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。     

农杆菌介导的携带egfp基因载体转化寡雄腐霉

【目的】寡雄腐霉(Pythium oligandrum Drechsler)是一种对动、植物和环境无害,兼具杀菌和增产效果的生防真菌。通过研究建立农杆菌介导的寡雄腐霉遗传转化体系。【方法】选用EHA105、AGL-1、LBA4404三种农杆菌菌株对寡雄腐霉进行遗传转化研究,通过对影响遗传转化效果的条件参数试验优化,确立适宜寡雄腐霉遗传转化的农杆菌菌株及转化条件,建立农杆菌介导的寡雄腐霉遗传转化体系。【结果】经研究发现,所选3种农杆菌菌株中EHA105菌株对寡雄腐霉的遗传转化效果最好,其次是AGL-1菌株,LBA4404菌株转化效果不好。EHA105菌株经IM(含300μmol/L AS)诱导培养至OD_(600)=0.6时,与浓度为10~6–10~7个/m L的寡雄腐霉孢子悬浮液以1–10:1的比例混合,在25–26°C以液体振荡的方式避光共培养72 h(pH 5.0,含300μmol/L AS),寡雄腐霉菌体液体振荡恢复培养24 h,涂布抗性选择平板筛选寡雄腐霉转化子,即可得到寡雄腐霉基因工程菌株,其转化率可达到130个转化子/106个孢子。【结论】本研究首次构建了农杆菌介导的寡雄腐霉遗传转化体系,研究结果可为寡雄腐霉的生防机制及分子育种研究提供技术支撑。     

农杆菌介导的斑玉蕈遗传转化

【目的】将农杆菌介导的转化应用于重要的工厂化栽培食用菌斑玉蕈中,建立稳定的农杆菌介导的斑玉蕈遗传转化技术。【方法】将构建的双元载体pYN6982转入农杆菌LBA4404菌株中,以斑玉蕈SIEF3133菌株打碎的双核菌丝为受体材料,利用根癌农杆菌介导的转化方法进行斑玉蕈转化试验。【结果】经潮霉素抗性筛选、PCR鉴定以及有丝分裂稳定性试验验证,表明潮霉素磷酸转移酶基因(hph)已经整合到斑玉蕈的基因组中;转基因斑玉蕈菌丝在荧光显微镜下可以观测到绿色荧光,表明增强型绿色荧光蛋白基因(egfp)已经在转基因斑玉蕈菌株中获得了表达;通过PCR检测,随机挑选的8个转基因斑玉蕈菌株中有2个可以扩增出载体转移DNA(T-DNA)边界重复序列外的卡那霉素基因(kan)序列。【结论】获得了稳定遗传和表达的斑玉蕈转基因菌株,建立了农杆菌介导的斑玉蕈遗传转化方法。农杆菌介导的斑玉蕈遗传转化中,存在载体T-DNA边界重复序列之外的DNA序列转移到转基因斑玉蕈中的现象,有待进一步研究。     

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。     

橡胶树胶孢炭疽菌T-DNA插入突变体库构建及其致病缺陷转化子筛选

【目的】进一步研究橡胶树胶孢炭疽菌致病分子机理。【方法】通过含ILV1基因(具氯嘧磺隆抗性)的pSULF.gfp双元载体农杆菌AGL-1介导进行橡胶树胶孢炭疽菌遗传转化,利用氯嘧磺隆抗性标记筛选转化子,对转化子PCR验证及荧光显微观察;采用离体古铜期橡胶树叶无伤接种法进行致病性缺陷转化子筛选,并对转化子进行遗传稳定性检测。【结果】获得含3 721个转化子的T-DNA插入突变体库,转化效率为150 400个转化子/106孢子,从3 721个转化子中筛选得到致病性缺陷转化子25个;随机选取20个转化子进行遗传稳定性测定,在不含氯嘧磺隆PDA平板上继代培养10次后仍保持氯嘧磺隆抗性,且表型稳定,表明插入外源基因能够稳定遗传。【结论】可以利用根癌农杆菌介导橡胶孢炭疽菌转化,构建橡胶树胶孢炭疽菌T-DNA插入突变体库,筛选致病缺陷突变菌,为进一步研究该菌致病相关基因提供材料。     

根癌农杆菌介导的黑曲霉遗传转化体系的建立及优化

基于根癌农杆菌介导的遗传转化方法的独特优点,研究黑曲霉转化过程中各主要影响因素,建立高效的黑曲霉遗传转化方法。构建双元载体pBI-hph,通过电转导入农杆菌LBA4404中,以黑曲霉TCCC41056为受体菌株,利用潮霉素B基因作为筛选标记,对影响转化效率的孢子悬液的新鲜程度及浓度、农杆菌菌液浓度、共培养时间、共培养温度这五个条件进行分析,建立根癌农杆菌介导的黑曲霉遗传转化体系。实验结果表明,上述条件对黑曲霉的转化效率有较大的影响,通过优化,黑曲霉转化效率可达83个转化子/10⁷分生孢子;整合到黑曲霉基因组的外源基因可以稳定遗传,在转接10代后遗传性能仍保持稳定,并在众多转化子中筛选得到了糖化酶活力提高18%的黑曲霉突变株。根癌农杆菌介导的黑曲霉转化体系的建立,为进一步研究黑曲霉的功能基因以及开发黑曲霉表达系统提供了有力的手段。     

农杆菌介导的香蕉枯萎病菌4号生理小种转化体系的优化

香蕉枯萎病是世界范围内香蕉种植区最为严重的病害之一,严重威胁和影响着香蕉产业的发展.本文针对香蕉枯萎病病原菌的4号生理小种,建立了农杆菌介导的转化体系,确定了影响转化效率主要因子的优化体系是:农杆菌在IM培养基诱导前农杆菌OD_(600)为0.15、农杆菌经IM液体培养基诱导的时间为7 h、乙酰丁香酮(AS)浓度为150 μmol/L、Focr4孢子浓度为1×10~6个/mL、共培养时间为48 h、培养温度为25℃、诱导培养基pH值为5.5.在此条件下,转化效率能达到700～800个转化子/10~6个香蕉枯萎病菌孢子.PCR验证表明外源的T-DNA已经成功随机地整合到该病原菌基因组中.目前,应用该转化体系已获得2 300多个转化子,为后续克隆相关致病基因打下了良好基础.     

杨树腐烂病菌(Cytospora chrysosperma)原生质体遗传转化体系的构建

【目的】通过分析不同酶解条件对金黄壳囊孢菌[Cytospora chrysosperma(Pers.)Fr.]原生质体释放的影响,建立高效制备原生质体及其遗传转化体系的方法,为开展杨树腐烂病菌的致病分子机制研究奠定基础。【方法】以杨树腐烂病菌菌株CFCC 89981为受体,在细胞壁降解酶作用下产生用于转化所需的原生质体,通过PEG(Polyethylene glycol)介导将g GFP DNA导入杨树腐烂病菌的原生质体中获得转化子。经PCR扩增、Southern blot和荧光观察验证g GFP DNA插入到杨树腐烂病菌基因组中并表达出GFP(Green fluorescent protein)蛋白。【结果】以p H 5.5的1.2 mol/L KCl为稳渗剂,杨树腐烂病菌菌丝经Driselase和Lysing enzymes共同酶解4 h可获得1.2×108个/m L原生质体,再生率可达63.74%±9.73%,FDA(Fluorescein diacetate)溶液染色结果显示98%左右的原生质体具有较高的活力。利用PEG介导的遗传转化方法,转化效率可达76个/μg DNA。PCR检测和Southern blot均可在转化子基因组中检测到GFP基因片段,且荧光检测转化子的菌丝均呈绿色荧光,表明GFP基因在杨树腐烂病菌中表达。此外,GFP转化子在无潮霉素抗性的PDA培养基中多代转接后仍稳定遗传并表达GFP蛋白。【结论】通过筛选酶解条件,获得高质量、高活性的杨树腐烂病菌原生质体,并利用PEG介导的转化方法建立了高效稳定的原生质体遗传转化体系。该体系的建立为杨树腐烂病菌的后续研究奠定了技术基础。     

农杆菌介导的胀果甘草愈伤组织遗传转化

目的:旨在建立一个农杆菌介导的甘草愈伤组织遗传转化的可行方案,并对转化条件进行优化。方法:选择EHA105和LBA4404两种根癌农杆菌菌株,热激法转入含有绿色荧光蛋白GFP基因的植物表达载体pBI121-gfp,挑选转化的农杆菌用于侵染胀果甘草愈伤组织。设置不同培养时间的愈伤组织作为受体材料和农杆菌不同侵染时间两组条件,经共培养后的甘草愈伤组织进行荧光检测。结果:愈伤组织在含有100mg/l卡那霉素的继代培养基上进行筛选培养,得到了具有卡那霉素抗性的转化甘草愈伤组织,转化愈伤组织经继代培养基后在紫外光下仍可见绿色荧光,PCR检测转化愈伤组织基因组中含有gfp基因。结论:试验建立了农杆菌介导的甘草愈伤组织的遗传转化体系,为目的基因导入甘草细胞利用基因工程手段调控甘草次生代谢产物生物合成的研究奠定了基础。     

根癌农杆菌介导的赭曲霉遗传转化体系的建立

赭曲霉在工业上用于甾体C-11α羟基化。为了对现有赭曲霉生产菌株进行遗传改造,以农杆菌LBA4404作为侵染菌株,以赭曲霉(TCCC41060)作为受体菌株,以潮霉素B基因作为筛选标记,成功建立了根癌农杆菌介导的赭曲霉遗传转化体系并对其转化效率的主要影响因素,如农杆菌浓度、孢子浓度、共培养时间和温度等进行了分析。在最佳条件下,转化效率可以达到57个转化子/108个分生孢子。随机挑选的8个阳性转化子10代连续培养结果表明所获得的转化子能稳定遗传。该遗传转化体系的建立为赭曲霉菌株的定向遗传改造提供了重要的操作平台。     

Effects of MULTIFOLIATE-PINNA, AFILA, TENDRIL-LESS and UNIFOLIATA genes on leafblade architecture in Pisum sativum

In order to dissect the genetic regulation of leafblade morphogenesis, 16 genotypes of pea, constructed by combining the wild-type and mutant alleles of MFP, AF, TL and UNI genes, were quantitatively phenotyped. The morphological features of the three domains of leafblades of four genotypes, unknown earlier, were described. All the genotypes were found to differ in leafblade morphology. It was evident that MFP and TL functions acted as repressor of pinna ramification, in the distal domain. These functions, with and without interaction with UNI, also repressed the ramification of proximal pinnae in the absence of AF function. The expression of MFP and TL required UNI function. AF function was found to control leafblade architecture multifariously. The earlier identified role of AF as a repressor of UNI in the proximal domain was confirmed. Negative control of AF on the UNI-dependent pinna ramification in the distal domain was revealed. It was found that AF establishes a boundary between proximal and distal domains and activates formation of leaflet pinnae in the proximal domain.     

Presence of Rhizobium Etli bv . Phaseoli and Rhizobium Gallicum bv . Gallicum in Egyptian Soils

Seven bean rhizobial strains EBRI 2, 3, 21, 24, 26, 27 and 29 identified as Rhizobium etli, and EBRI 32 identified as Rhizobium gallicum, isolated from Egyptian soils and which nodulated Phaseolus vulgaris efficiently, were subjected to hybridization with a nifH probe in order to estimate the copy number of this gene. Seven strains (EBRI 2, 3, 21, 24, 26, 27 and 29) which were only able to nodulate Phaseolus vulgaris, contained three copies of the nifH gene, consistent with their identification as Rhizobium etli bv. phaseoli. Only one strain (EBRI 32) which nodulated both Phaseolus vulgaris and Leucaena leucocephala, had one copy of nifH gene. This confirmed the classification of this strain as Rhizobium gallicum bv. gallicum.     

Genome Analysis inNeurospora crassa;Cloning of Four Lociarginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) and Associated Chromosome Walking

We have cloned fourNeurospora crassagenes by complementation analysis. Cloned genes include thearginine-1(arg-1),methionine-6(met-6),unknown-7(un-7), andribosome production-1(rip-1) loci. Chromosome walks were initiated in ordered cosmid libraries from the cloned loci. A total of about 700 kb of theNeurosporagenome is covered in these walks.     

Characterisation of cell wall polysaccharides, arabinogalactans-proteins (AGPs) and phenolics of Cola nitida, Cola acuminata and Garcinia kola seeds

Many Cola plant species are endemic to West and Central Africa. Cola acuminata and Cola nitida are used as masticatory when fresh, while the dried nuts are used for beverages and pharmaceutical purposes in Europe and North America. Garcinia kola seeds, that serve as a substitute for the true kola nuts, are used in African traditional medicine for the treatment of various diseases, including colic, headache and liver cirrhosis. Seeds extracts of G. kola are also known for their anti-inflammatory, antimicrobial and antiviral properties. To gain information on the chemical properties of the kolas, we have isolated and analyzed cell wall polysaccharides, arabinogalactan-proteins and phenolic substances from the seeds of the three kola species. The sugar composition of cell wall material of C. acuminata, C. nitida and G. kola revealed that Gal (up to 30%), Ara, GalA and Glc as the predominant monosaccharides, representing approximately 90% by mol of the total hydrolysable sugar present in this material. In Ammonium oxalate cell wall fraction, GalA was found to be the major sugar present in all kola species. In the alkali-soluble fraction, there were significant differences in the level of Glc and Gal. The level of Glc was high in C. acuminata and C. nitida while the level of Gal and Xyl were high in C. nitida and G. cola. Isolation and quantification of arabinogalactan-proteins demonstrate that G. kola seeds contained four to eight times more of these proteoglycans than the seeds of the other two species. Finally, analysis of soluble phenolic substances shows that caffeine and catechin were largely represented in C. acumina and C. nitida seeds, with caffeine accounting for 50% of all soluble phenolics. These findings indicate that the three Kola seeds are highly enriched in pectins and proteoglycans and that C. acuminata and C. nitida can be used as a possible source of caffeine and catechin.     

The phylogenetic placement of the Leucogastrales, including Mycolevis siccigleba (Cribbeaceae), in the Albatrellaceae using morphological and molecular data

The morphology of many hypogeous fungi converges on a homogeneous reduced form, suggesting that disparate lineages are subject to a uniform selection pressure. The primary goal of this study was to evaluate the morphology and infer the phylogeny of the Leucogastrales with Mycolevis siccigleba using a Bayesian methodology. A comprehensive morphological assessment was used for an a priori phylogenetic inference to guide the sequencing effort. All structures except spore ornamentation pointed to the Albatrellaceae as the most likely sister taxon. Polyporoletus sublividus, a close relative of Albatrellus, produces ornamented basidiospores with a similar structure to M. siccigleba basidiospores. The ITS from 30 taxa was used for the molecular phylogenetic analysis. P. sublividus was found sister to Mycolevis. Leucophleps spinispora and L. magnata formed a group sister to the Polyporoletus/Mycolevis group, whereas Leucogaster was polyphyletic with respect to the core of the Leucogastrales and sister to A. caeruleoporus. This relationship was expected as previously undescribed chlamydospores produced by members of Albatrellus had a similar morphology to the basidiospores of L. rubescens.     

Fluorescent labeling of (Na + K)-ATPase by 5-iodoacetamidofluorescein

5-Iodoacetamidofluorescein (5-IAF) covalently labels dog kidney (Na⁺ + K⁺)-ATPase with approximately 2 moles incorporated per mole of enzyme. ATPase and K⁺-phosphatase activities are fully retained after reaction, and the kinetic parameters for Na⁺, K⁺, Mg²⁺, ATP and p-nitrophenyl phosphate are likewise not significantly affected. The fluorescence of the bound 5-IAF is increased by ATP, Na⁺, and Mg²⁺, and decreased by K⁺. These fluorescence changes likely reflect ligand-induced stabilization of the E₁ or E₂ states of the enzyme.     

Annular and non-annular binding sites on the (Ca + Mg)-ATPase

Quenching of the fluorescence of the (Ca²⁺ + Mg²⁺)-ATPase purified from muscle sarcoplasmic reticulum can be used to measure relative binding constants of hydrophobic compounds to the phospholipid-protein interface. We show that the binding constant for cholesterol is considerably less than that for phosphatidylcholine, so that cholesterol is effectively excluded from the phospholipid annulus around the ATPase. However, dibromocholestan-3β-ol causes quenching of the fluorescence of the ATPase, and so has access to other, non-annular sites. We suggest that these non-annular sites could be at protein/protein interfaces in ATPase oligomers. Oleic acid can bind at the phospholipid/protein interface, although its binding constant is less than that for a phosphatidylcholine, and it can also bind at the postulated non-annular sites. The effects of these compounds on the activity of the ATPase depend on the structure of the phospholipid present in the systems.     

A new fungal genus, Teracosphaeria, with a phialophora-like anamorph (Sordariomycetes, Ascomycota)

The new genus and species Teracosphaeria petroica is described for a perithecial ascomycete and its anamorph occurring on decayed wood collected in New Zealand. The fungus produces immersed, non-stromatic ceratosphaeria-like perithecia in nature, with hyaline, septate ascospores produced in unitunicate, non-amyloid asci. The anamorph produced in vitro is phialophora-like with lightly pigmented phialides terminating in flaring, deep collarettes that are often noticeably brown with conspicuous periclinal thickening. Phylogenetic analysis of LSU rDNA sequence data indicates that this fungus is distinct from morphologically similar fungi classified in the Chaetosphaeriales, the Trichosphaeriales or the Magnaporthaceae. It forms a monophyletic group with recently described, chaetosphaeria-like ascomycetes, such as the pyrenomycete genus Mirannulata, and shows affinity with the anamorphic species Dictyochaeta cylindrospora. The usefulness of describing anamorph genera for morphologically reduced anamorphs, when anamorph characteristics are actually part of the holomorph diagnosis, is discussed. An apparently contradictory example of the so-called Cordana and Pseudobotrytis anamorphs of Porosphaerella spp. is also discussed.