苹果遗传转化的研究进展

苹果的遗传转化技术通过分子手段改良苹果,有助于缩短其育种周期.最近十年,在该领域的研究取得很大进展,涉及到一些重要的苹果基因型及有用的外源基因.迄今,苹果的遗传转化主要采用农杆菌介导法,侵染与转化材料的再生是影响其转化效率的关键过程,了解其影响因素,寻找有利因素以提高转化效率是目前苹果遗传转化研究的重点。 Abstract:Techniques of apple genetic transformation can improve apple trees and shorten the breeding cycle by molecular methods.In the last years,great progress has been made in this field,which involves a number of important apple genotypes developed.So far,apple genetic ransformation mainly adopts Agrobacterium-mediated technique,involving infection and transgenic tissue regeneration,which are important steps to affect the transformation efficiency.The initial work to the date is to know and to search for the factors which can increase the efficiency.     

苹果主栽品种高效遗传转化系统的建立及其影响因子的研究

研究影响农杆菌介导的苹果主栽品种遗传转化的若干因子效应,建立起新乔纳金苹果简单、高效的遗传转化系统。外源基因向新乔纳金苹果细胞转移的效率明显高于皇家嗄拉和富士苹果。菌株ＥＨＡ１０５介导基因转移的能力比菌株ＬＢＡ４４０４明显高。用乙酰丁香酮、磷酸饥饿、低ｐＨ值等ｖｉｒ基因诱导因子处理菌株ＥＨＡ１０５的细胞,未能提高转化效率。叶片在菌液中的浸泡时间不仅影响基因转移效率,而且影响头孢霉素对外植体上农杆菌生长的抑制效果。共培养时,叶片远轴面与培养基接触比近轴面与培养基接触的转化效率高。     

影响苹果离体叶片早期转化效率因素的研究

利用gus为报告基因,通过统计gus基因瞬时表达的GUS 位点数和形成的愈伤组织,快速检测外源基因是否导入植物细胞及转化效率,以及对苹果转化早期阶段的影响因素。结果表明,A281对苹果叶片的浸染率比LBA4404浸染率要高,转化效率要好;叶片近轴面接触培养基转化效率较好,显著高于远轴面;整叶划痕为最佳切割方式,有利于提高苹果叶片转化率;苹果基因型、菌液浓度和共培养天数对不同基因型苹果的转化效率影响很大。     

植物基因转化技术在苹果遗传改良中的应用

近年来,苹果基因转化技术研究取得了较大进展,本文从转化体系,选择标记,报告基因及目的基因的遗传学行为等方面综述了苹果遗传转化研究发展现状,着重论述了其在苹果遗传改良中的应用,并对植物基因转化技术在莱果上的应用前景和应注意的几个问题进行了讨论。     

向日葵遗传转化研究进展

综述了PEG、基因枪和农杆菌介导的转化方法应用于向日葵遗传转化研究所取得的进展,并对影响向日葵遗传转化效率的因素和研究前景进行了探讨。     

高粱遗传转化研究进展

高粱是世界上仅次于小麦、水稻、玉米和大豆的重要作物之一,然而由于其高效、稳定的遗传转化体系的建立较难,限制了其转基因研究进程.近年来,随着转基因技术的不断发展和完善,高粱转基因研究也取得了飞速的发展.从高粱遗传转化再生系统中外植体的选择、转化方法、影响转化和基因表达效率的因素等几方面进行了综述,并总结转基因高粱研究进展.     

农杆菌介导的番茄遗传转化的研究进展

番茄(Lycopersicon esculentum)传转化体系对其功能基因的研究和基因工程育种有重要影响,对农杆菌(Agrobacterium tumefaciens)介导的番茄遗传转化的研究进展进行了综述,主要包括影响番茄遗传转化效率的几个因素,如番茄的基因型、外植体状态、预培养和侵染过程、分化培养基中的激素和抗生...     

影响花粉管通道法介导的棉花遗传转化的外部因素

该文总结了近年来为了提高花粉管通道法介导的棉花遗传转化效率在影响棉花遗传转化的外部因素方面的研究进展。     

影响根癌农杆菌介导的木霉菌遗传转化因素分析

采用根癌农杆菌介导的转化方法,以木霉菌分生孢子为受体材料,对影响转化效率的主要因素进行了分析。结果表明,农杆菌菌株类型、初始菌液量、分生孢子浓度、共培养时间以及乙酰丁香酮的诱导等因素对转化效率都具有重要的影响。通过对这些因素的分析,基本得出了根癌农杆菌转化系统对木霉菌遗传转化的特点和规律,为将该转化系统用于其它丝状真菌的遗传转化提供了重要参考。     

影响根癌农杆菌介导的木霉菌遗传转化因素分析*

采用根癌农杆菌介导的转化方法,以木霉菌分生孢子为受体材料,对影响转化效率的主要因素进行了分析。结果表明,农杆菌菌株类型、初始菌液量、分生孢子浓度、共培养时间以及乙酰丁香酮的诱导等因素对转化效率都具有重要的影响。通过对这些因素的分析,基本得出了根癌农杆菌转化系统对木霉菌遗传转化的特点和规律,为将该转化系统用于其它丝状真菌的遗传转化提供了重要参考。     

An artificial miRNA as a new tool to silence and explore gene functions in apple

Artificial miRNA (amiRNA) is a powerful technology to silence genes of interest. It has a high efficiency and specificity that can be used to explore gene function through targeted gene regulation or to create new traits. To develop this gene regulation tool in apple, we designed two amiRNA constructs based on an apple endogenous miRNA backbone previously characterized (Md-miR156h), and we checked their efficiency on an easily scorable marker gene: the phytoene desaturase gene (MdPDS in apple). Two pairs of miRNA:miRNA* regions were designed (named h and w). The monocistronic Md-miR156h with these MdPDS targets was placed under the control of the CaMV 35S promoter to generate the two plasmids: pAmiRNA156h-PDSh and pAmiRNA156h-PDSw. Two Agrobacterium-mediated transformation experiments were performed on the cultivar ‘Gala’. A total of 11 independent transgenic clones were obtained in the first experiment and 5 in the second. Most transgenic lines had a typical albino and dwarf phenotype. However, six clones had a wild type green phenotype. Molecular analyses indicated clear relationships between the degree of albino phenotype, the level of MdPDS gene expression and the amount of mature amiRNAs. This study demonstrated for the first time in apple the functionality of an artificial miRNA based on an endogenous miRNA backbone. It provides important opportunities for apple genetic functional studies as well as apple genetic improvement projects.

     

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。     

Molecular selection in apple for resistance to scab caused by Venturia inaequalis

Large-scale marker-assisted selection requires highly reproducible, consistent and simple markers. The use of genetic markers is important in woody plant breeding in general, and in apple in particular, because of the high level of heterozygosity present in Malus species. We present here the transformation of two RAPD markers, which we found previously to be linked to the major scab resistance gene Vf, into more reliable and reproducible markers that can be applied directly to apple breeding. We give an example of how the use of such markers can speed up selection for the introduction of scab resistance genes into the same plant, reducing labour and avoiding time-consuming test crosses. We discuss the nature and relationship of the scab resistance gene Vf to the one present in Nova Easygro, thought to be Vr.     

Factors affecting the early gene transfer step in the development of transgenic ‘Fuji’ apple plants

This study was conducted to determine the optimal transformation conditions during early gene transfer steps necessary to improve the efficiency of transformation mediated by Agrobacterium tumefaciens in Fuji apple plants. The use of 200 μM acetosyringone in the co-culture medium resulted in 4.7% transformation efficiency, compared with different concentrations of 0–400 μM. A 4-day co-culture period gave 1.9% transformation efficiency, compared with different co-culture periods of 1–5 days. There was no significant difference in transformation efficiency with different explant placement orientations on co-culture medium. A comparison of young leaves from plants cultured in rooting medium for 4 weeks with those cultured for 8 weeks showed that an 8-week culture period resulted in higher transformation efficiency. We therefore concluded that the efficiency of Fuji apple transformation depends on improving factors related to the gene transfer step.     

纳米载体介导的植物遗传转化研究现状和前景

高效遗传转化技术是植物重要性状功能基因鉴定的前提和转基因育种的基础.随着纳米生物技术的发展,以纳米载体介导的植物转基因技术已显示出巨大的应用潜力.综述了国内外应用于植物纳米载体的类型、与外源基因的结合方式以及传输细胞的原理,重点阐述了影响纳米基因载体性能与转化效率的重要因素,以及纳米载体介导外源基因转化植物细胞的方法,...     

丝状真菌遗传筛选系统的研究及其应用

随着基因组时代的发展,主要丝状真菌基因组测序基本完成而被广泛应用于工业、农业、医药等领域。然而丝状真菌的遗传转化效率极低,为了保证在大量非转化子背景下能筛选到目标转化子,恰当的筛选标记显得尤为重要。目前,在丝状真菌的遗传转化过程中常用的筛选标记可分为两类:药物抗性筛选标记和营养缺陷型筛选标记。但两者均具有一定的局限性,为此科研人员利用最新研究的基因组编辑技术对筛选标记加以修饰改造,以更好地用于遗传筛选。本文综述了目前常用的遗传筛选系统的分子机制及运用基因组编辑技术改造的新型筛选标记理论,为丝状真菌在各领域更广泛的应用奠定基础。     

Reasons for lower transformation efficiency in indica rice using Agrobacterium tumefaciens-mediated transformation: lessons from transformation assays and genome-wide expression profiling

Agrobacterium tumefaciens-mediated genetic transformation has been routinely used in rice for more than a decade. However, the transformation efficiency of the indica rice variety is still unsatisfactory and much lower than that of japonica cultivars. Further improvement on the transformation efficiency lies in the genetic manipulation of the plant itself, which requires a better understanding of the underlying process accounting for the susceptibility of plant cells to Agrobacterium infection as well as the identification of plant genes involved in the transformation process. In this study, transient and stable transformation assays using different japonica and indica cultivars showed that the lower transformation efficiency in indica rice was mainly due to the low efficiency in T-DNA integration into the plant genome. Analyses of the global gene expression patterns across the transformation process in different varieties revealed major differences in the expression of genes responding to Agrobacterium within the first 6 h after infection and more differentially expressed genes were observed in the indica cultivar Zhenshan 97 (ZS), with a number of genes repressed early during infection. Microarray analysis revealed an important effect of plant defense response on Agrobacterium-mediated transformation. It has been shown that some genes which may be necessary for the transformation process were down-regulated in the indica cultivar ZS. This dataset provided a versatile resource for plant genomic research to understand the regulatory network of transformation process, and showed great promise for improving indica rice transformation using genetic manipulation of the rice genome.     

Genetic architecture of apple fruit quality traits following storage and implications for genetic improvement

Accurate prediction of genetic potential and response to selection in breeding requires knowledge of genetic parameters for important selection traits. Data from breeding trials can be used to obtain estimates of these parameters so that predictions are directly relevant to the improvement program. Here, a factor allocation diagram was developed to describe the sampling design used to assess the quality of fresh and post-storage (2 months) fruit from advanced selection trial in an apple breeding program from which models for analyses were developed. Genetic variation was the largest source of variation for the fruit size, red colour type, proportion of red skin colour and lenticels, and instrumentally assessed fruit diameter, mass, puncture force and titratable acidity. In contrast, residual variation was the largest for fruit shape, juiciness, sweetness, aromatic flavour, eating and overall quality, and instrumental crispness. Genetic effects for traits were generally stable over fixed effects, except for a significant interaction with storage duration for firmness. Genetic correlations among traits were generally weak except between fruit mass (and diameter) and sensory size (0.98), titratable acidity and sensory acidity (0.97), puncture force and sensory firmness (0.96–0.90), crispness and juiciness (0.87), sweetness and aromatic flavour (0.84) and instrumental and sensory crispness (0.75). Predictions of the performance for seven commercial cultivars are presented. This study suggests that the Washington State apple production area can be treated as a single target environment and sufficient diversity exists to generate new elite cultivars. In addition, options for evaluating the efficiency of apple breeding are discussed.