利用Red系统快速敲除家蚕核型多角体病毒orf60基因

用Red重组系统和最近构建的家蚕核型多角体病毒（BmNPV）bacmid在大肠杆菌BW25113中快速地敲除BmNPV orf60基因。从大肠杆菌BmDH10Bac中提取BmNPV bacmid,将其电转化到含有质粒pKD46（能表达Red重组酶）的大肠杆菌菌株BW25113中,获得了可用于BmNPV基因打靶的菌株BW25113-Bac。设计一对长63bp的引物（5′端为orf60基因的左右同源臂,长45bp;3′端长18bp,为氯霉素抗性基因（cat）的首尾序列）,以pKD3质粒（含cat）为模板,PCR扩增携带orf60左右同源臂的cat,即打靶线性化片段。将该线性化片段电转入BW25113-Bac菌株,在Red重组酶的作用下,线性化片段与BmNPV bacmid中的orf60基因发生同源重组。设计3对特异引物,用PCR方法证明cat成功地替换了BmNPV orf60基因。重组bacmid DNA转染BmN细胞后,Western blot分析未检测到orf60基因的表达。     

利用Red系统快速敲除家蚕核型多角体病毒orf60基因

用Red重组系统和最近构建的家蚕核型多角体病毒（BmNPV）bacmid在大肠杆菌BW25113中快速地敲除BmNPV orf60基因。从大肠杆菌BmDH10Bac中提取BmNPV bacmid,将其电转化到含有质粒pKD46（能表达Red重组酶）的大肠杆菌菌株BW25113中,获得了可用于BmNPV基因打靶的菌株BW25113-Bac。设计一对长63bp的引物（5′端为orf60基因的左右同源臂,长45bp;3′端长18bp,为氯霉素抗性基因（cat）的首尾序列）,以pKD3质粒（含cat）为模板,PCR扩增携带orf60左右同源臂的cat,即打靶线性化片段。将该线性化片段电转入BW25113-Bac菌株,在Red重组酶的作用下,线性化片段与BmNPV bacmid中的orf60基因发生同源重组。设计3对特异引物,用PCR方法证明cat成功地替换了BmNPV orf60基因。重组bacmid DNA转染BmN细胞后,Western blot分析未检测到orf60基因的表达。     

大肠杆菌Zn2+敏感突变株构建及其功能验证

【背景】Zn²⁺在细胞解毒及许多生理过程中发挥着关键作用,Zn²⁺转运蛋白已逐渐引起人们的重视。在大肠杆菌中,zntA和zitB是2个外排Zn²⁺的关键基因。【目的】构建大肠杆菌Zn²⁺敏感突变株,并对其功能进行验证。【方法】以Escherichia coli DH5α为出发菌株,利用λ Red重组系统,通过携带卡那霉素抗性基因的同源重组片段敲除zntA基因。在单基因敲除菌株基础上,利用携带庆大霉素抗性基因的同源重组片段敲除zitB基因,获得一株敲除了zntA和zitB的双基因敲除菌株KZAB04。通过功能互补实验检测基因敲除菌株及对照菌株对不同浓度Zn²⁺的敏感程度。【结果】基因敲除菌株KZAB04比出发菌株E.coli DH5α具有更高的Zn²⁺敏感性。【结论】大肠杆菌Zn²⁺敏感突变株构建成功。该菌株的构建为zntA和zitB基因功能的研究提供了必要条件,同时也为其他Zn²⁺转运蛋白基因的功能鉴定与分析奠定了基础。     

基于谷氨酸消旋酶基因(murI)构建杀香鱼假单胞菌减毒活疫苗株的染色体-质粒平衡致死表达系统

【目的】构建一种基于谷氨酸消旋酶(MurI)基因的染色体-质粒平衡致死系统,用于杀香鱼假单胞菌减毒活疫苗株(Pseudomonas plecoglossicida ΔtssD-1, Pp ΔtssD-1)中表达外源抗原,为开发多联活疫苗提供新的思路和方法。【方法】利用同源重组技术,将亲本株Pp ΔtssD-1中的murI基因敲除,构建murI基因缺失突变株;将广宿主穿梭质粒pBBR1MCS-2的卡那霉素抗性基因替换为murI基因,构建平衡致死质粒(即无抗性回补质粒);在平衡致死质粒的多克隆位点处插入绿色荧光蛋白以检测外源抗原是否稳定表达,对重组菌株进行生物学特性分析,包括生长曲线、质粒稳定性和外源抗原表达水平。【结果】murI基因缺失株在不含D-谷氨酸的LB培养基上无法生长;无抗性回补株在不含D-谷氨酸的LB培养基上恢复了生长能力,但生长速度低于亲本株;经鉴定外源抗原可在无抗性质粒中稳定表达,并可在荧光显微镜下观察到明显的绿色荧光信号;此外,平衡致死质粒在重组菌株中具有良好的遗传稳定性。【结论】本研究以murI为靶点构建了新型的染色体-质粒平衡致死系统,可在无抗性筛选条件下在Pp ΔtssD-1中表达外源抗原,为开发多联活疫苗提供了新的策略和方法。     

Cre-LoxP系统在炭疽芽胞杆菌基因敲除中的应用及eag基因的敲除

将Cre-LoxP系统应用于Bacillus anthracis中并成功敲除eag基因．以B.anthracis基因组为模板扩增得到上下游同源臂,联合两端带有LoxP位点的壮观霉素抗性基因片段构建好同源重组载体,转化B．anthracis AP422,通过一系列筛选得到带有抗性标记的重组菌．然后,通过转入Cre重组酶表达质粒,去除抗性标记,得到eag基因缺失的重组菌,并在DNA水平、RNA水平和蛋白质水平进行了系统的鉴定．最终建立了Cre-LoxP系统在B．anthracis中的应用方法,并成功敲除eag基因.     

金黄色葡萄球菌中PVL基因与噬菌体DNA的同源性*

为了探讨金黄色葡萄球菌PVL基因与噬菌体的相关性 ,从 4株含有PVL基因的菌株中分离出DNA ,用HindⅢ或EcoRⅠ酶切后 ,分别与PVL和LukM-lukF-PV探针进行Southern印迹杂交 ,以及对含有PVL基因及其下游区域的片段克隆、测序和同源性分析。结果表明3株菌的PVL基因及其下游区域的序列与V8菌株噬菌体∮PVL的PVL基因及其下游噬菌体attsite的序列     

阿特拉津降解菌Arthrobacter sp. AG1降解基因研究

菌株Arthrobacter sp. AG1能以4000 mg/L的阿特拉津（AT）为唯一碳源、氮源和能源生长。通过设计特异引物从AG1中扩增出阿特拉津氯水解酶基因trzN的全序列,该基因与已报道的trzN基因序列相似性为99%。AG1菌株中含有两个大于100kb的质粒,Southern杂交结果显示trzN和atzB基因均位于其中较大的一个质粒pAG1上。将AG1菌株在LB液体培养基中转接三代后,发现34%的细菌细胞丢失了降解活性,但却未发现丢失质粒,PCR扩增结果表明突变子丢失了trzN基因,但atzB和atzC基因未丢失,说明降解活性的缺失是trzN基因片段从质粒上丢失的结果,表明trzN基因在环境中存在水平转移现象,暗示菌株AG1中的阿特拉津降解基因是基因的水平转移重组的结果。     

阿特拉津降解菌Arthrobacter sp. AG1降解基因研究

菌株Arthrobacter sp. AG1能以4000 mg/L的阿特拉津（AT）为唯一碳源、氮源和能源生长。通过设计特异引物从AG1中扩增出阿特拉津氯水解酶基因trzN的全序列,该基因与已报道的trzN基因序列相似性为99%。AG1菌株中含有两个大于100kb的质粒,Southern杂交结果显示trzN和atzB基因均位于其中较大的一个质粒pAG1上。将AG1菌株在LB液体培养基中转接三代后,发现34%的细菌细胞丢失了降解活性,但却未发现丢失质粒,PCR扩增结果表明突变子丢失了trzN基因,但atzB和atzC基因未丢失,说明降解活性的缺失是trzN基因片段从质粒上丢失的结果,表明trzN基因在环境中存在水平转移现象,暗示菌株AG1中的阿特拉津降解基因是基因的水平转移重组的结果。     

单增李斯特菌vip基因缺失

单增李斯特菌(Listeria monocytogenes)是广泛存在于自然界及食物中的食源性致病菌,作为胞内寄生菌,它可以引起强烈的细胞免疫,是潜在的优良疫苗载体。vip是单增李斯特菌的毒力基因,与其侵袭能力密切相关。因此构建vip基因敲除株可为单增李斯特菌疫苗载体的研发打下重要基础。从单增李斯特菌EGDe基因组中扩增出vip基因上、下游序列,连接到穿梭载体pKSV7中得到敲除载体pKSV7-Δvip,将其以电穿孔的方式转入单增李斯特菌后,通过同源重组利用氯霉素和温度双重压力筛选得到vip基因的敲除突变株,并对敲除菌株的生长曲线进行分析发现vip敲除对细菌的生长没有显著影响,为进一步研究vip基因功能、单增李斯特的致病机制和疫苗载体的研发提供参考。     

苏云金芽胞杆菌cry1Ac与烟草几丁质酶tchiB双价基因克隆表达及其杀虫增效作用研究

将苏云金芽胞杆菌(Bacillus thuringiensis,Bt)4.0718菌株质粒上的cry1Ac基因和烟草几丁质酶tchiB基因 (去掉信号肽或去信号肽再加肠激酶位点)构建了重组基因。经过双酶切和亚克隆,将带有cry1Ac基因上游启动序列和下游终止序列的重组基因片段克隆至穿梭载体pHT315,分别构建重组质粒pHUAccB6、pHUAccB7,在大肠杆菌中扩增后,将两个重组质粒分别电转化苏云金芽胞杆菌无晶体突变株XBU001中,获得重组菌株HAccB6和HAccB7。经液体双相胞晶分离提取离心后,将晶体和上清液分别进行SDS-PAGE分析,双价基因重组与cry1Ac基因在无晶体突变株中表达量相比较,几丁质酶活性提高5.2倍,双价重组蛋白表达量显著提高,主要产生130kDa蛋白条带。经定量分析：双价重组目的晶体蛋白占总蛋白量的61.38%;Cry1Ac蛋白占总蛋白量的42%。发酵上清液经60％硫酸铵沉淀,显示出一条分子量为18kDa新蛋白条带。经原子力显微镜和电子显微镜观察,表达后的重组蛋白呈菱形或椭原形晶体,其规格约为1.5×3.0μm;经生测分析,重组菌株HAccB6和 HAccB7毒力相近,与HAc菌株比较毒力提高4.5倍,对棉铃虫（Helicourpa armigora）具有高效杀虫活性,其3d LC₅₀值分别为9.1μg/mL和11.34μg/mL。研究结果表明,烟草几丁质酶与cry1Ac双价基因重组表达产物具有杀虫增效作用。     

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.     

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.     

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.     

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.     

The phylogenetic placement of Ostropales within Lecanoromycetes (Ascomycota) revisited

Results of molecular studies regarding the phylogenetic placement of the order Ostropales and related taxa within Lecanoromycetes were thus far inconclusive. Some analyses placed the order as sister to the rest of Lecanoromycetes, while others inferred a position nested within Lecanoromycetes. We assembled a data set of 101 species including sequences from nuLSU rDNA, mtSSU rDNA, and the nuclear protein-coding RPB1 for each species to examine the cause of incongruencies in previously published phylogenies. MP, minimum evolution, and Bayesian analyses were performed using the combined three-region data set and the single-gene data sets. The position of Ostropales nested in Lecanoromycetes is confirmed in all single-gene and concatenated analyses, and a placement as sister to the rest of Lecanoromycetes is significantly rejected using two independent methods of alternative topology testing. Acarosporales and related taxa (Acarosporaceae group) are basal in Lecanoromycetes. However, if the these basal taxa are excluded from the analyses, Ostropales appear to be sister to the rest of Lecanoromycetes, suggesting different ingroup rooting as the cause for deviating topologies in previously published phylogenies.     

Prosthecium species with Stegonsporium anamorphs on Acer

Data from microscopic morphology, single-spore cultures, and DNA analyses of teleomorphs and anamorphs support the recognition of five species of Prosthecium with Stegonsporium anamorphs on Acer: P. acerinum sp. nov., the teleomorph of S. acerinum; P. acerophilum comb. nov., formerly known as Dictyoporthe acerophila; P. galeatum comb. nov., originally described as Massaria galeata; P. opalus sp. nov.; and P. pyriforme sp. nov., the teleomorph of S. pyriforme s. str. The morphology of both type specimens and freshly collected material was investigated. The teleomorphs have brown ellipsoidal ascospores with five distosepta and often a longitudinal distoseptum. The anamorphs of all species described here belong to Stegonsporium; their connection to the Prosthecium teleomorphs was demonstrated by morphology and DNA sequences of single spore cultures derived from both ascospores and conidia. The anamorphs and teleomorphs of all five Prosthecium species are described and illustrated by LM images, and a key to these species is provided. As perceived from this work, S. pyriforme is restricted to Europe and does not occur in North America, whereas S. acerinum is restricted to North America, not found in Europe. The host associations given in the literature are revised and evidence is provided that only A. opalus, A. pseudoplatanus, and A. saccharum are confirmed hosts of Prosthecium with Stegonsporium anamorphs. Molecular phylogenetic analyses of tef1, ITS rDNA, and partial nuLSU rDNA sequences confirm that the species with Stegonsporium anamorphs are closely related to P. ellipsosporum, the generic type species. Stilbospora macrosperma is confirmed as the anamorph of P. ellipsosporum by DNA data of single spore isolates obtained from both ascospores and conidia.     

Nomenclatural changes resulting from the transfer of tropical African Sarcostemma to Cynanchum ( Apocynaceae : Asclepiadoideae )

Summary  Four species of tropical African Sarcostemma are transferred to Cynanchum together with two subspecies of S. viminale. In addition, Sarcostemma mulanjense is reduced to subspecific rank under C. viminale.     

Hydroxylation of steroids by Fusarium oxysporum, Exophiala jeanselmei and Ceratocystis paradoxa

The potential of Fusarium oxysporum var. cubense UAMH 9013 to perform steroid biotransformations was reinvestigated using single phase and pulse feed conditions. The following natural steroids served as substrates: dehydroepiandrosterone (1), pregnenolone (2), testosterone (3), progesterone (4), cortisone (5), prednisone (6), estrone (7) and sarsasapogenin (8). The results showed the possible presence of C-7 and C-15 hydroxylase enzymes. This hypothesis was explored using three synthetic androstanes: androstane-3,17-dione (9), androsta-4,6-diene-3,17-dione (10) and 3α,5α-cycloandrost-6-en-17-one (11). These fermentations of non-natural steroids showed that C-7 hydroxylation was as a result of that position being allylic. The evidence also pointed towards the presence of a C-15 hydroxylase enzyme.The eleven steroids were also fed to Exophialajeanselmei var. lecanii-corni UAMH 8783. The results showed that the fungus appears to have very active 5α and 14α-hydroxylase enzymes, and is also capable of carrying out allylic oxidations.Ceratocystis paradoxa UAMH 8784 was grown in the presence of the above-mentioned steroids. The results showed that monooxygenases which effect allylic hydroxylation and Baeyer–Villiger rearrangement were active. However, redox reactions predominated.     

转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响

【目的】为探究转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育及其捕食功能的影响。【方法】以转Cry1Ac/1Ab基因棉与其亲本常规棉为实验材料,利用取食不同棉花品种叶片的棉铃虫饲喂异色瓢虫幼虫。【结果】与常规亲本棉相比,取食饲喂转基因棉花叶片的初孵棉铃虫幼虫的异色瓢虫幼虫从1龄发育至化蛹期时间延长0.77 d,但差异不显著;除1龄幼虫体重增加(0.0773 mg)外,其余各龄期幼虫体重均有所下降,但差异均不显著;异色瓢虫1、2、3、4龄幼虫对初孵棉铃虫捕食量均随棉铃虫密度的增加而增加,捕食功能反应均符合HollingⅡ圆盘方程。【结论】转Cry1Ac/1Ab基因棉花对异色瓢虫生长发育无显著影响,饲喂取食转Cry1Ac/1Ab基因棉花的棉铃虫对异色瓢虫捕食功能无显著差异。