈伤组织为受体,C58c1农杆菌菌株为供体,将含有半夏凝集素基因的重组质粒pBIpta转入了小麦,经G418 25 mg/L抗性筛选、PCR检测和荧光定量PCR检测共获得转基因植株3株,外源基因的插入拷贝数分别为2、1、3。同时对转基因小麦的T₁代植株进行了PCR检测和抗虫性分析,表明半夏凝集素基因在转基因植株的后代中得到了遗传并有一定的抗蚜虫作用。     

基因枪介导小麦成熟胚遗传转化的影响因素

小麦成熟胚作为转化受体可克服小麦幼胚存在的受季节和幼胚发育阶段限制的缺点。以湖北省小麦品种‘鄂麦12’和模式品种‘Bobwhite’为材料,成熟胚为转化受体,优化基因枪转化法的轰击压力、轰击距离、选择剂等因素,建立以小麦成熟胚为转化受体的高效转化系统。结果表明:小麦成熟胚作为转化受体时,适宜轰击压力和轰击距离组合是900 psi、6 cm;成熟胚对选择剂G418的敏感性强于幼胚,轰击后需要延长恢复时间,选择剂G418的适合浓度为20～40 mg/L。在以上优化条件下小麦成熟胚转化频率达0.3%～0.9%,已初步建立基因枪介导的小麦成熟胚遗传转化系统。     

农杆菌介导的玉米遗传转化研究进展

农杆菌介导的转基因法是目前玉米遗传转化的主流方法之一。目前,模式玉米种质幼胚的转化体系已程式化,且开发了新筛选基因和获得不含筛选基因转基因玉米的方法,但是大多数育种骨干自交系转化频率低和转化受体基本上是幼胚。从农杆菌、受体及培养条件多方面各种因素对问题进行分析,多数研究认为针对特定基因型和受体材料建立好的受体再生系统,结合高效率农杆菌转化体系,获得多目的基因聚合(无其它外源片段)的转基因玉米将是农杆菌介导玉米转化体系研究的最终目标。本文主要从农杆菌介导(转基因)法应用于玉米遗传转化的历史、现状、问题等方面进行综述,为同领域的研究者提供一定的参考。     

水稻幼胚电激转化及转基因植株再生

幼胚的遗传转化对研究植物胚胎发育相关基因的表达与调控具有重要意义 ,也为植物遗传改良提供新的技术。本研究借助一种自制的特殊装置 ,采用电激法将GFP基因转入 2 - 3天水稻幼胚 ,得到瞬时表达 ,4- 6天水稻幼胚经电激后再生了植株 ,并在愈伤组织阶段及R0植株中检测到GFP荧光的转基因植株 ,从而建立了水稻幼胚的遗传转化实验系统。在电容为 5 0 0 μF、电压为 30 0V/cm ,浓度为 10 0 μg/mL的条件下 ,幼胚GFP的电激转化频率可达 35 %。在pH 5 .8的电激缓冲液中 ,最高转化频率可达 40 %。在三种不同的启动子实验中 ,以Ubi启动子的转化频率最高。     

水稻幼胚电激转化及转基因植株再生

幼胚的遗传转化对研究植物胚胎发育相关基因的表达与调控具有重要意义, 也为植物遗传改良提供新的技术.本研究借助一种自制的特殊装置,采用电激法将GFP基因转入2-3天水稻幼胚,得到瞬时表达, 4-6天水稻幼胚经电激后再生了植株,并在愈伤组织阶段及R0植株中检测到GFP荧光的转基因植株,从而建立了水稻幼胚的遗传转化实验系统.在电容为500 μF、电压为300 V/cm,浓度为100 μg/mL的条件下,幼胚GFP的电激转化频率可达35%. 在pH 5.8的电激缓冲液中,最高转化频率可达40%.在三种不同的启动子实验中,以Ubi启动子的转化频率最高.     

负压处理对农杆菌介导小麦成熟胚转化效率的影响

目的:提高小麦成熟胚遗传转化效率,为建立快速高效的小麦成熟胚遗传转化体系奠定基础.方法:以普通小麦HB341、SN2618、TS021和栽培二粒小麦成熟种子为试材,采用整粒切胚诱愈的方法,通过农杆菌GV3101/pBI121::gus介导,研究负压处理对小麦成熟胚遗传转化的影响.结果:接种侵染过程中的负压处理,对小麦成熟胚愈伤组织的诱导没有显著影响,但对小麦成熟胚的遗传转化却有显著性影响.负压处理10、20、30次,转化率分别较对照组提高了9.37倍、10.90倍和10.03倍,最高转化率可达12.50%.同时负压处理能够提高小麦成熟胚转化效率,具有普遍意义.结论:该研究为快速高效小麦成熟胚转化体系的建立提供了依据.     

可育的抗除草剂溴苯腈转基因小麦

报道了采用微粒轰击（Ｍｉｃｒｏｐｒｏｊｅｃｔｉｌｅ ｂｏｍ ｂａｒｄｍ ｅｎｔ） 幼胚将除草剂抗性基因导入小麦（Ｔｒｉｔｉｃｕｍａｅｓｔｉｖｕｍ Ｌ．）的转化研究。实验共使用了１３ 个小麦品种, 从开花后１４～１８ ｄ 的籽粒中剥取幼胚, 植物表达质粒含有ＣａＭＶ ３５Ｓ启动子控制的除草剂溴苯腈抗性基因ｂｘｎ 以及筛选标记基因ＮＴＰⅡ。采用高压放电基因枪,用质粒ＤＮＡ 包被的钨粒轰击预培养３ ｄ 的幼胚。在含有卡那霉素类似物ｇｅｎｅｔｉｃｉｎ Ｇ４１８ｓｕｌｐｈａｔｅ 的ＭＳ培养基上, 经过多步骤筛选和分化, 从８００ 多个幼胚中获得了１６ 株转化苗。除草剂抗性鉴定和Ｓｏｕｔｈｅｒｎ 杂交分析证明, 其中４ 株为转基因植物,具有溴苯腈抗性, 并且自交可育。转化工作从分离幼胚到转化苗鉴定完毕, 最短时间为６ 个月, 因此, 该方法是一项快速有效的基因导入技术     

胚龄和激素对小麦幼胚组织培养的影响

以扬麦158为试验材料,通过田间取样室内培养的方法研究了胚龄和激素对小麦幼胚组织培养的作用。结果表明,幼胚组织培养最适宜的胚龄为14—16d;适宜的2,4-D浓度为1．5-2．5mg/L;适宜的IAA浓度为2．0-3．0mg/L;适宜的6-BA浓度为0．1-0．8mg/L;适宜的KT浓度为0．5—1．5mg/L。因此,胚龄和激素对于小麦幼胚组织培养具有明显的调节作用．在组织培养实践中,充分认识和综合协调这些因素对小麦幼胚组织培养的作用,可以提高组织培养效率,使其更加有利于生物学研究、遗传转化和快速育种等工作。     

Bacterial gene transfer by natural genetic transformation in the environment.

Natural genetic transformation is the active uptake of free DNA by bacterial cells and the heritable incorporation of its genetic information. Since the famous discovery of transformation in Streptococcus pneumoniae by Griffith in 1928 and the demonstration of DNA as the transforming principle by Avery and coworkers in 1944, cellular processes involved in transformation have been studied extensively by in vitro experimentation with a few transformable species. Only more recently has it been considered that transformation may be a powerful mechanism of horizontal gene transfer in natural bacterial populations. In this review the current understanding of the biology of transformation is summarized to provide the platform on which aspects of bacterial transformation in water, soil, and sediments and the habitat of pathogens are discussed. Direct and indirect evidence for gene transfer routes by transformation within species and between different species will be presented, along with data suggesting that plasmids as well as chromosomal DNA are subject to genetic exchange via transformation. Experiments exploring the prerequisites for transformation in the environment, including the production and persistence of free DNA and factors important for the uptake of DNA by cells, will be compiled, as well as possible natural barriers to transformation. The efficiency of gene transfer by transformation in bacterial habitats is possibly genetically adjusted to submaximal levels. The fact that natural transformation has been detected among bacteria from all trophic and taxonomic groups including archaebacteria suggests that transformability evolved early in phylogeny. Probable functions of DNA uptake other than gene acquisition will be discussed. The body of information presently available suggests that transformation has a great impact on bacterial population dynamics as well as on bacterial evolution and speciation.     

Transformation of nuclear and plastomic plant genomes by biolistic particle bombardment

Microprojectile bombardment is a powerful method for the transformation of various organisms and tissues. For plants, the biolistic approach is primarily used for transformation of cereals and other monocotyledons, as well as for dicotyledonous plants shown to be recalcitrant to Agrobacterium-based transformation of organellar genomes, and transformation of plant and algal chloroplasts has recently been reported. In this protocol paper we provide methods for nuclear and plastomic transformation of plants using the biolistic technique.     

Chromosome abnormalities associated with Phl and acturial survivorship curve in chronic myeloid leukemia. Probabilistic interpretation of blastic transformation of CML

Sixty-six patients with chronic myelogenous leukemia, all with Philadelphia chromosome, have been studied for chromosomic abnormalities associated (CAA) to Ph', as well as for actuarial curve of survivorship. Patients dying from another disease were excluded from this study. Frequency of cells with CAA was measured and appeared strongly higher after blastic transformation than during myelocytic state; probability to be a blastic transformation is closely correlated with this frequency. On the other hand, actuarial curve of survivorship is very well represented by an exponential curve. This suggests a constant rate of death during disease evolution, for these patients without intercurrent disease. As a mean survivance after blastic transformation is very shorter than myelocytic duration, a constant rate of blastic transformation could be advanced: it explains possible occurrence of transformation as soon as preclinic state of a chronic myelogenous leukemia. Even if CAA frequency increases after blastic transformation, CAA can occur a long time before it and do not explain it: submicroscopic origin should be searched for the constant rate of blastic transformation would express the risk of a genic transformation at a constant rate during myelocytic state.     

Optimization of sorghum transformation parameters using genes for green fluorescent protein and beta-glucuronidase as visual markers

Early and reliable detection of plant transformation events is essential for establishing efficient transformation protocols. We have compared the effectiveness of using the gene encoding a green fluorescent protein (GFP) and a beta-glucuronidase (gus) as reporter genes for early detection of transgene expression in explants subjected to biolistic bombardment and Agrobacterium-mediated transformation. The results indicate that gfp gene is superior to gus gene in following transgene expression in transiently transformed materials in both methods of transformation. Using GFP as the screenable marker, we have optimized sorghum transformation with respect to the conditions for transformation, type of explants, promoters, and inbreds. These optimized conditions have been used to obtain stably transformed explants for subsequent regeneration.     

利用植物悬浮培养细胞进行蛋白质快速定位分析的方法

稳定遗传表达分析是一种植物中常用的整体解析基因的方式。有多种转化方式可供选择,也可根据所需要的获得的转基因植物材料选择受体材料。但是由于稳定遗传转化周期较长且大部分材料不适合于进行荧光观察,所以在一些基因的研究中逐渐被瞬时表达分析系统。虽然瞬时表达分析用时短,但是转化效率受到多方面的限制,转化材料无法保存。目前由于植物悬浮培养细胞材料均一,增殖迅速并且可以满足大批量研究需求逐渐成为植物研究中的热点材料。以此同时,在亚细胞定位方面,悬浮培养细胞还是良好的应用材料。采用农杆菌介导法进行植物悬浮培养细胞的转化中方法较为成熟,但是获得纯净的转基因细胞系的转化周期较长。在本研究中针对上述问题我们建立了一种转化时间短,转化效率高的植物悬浮培养细胞稳定遗传转化体系。同时将这个体系应用到基因的亚细胞定位当中进行蛋白质快速定位分析。     

Box–Cox transformation for QTL mapping

The maximum likelihood method of QTL mapping assumes that the phenotypic values of a quantitative trait follow a normal distribution. If the assumption is violated, some forms of transformation should be taken to make the assumption approximately true. The Box–Cox transformation is a general transformation method which can be applied to many different types of data. The flexibility of the Box–Cox transformation is due to a variable, called transformation factor, appearing in the Box–Cox formula. We developed a maximum likelihood method that treats the transformation factor as an unknown parameter, which is estimated from the data simultaneously along with the QTL parameters. The method makes an objective choice of data transformation and thus can be applied to QTL analysis for many different types of data. Simulation studies show that (1) Box–Cox transformation can substantially increase the power of QTL detection; (2) Box–Cox transformation can replace some specialized transformation methods that are commonly used in QTL mapping; and (3) applying the Box–Cox transformation to data already normally distributed does not harm the result.     

Computer Tool For Evaluation of Anaerobic Microbial PCB Transformations

Several researchers have demonstrated the transformation of polychiorinated biphenyls (PCBs) by both aerobic and anaerobic bacteria. This transformation, or conversion, is characteristic and often dependent on PCB congener structure and, in addition, dictates the products or extent of degradation. Because transformation is linked to microbial activities, bioremediation has been hailed as a possible solution for PCB-contaminated soils and sediments, and several demonstration activities have verified laboratory results. This article presents results from mathematical modeling of anaerobic microbial PCB transformation. Because transformation can be influenced by both starting composition of the PCBs and microbial activity, this article systematically evaluates several of the most common transformation patterns. The predicted data are also compared with experimental results. For example, the correlation between laboratory-observed and predicted products was, in some cases, as good as 0.96 (perfect correlation = 1.0). In addition to predicting extent of transformation, the water solubility and the possible human effects of the PCBs are discussed through the use of documented dioxin-like toxicity and accumulation in humans before and after transformation.     

Cell transformation as aberrant differentiation：Environmentally dependent spontaneous transformation of NIH 3T3 cells

NIH 3T3 cells, a mouse fibroblast cell line used as routine target cells for transfection experiments, undergo spontaneous transformation in our experiments after they form a confluent sheet in medium containing fetal bovine serum (FBS) or lower coneentration of calf serum (CS). The transformation takes the form of foci of multiplying cells among the surrounding cells which have stopped cell division. However, no focus of transformed cells could be seen in medium containing high concentration (10％) of CS. Further experiments indicated that the frequency of transformation is highly dependent on the concentration of serum and the transformation in CS is changeable when the cells are passaged in FBS. 3~H-thymidine autoradiography has been proved to be a sensitive measurement indicator for focus formation. Our results suggest that the high frequency of transformation and its dependence on confluency as well as on medium composition are characteristics of cell differentiation rather than mutation. The role of the NIH 3T3 cell line as a cancer-initiated cell population and its accelerated transformation by ras oncogene might be considered as a form of tumor promotion is discussed.     

Advances and remaining challenges in the transformation of barley and wheat

Highly efficient and cost-effective transformation technologies are essential for studying gene function in the major cereal crops, wheat and barley. Demand for efficient transformation systems to allow over-expression, or RNAi-mediated silencing of target genes, is greatly increasing. This is due to technology advances, such as rapid genome sequencing, enhancing the rate of gene discovery and thus leading to a large number of genes requiring functional analysis through transformation pipelines. Barley can be transformed at very high efficiency but the methods are genotype-dependent. Wheat is more difficult to transform, however, recent advances are also allowing the development of high-throughput transformation systems in wheat. For many gene function studies, barley can be used as a model for wheat due to its highly efficient transformation rates and smaller, less complex genome. An ideal transformation system needs to be extremely efficient, simple to perform, inexpensive, genotype-independent, and give the required expression of the transgene. Considerable progress has been made in enhancing transformation efficiencies, controlling transgene expression and in understanding and manipulating transgene insertion. However, a number of challenges still remain, one of the key ones being the development of genotype-independent transformation systems for wheat and barley.