抗氧化剂对农杆菌介导的大豆下胚轴GUS基因瞬时表达的影响

 大豆（Glycine max）下胚轴作为大豆遗传转化的外植体材料,能快速高频再生不定芽。然而,在遗传转化过程中褐化影响基因转化效率。在该研究中,我们用含有GUS染色基因和hpt II（Hygromycin phosphotransferase II）筛选基因的农杆菌(Agrobacteri um tumefaciens) LBA4404侵染大豆下胚轴,并用组织化学定位法测定了GUS基因的瞬时表达,以确定大豆的优化基因转化条件。 结果显示,在共培养基中加入硫代硫酸钠、L_半胱氨酸以及二硫苏糖醇等抗氧化剂,可以有效地抑制大豆下胚轴在组培过程中褐化的发生,并大幅度提高农杆菌在下胚轴的瞬时表达率。这些结果说明抗氧化剂可以降低这种影响并有效提高基因转化效率。     

农杆菌介导的玉米幼胚遗传转化体系的优化

本实验以玉米品种HiⅡ(PA*PB和PB*PA)的幼胚为材料,用根瘤农杆菌菌株EHA105和LBA4404对幼胚进行转化,将PTF102-GUS导入玉米中,借助GUS基因的瞬时表达率,对影响农杆菌介导玉米幼胚转化的部分因素进行优化。研究表明使用EHA105侵染HiⅡ幼胚;农杆菌浓度在OD550=0.3,侵染时间在10min;幼胚大小为1.0mm时,GUS染色瞬时表达率较高。     

农杆菌介导韭菜遗传转化相关因素的研究

以"汉中冬韭"韭菜品种为试验材料,用含有pCAMBIA3301质粒的根癌农杆菌菌株EHA105对影响韭菜遗传转化效率的多种因素进行研究。结果表明,诱导40 d的愈伤组织,GUS基因瞬时表达率达到93%,且最适宜于不定芽的分化;当乙酰丁香酮(AS)的浓度为100μmol/L时,愈伤的GUS表达率达到91%,植株再生率为7.9%,AS浓度增加时其值也不会增加;菌液OD600值为0.6侵染10 min时,与其他组合相比,外植体受伤程度小,GUS表达率及再生率最高;侵染后的愈伤共培养3 d后,农杆菌生长较少,GUS表达率为91.1%,而再生率达到7.2%,为最佳的共培养时间。通过试验得到韭菜遗传转化因素的最佳条件,为今后的遗传转化提供一些参考。     

农杆菌介导的大豆子叶节非组织培养遗传转化体系优化

本研究以GUS基因在子叶节区的瞬时表达为依据,通过探讨影响农杆菌转化效率的因素,优化了大豆子叶节非组织培养遗传转化体系;利用该体系对冀豆16号进行Bar基因的遗传转化,并使用针刺法对转基因植株进行草铵膦筛选.结果表明,侵染液中附加3％蔗糖、OD600=0.6、以脱脂棉作为菌液附着介质同时不添加表面活性剂Silwet L-77、侵染1次的GUS阳性率最高达到62.13％.草铵膦抗性植株经PCR检测获得T0阳性植株10个,转化率为2.5％.经PCR和RT-PCR鉴定共获得3株T1阳性植株,初步证明目的基因已整合到大豆基因组中.     

农杆菌介导的刺芹侧耳遗传转化体系的建立

以刺芹侧耳菌丝球为受体,潮霉素(Hyg)为筛选标记,应用农杆菌介导法对刺芹侧耳菌丝进行了遗传转化研究。潮霉素敏感性测试结果表明,刺芹侧耳Hyg耐受浓度为50mg/L。农杆菌介导的刺芹侧耳菌丝最佳遗传转化体系为:菌液浓度OD600=0.6–0.7,侵染时间30–35min,共培养时间2d,侵染液和共培养培养基中乙酰丁香酮(AS)浓度为1mg/m L;经潮霉素抗性筛选、PCR鉴定和GUS活性的组织化学分析,表明外源基因GUS已转入到刺芹侧耳菌丝中,并获得表达。本实验成功地建立了稳定的农杆菌介导的刺芹侧耳遗传转化体系。     

优良大麦品种花30幼胚遗传转化体系的优化

以大麦花培基因型花30的幼胚为外植体,设置不同的培养基类型、不同激素配比及碳源,研究其对幼胚愈伤组织诱导及绿苗分化的影响,以此建立和优化一个适于优良大麦品种遗传转化的高效组织培养体系。结果表明:在N6、MS和B5的组合改良培养基下,以蔗糖为碳源,附加2mg/L 2,4-D、1mg/L ABA时,有最高的愈伤组织诱导率,且愈伤质量最好。Cu2+的添加具有抑制幼胚直接发芽成苗和改善愈伤组织质量的双重功效。添加2mg/L 6-BA对愈伤组织的分化效果比较理想。为了提高农杆菌介导转化大麦外源基因的瞬时表达率和优化遗传转化体系,利用花30幼胚产生的愈伤组织为受体材料,通过检测GUS基因的瞬时表达情况,研究了农杆菌介导的大麦遗传转化中菌液的浓度、侵染时间以及共培养天数对遗传转化的影响,结果表明:当菌液浓度OD600=0.5的条件下,侵染15min,共培养2d表现出最佳的GUS瞬时表达率。     

根癌农杆菌介导的大豆遗传转化

农杆菌介导法是大豆遗传转化的重要方法之一 ,许多实验室应用该方法得到了转基因大豆 ,但目前使用该方法进行转化的效率还比较低 ,尚需深入研究。农杆菌菌株、大豆基因型、组织培养条件、T-DNA的转移效率和转化后的筛选模式都会影响大豆转化的效率。概述了近年来根癌农杆菌介导的大豆遗传转化的一些重要成果 ,以及转化过程中大豆的易感性与农杆菌的转化能力、乙酰丁香酮促进vir基因活化、转化的受体系统和巯基混合物减轻受体材料的褐化、提高T DNA的转移效率等几个重要因素的研究进展 ,并介绍了转化中常用的几个筛选标记基因 (nptⅡ、hpt、bar基因和突变的ahas基因 )及通过共转化法去除标记基因的方法 ,同时对今后研究的重点进行了讨论.     

农杆菌菌株及其侵染浓度和时间对基于菜豆黄矮病毒表达载体瞬时表达外源基因的影响

以3种常见的农杆菌菌株(GV3101、EHA105、LBA4404)和基于菜豆黄矮病毒的复制型植物表达载体为材料,利用农杆菌介导的瞬时转化技术,将外源绿色荧光蛋白(GFP)基因导入本氏烟(Nicotiana benthamiana L.)叶片中实现瞬时表达,并对不同农杆菌菌株、侵染浓度及侵染时间对于瞬时表达水平的影响进行比较。结果显示,3种不同的农杆菌菌株在介导转化本氏烟叶片瞬时表达GFP积累水平之间存在显著差异,其中EHA105菌株转化效率最高,LBA4404次之,GV3101最低。此外,GV3101、EHA105和LBA4404最适侵染浓度的OD600值分别为0. 5、0. 3和0. 3;最佳侵染时间均为第4 d。研究结果表明农杆菌菌株染色体结构和Ti质粒的差异是影响瞬时转化过程中农杆菌侵染浓度及其外源基因瞬时表达效率的重要因素。     

农杆菌介导巨桉Eg5高效遗传转化

以巨桉(Eucalyptus grandis)无性系Eg5叶片为外植体, 探讨了农杆菌(Agrobacterium tumefaciens)侵染时间、共培养pH值和共培养时间对瞬时转化效率的影响, 分析了不同筛选策略对遗传转化植株筛选效果的影响。结果表明, 外植体侵染45分钟, 共培养pH值为5.8, 共培养3天所得到的瞬时转化效率最高; 逐步提高卡那霉素(Km)浓度筛选转基因植株有效, 筛选率达到15%, 转化率达到0.26%。经过GUS染色分析和PCR检测, 证实为转基因植株。     

甘露醇在根癌农杆菌转化石斛兰中的作用

以淡绿色细小且致密的石斛兰原球茎为材料,研究甘露醇在根癌农杆菌转化石斛兰中的作用.结果表明:原球茎侵染前经过附加0.3 mol/L甘露醇的高渗固体培养基中进行前处理6 h,GUS瞬时表达率为60.00%,而对照的GUS瞬时表达率为0.0.甘露醇高渗处理对石斛兰根癌农杆菌的转化具有提高转化效率的作用.     

Proteomic and genomic characterization of Kunitz trypsin inhibitors in wild and cultivated soybean genotypes

In this study, we investigated protein and genetic profiles of Kunitz trypsin inhibitors (KTIs) in seeds of 16 different soybean genotypes that included four groups consisting of wild soybean (Glycine soja), the cultivated soybean (G. max) ancestors of modern N. American soybean cultivars (old), modern N. American soybean (elite), and Asian cultivated soybean landraces that were the immediate results of domestication from the wild soybean. Proteins were well separated by two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and stained protein cut from a 2D-PAGE indicated that KTI exists as multiple isoforms (spots) in soybean. Protein spots of KTI were identified and characterized using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF-MS). Although overall distribution patterns of the KTI protein spots appeared similar, the number and intensity of the protein spots between wild and cultivated genotypes varied. Three KTI peptides were identified in three of the wild genotypes, PI 393551, PI 407027 and PI 407282, in which KTI3 peptide showed highest intensity. The remaining wild genotype, PI 366120, showed four protein spots. In contrast, the ancestors, modern and Asian landrace genotypes showed only two protein spots corresponding to KTI. On the basis of DNA blot analysis, there is one copy of the KTI3 gene in all 16 genotypes. Polymorphism was detected in one of the wild genotypes (PI 366120) both in proteomic and genomic analyses. Our data suggest that the major variation of protein profiles were between wild and cultivated soybean genotypes rather than among genotypes in the same group. Genetic variation of KTI1, KTI2 and KTI3-related genes were detected within and between groups.     

高羊茅和黑麦草农杆菌介导转化体系的研究

利用C58C1农杆菌菌系（携带的表达载体上含GUS基因和nptII基因）感染4个草坪草品种追寻者、爱神特、腾跃和守门员成熟胚来源的愈伤组织,共培养后部分愈伤组织进行X-Gluc组织化学染色检测,其余愈伤组织在含G418 10-25 mg/L的MS改良培养上先后筛选抗性愈伤组织和分化抗性再生植株,对移栽成活的144棵抗性再生植株分别进行了ELISA检测、PCR检测和组织化学染色检测。愈伤组织阶段X-Gluc染色检测结果表明,4个草坪草品种GUS基因瞬间表达率8.6%～46.9%,爱神特愈伤组织对农杆菌侵染最为敏感,其次是腾跃和守门员,追寻者最不敏感;ELISA检测结果表明,45株呈现阳性,证明nptII基因已转入草坪草并已表达;PCR检测结果与ELISA检测结果一致,表明nptII基因确实已经整合到了草坪草基因组中,且没有发生沉默现象;转基因植株X-Gluc染色检测结果表明,GUS基因在43株中得到了稳定表达,在2株中发生了沉默现象。4个草坪草品种抗性再生植株分化率0～43.5%,转化率0～21.5 %。结果还表明,GUS基因瞬间表达率与稳定转化率在草坪草上很不一致,不能作为衡量基因型转化效果的指标。     

A single origin and moderate bottleneck during domestication of soybean (Glycine max): implications from microsatellites and nucleotide sequences

Background and Aims

It is essential to illuminate the evolutionary history of crop domestication in order to understand further the origin and development of modern cultivation and agronomy; however, despite being one of the most important crops, the domestication origin and bottleneck of soybean (Glycine max) are poorly understood. In the present study, microsatellites and nucleotide sequences were employed to elucidate the domestication genetics of soybean.

Methods

The genomes of 79 landrace soybeans (endemic cultivated soybeans) and 231 wild soybeans (G. soja) that represented the species-wide distribution of wild soybean in East Asia were scanned with 56 microsatellites to identify the genetic structure and domestication origin of soybean. To understand better the domestication bottleneck, four nucleotide sequences were selected to simulate the domestication bottleneck.

Key Results

Model-based analysis revealed that most of the landrace genotypes were assigned to the inferred wild soybean cluster of south China, South Korea and Japan. Phylogeny for wild and landrace soybeans showed that all landrace soybeans formed a single cluster supporting a monophyletic origin of all the cultivars. The populations of the nearest branches which were basal to the cultivar lineage were wild soybeans from south China. The coalescent simulation detected a bottleneck severity of K′ = 2 during soybean domestication, which could be explained by a foundation population of 6000 individuals if domestication duration lasted 3000 years.

Conclusions

As a result of integrating geographic distribution with microsatellite genotype assignment and phylogeny between landrace and wild soybeans, a single origin of soybean in south China is proposed. The coalescent simulation revealed a moderate genetic bottleneck with an effective wild soybean population used for domestication estimated to be ≈2 % of the total number of ancestral wild soybeans. Wild soybeans in Asia, especially in south China contain tremendous genetic resources for cultivar improvement.     

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.     

不同转化条件对3种农杆菌GFP基因在本氏烟草中瞬时表达的影响

以本氏烟草（Nicotiana benthamiana）为植物材料,分析了不同农杆菌菌株（LBA4404菌株、EHA105菌株、GV3101菌株）、菌液浓度以及侵染时间在瞬时转化过程中对报告基因GFP荧光表达量的影响。结果显示,不同的农杆菌菌株瞬时表达外源基因的最适浓度和时间均有所不同：LBA4404菌株在菌悬液OD₆₀₀值为0.8时所介导的瞬时表达效率最高;而EHA105和GV3101菌株在菌悬液OD₆₀₀值为0.6时可达到最高瞬时表达效率。LBA4404菌株所介导的瞬时表达在农杆菌注射后第2天时表达量最高,而EHA105和GV3101菌株所介导的瞬时表达在农杆菌注射后第4天时表达量最高。不同菌株间比较分析表明,LBA4404菌株所介导的瞬时表达效率最高。上述结果表明,农杆菌菌株以及浓度和侵染时间等转化条件均是影响瞬时表达效率的重要因素。     

Proteomic and genetic analysis of glycinin subunits of sixteen soybean genotypes.

We investigated proteomic and genomic profiles of glycinin, a family of major storage proteins in 16 different soybean genotypes consisting of four groups including wild soybean (Glycine soja), unimproved cultivated soybean landraces from Asia (G. max), ancestors of N. American soybean (G. max), and modern soybean (G. max) genotypes. We observed considerable variation in all five glycinin subunits, G1, G2 G3, G4 and G5 using proteomics and genetic analysis. Two-dimensional polyacrylamide gel electrophoresis (2D-PAGE) and mass spectrometry (MS) analysis showed that the wild genotypes had a range of 25-29 glycinin protein spots that included both acidic and basic polypeptides followed by the ancestors with 24-28, modern cultivars with 24-25, and landraces with 17-23 protein spots. Overall, the wild genotypes have a higher number of protein spots when compared to the other three genotypes. Major variation was observed in acidic polypeptides of G3, G4 and G5 compared to G1 and G2, and minor variation was observed in basic polypeptides of all subunits. Our data indicated that there are major variations of glycinin subunits between wild and cultivated genotypes rather than within the same groups. Based on Southern blot DNA analysis, we observed genetic polymorphisms in group I genes (G1, G2, and G3) between and within the four genotype groups, but not in group II genes (G4 and G5). This is the first study reporting the comparative analysis of glycinin in a diverse set of soybean genotypes using combined proteomic and genetic analysis.     

大豆转基因体系的研究进展

从大豆的转基因方法和受体系统两个方面概述大豆转基因体系的最新研究进展,并讨论了大豆遗传转化的主要障碍及可能的解决途径。作者认为,以根癌农杆菌介导大豆子叶节和基因枪轰击大豆未成熟子叶是较有效的大豆遗传化系统。目前,在大豆遗传转化研究中存在着大豆组织培养体系有待于进一步完善、遗传转化率较低、重复性差、大豆受体基因型单一等问题,建立新的、高效和稳定的大豆组织培养体系,提高生产上栽培大豆品种的组织培养能力,改遗传转化现有的单一基因为多基因,可能是解决上述问题的有效途径。     

根癌农杆菌介导的高效大豆遗传转化体系的建立

利用根癌农杆菌对来自大豆成熟种子的胚尖进行遗传转化,研究了影响农杆菌介导大豆转化的各种因素,建立了一套优化的大豆遗传转化体系。研究结果表明:菌株KYRT1比EHA105和LBA4404具有更强的侵染能力;较酸的共培养基(pH5.4)、较低的培养温度(22℃)均有利于提高转化效率;恢复培养和分步抗性筛选方式有利于提高抗性组织的存活率和分化率。同时应用这种优化的遗传转化体系,获得了7个大豆品系的转基因植株,转化频率为4.29%-18%。经过PCR和Southern分析证明外源的双价抗虫基因cryIA(c)和pta已经整合到大豆的基因组中。     

农杆菌介导GUS基因对多年生黑麦草转化的研究

通过检测愈伤组织中GUS基因的瞬间表达,研究农杆菌LBA4404/pCAMBIA1301介导多年生黑麦草的转化体系。通过对多年生黑麦草瞬间表达率的比较,确立了其遗传转化的最佳优化条件。研究发现,多年生黑麦草不同品种的转化率在25%~45%之间变化。多年生黑麦草遗传转化最佳优化条件是预培养10d的胚性愈伤组织、浓度为0.5~0.8OD的农杆菌菌液以及2d共培养时间。在共培养基中添加100μmol/L乙酰丁香酮能有效地提高植物瞬间表达率。两种侵染处理方法比较结果为滤纸滴加法比浸泡法更优。转化后对愈伤组织的干燥处理能抑制农杆菌过度繁殖,能改善愈伤状态,有利于提高转化率。