苏云金芽胞杆菌G03 spoIVF操纵子敲除对芽胞和晶体形成的影响

spoIVF是一个普遍存在于芽胞杆菌中的操纵子。在枯草芽胞杆菌中,它编码的两个蛋白是芽胞形成所必需的。采用基因重组技术敲除了苏云金芽胞杆菌G03菌株中的spoIVF操纵子,构建了spoIVF缺失株G03(spoIVF-)。研究表明:该突变株丧失了形成芽胞和晶体的能力。lacZ基因与cry1Aa基因的启动子融合表达分析发现:突变株中的cry1Aa基因的活性严重降低。利用载体pSTK携带spoIVF操纵子在突变株中的表达,使突变株部分恢复了产胞和形成杀虫晶体蛋白的能力。这说明spoIVF操纵子是所必需的,同时该操纵子还影响σ^E因子控制的cry1Aa基因表达。     

苏云金芽胞杆菌cry26Aa和cry28Aa基因共表达可在芽胞外壁内侧形成伴胞晶体

苏云金芽胞杆菌幕虫亚种的伴胞晶体在预芽胞外壁内侧形成,呈现晶体芽胞粘连的现象。根据已发表的cry26Aa1和cry28Aa1基因序列设计引物,从苏云金芽胞杆菌幕虫亚种T02中扩增得到cry26Aa和cry28Aa基因,通过穿梭载体将这两个基因分别和同时转化到苏云金芽胞杆菌无晶体突变株BMB171后,透射电镜下可在芽胞外壁内侧和外侧同时观察到伴胞晶体,而单独表达时可在芽胞外壁外侧观察到伴胞晶体。结果表明,伴胞晶体在芽胞外壁内侧表达不单独依赖于启动子的时空调控,可能还受到晶体蛋白相互作用的影响。     

苏云金芽胞杆菌cry26Aa和cry28Aa基因共表达可在芽胞外壁内侧形成伴胞晶体

苏云金芽胞杆菌幕虫亚种的伴胞晶体在预芽胞外壁内侧形成,呈现晶体芽胞粘连的现象。根据已发表的cry26Aa1和cry28Aa1基因序列设计引物,从苏云金芽胞杆菌幕虫亚种T02中扩增得到cry26Aa和cry28Aa基因,通过穿梭载体将这两个基因分别和同时转化到苏云金芽胞杆菌无晶体突变株BMB171后,透射电镜下可在芽胞外壁内侧和外侧同时观察到伴胞晶体,而单独表达时可在芽胞外壁外侧观察到伴胞晶体。结果表明,伴胞晶体在芽胞外壁内侧表达不单独依赖于启动子的时空调控,可能还受到晶体蛋白相互作用的影响。     

苏云金芽胞杆菌spoⅢD基因缺失突变株的特点

微镜观察和芽胞计数分析显示,突变株基本丧失了形成芽胞的能力,但依然形成晶体.SDS-PAGE结果显示,在SSM培养基中,突变株对伴胞晶体蛋白的形成量影响并不显著;在营养较富集的Luria-Bertani培养基中,突变株中伴胞晶体蛋白的形成量较野生型和互补株明显降低.利用载体pHT315携带spoⅢD操纵子互补突变株,互补株恢复了产生晶体和芽胞的能力.[结论]本研究证明spoⅢD基因是苏云金芽胞杆菌芽胞形成所必需,同时与晶体蛋白的表达相关.     

苏云金芽胞杆菌spoIIID基因缺失突变株的特点

摘要：【目的】构建苏云金芽胞杆菌spoIIID基因缺失突变株,并研究其与出发菌株的表型及性质差异。【方法】采用基因同源重组技术敲除了苏云金芽胞杆菌HD-73菌株中的spoIIID基因,构建了spoIIID缺失突变株,测定生长曲线,并通过扫描电子显微镜观察,芽胞计数分析及SDS-PAGE 蛋白电泳比较突变株与出发菌株的差异。构建遗传互补菌株,观察菌株性状的回复情况。【结果】通过温敏载体同源重组敲除技术获得了苏云金芽胞杆菌HD-73菌株spoIIID基因缺失突变株,生长曲线测定表明,突变株较出发菌株在平稳期后期生长较缓和;扫描电子显微镜观察和芽胞计数分析显示,突变株基本丧失了形成芽胞的能力,但依然形成晶体。SDS-PAGE结果显示,在 SSM培养基中,突变株对伴胞晶体蛋白的形成量影响并不显著;在营养较富集的Luria-Bertani培养基中,突变株中伴胞晶体蛋白的形成量较野生型和互补株明显降低。利用载体pHT315携带spoIIID操纵子互补突变株,互补株恢复了产生晶体和芽胞的能力。【结论】本研究证明spoIIID基因是苏云金芽胞杆菌芽胞形成所必需,同时与晶体蛋白的表达相关。     

苏云金芽胞杆菌HD-73菌株sigE基因敲除突变株的构建与特点

SigE因子是芽胞杆菌芽胞形成过程中起重要作用的sigma因子,它控制着众多芽胞形成相关基因的表达.在苏云金芽胞杆菌中,Cryl等杀虫晶体蛋白的表达也受SigE因子的控制.本研究利用同源重组技术构建了苏云金芽胞杆菌库斯塔克亚种标准菌株HD-73 sigE缺失突变株.研究表明:突变株衰亡期提前,丧失了形成芽胞和晶体的能力:cryIAa指导的β-半乳糖苷酶活性分析表明sigE基因对crylAa基因的转录有较大影响.利用载体pHT315携带sigE所在操纵子spoIIG及其启动子序列在突变株中表达,使突变株恢复了产生芽胞和形成杀虫晶体的能力,生长基本恢复正常,这些结果表明SigE因子是Bt库斯塔克亚种菌株产生芽胞和形成晶体所必需的.     

苏云金芽胞杆菌幕虫亚种晶胞粘连现象与晶体蛋白基因cry26Aa所在质粒有关

苏云金芽胞杆菌幕虫亚种T02菌株的伴胞晶体在芽胞外壁内侧形成,呈现晶胞粘连的现象。在此菌株中克隆了cry26 Aa和cry28 Aa两个基因,并对晶胞粘连现象与质粒的相关性做了系统研究。通过消除幕虫亚种T02菌株的质粒,得到了仅消除cry26 Aa所在质粒的菌株BMB1151和无质粒的菌株BMB1152。通过穿梭载体将cry26 Aa和cry28 Aa两个基因分别和同时转化无质粒突变株BMB1152并表达,形成的晶体与芽胞独立存在不能粘连,表明在幕虫亚种染色体背景下仅仅cry的表达不能形成晶胞粘连现象,从而推断晶胞粘连现象可能与幕虫亚种两个基因所在的质粒有关;进一步的研究发现将cry26 Aa在仅消除cry26 Aa所在质粒的突变株BMB1151中表达,形成的晶体与芽胞也分别独立存在不能粘连,从而进一步推断幕虫亚种晶胞粘连现象与cry26 Aa所在质粒有关。     

苏云金芽胞杆菌sigK 基因插入失活突变体的特点及其对cry3A基因启动子活性的影响

摘要: 【目的】构建苏云金芽胞杆菌(Bacillus thuringiensis,简称Bt) sigK 基因插入失活突变体,分析突变体特点并明确其对cry3A 基因启动子的影响。【方法】采用同源重组技术在苏云金芽胞杆菌HD-73 菌株sigK 基因中插入卡那霉素抗性基因,构建了sigK 基因插入失活突变体。通过生长曲线测定、扫描电子显微镜观察晶体、芽胞形成情况和芽胞计数及SDS-PAGE 等方法分析了突变体的特点; 构建了遗传恢复菌株对上述性状进行了功能验证; 利用启动子融合lacZ 技术检测了cry3A 基因启动子的转录活性。【结果】获得了苏云金芽胞杆菌HD-73 菌株sigK 基因突变体,生长曲线测定表明,突变体较出发菌株在稳定期后期生长较慢; 扫描电子显微镜观察和芽胞计数分析显示,突变体丧失了形成芽胞和晶体的能力; SDS-PAGE 分析表明突变体中伴胞晶体蛋白的表达量明显低于出发菌株和恢复菌株。利用载体pHT315 携带sigK 基因及其启动子在突变株中表达,所获得的遗传恢复菌株恢复了突变株产生芽胞和晶体的能力; sigK 基因的突变可以提高cry3A 基因启动子在产胞后期的转录活性,对cry3A 启动子指导的Cry 蛋白表达量没有显著影响。【结论】本研究证明sigK 基因为苏云金芽胞杆菌芽胞形成所必需,并影响伴胞晶体蛋白的产量; sigK 基因功能的丧失有利于cry3A 基因启动子在产胞后期的转录。     

带cry3Aa启动子的aiiA基因在苏云金芽胞杆菌中的表达

N 乙酰高丝氨酸内酯 (N acyl homoserinelactones,AHLs) ,是一类数量感知 (Quorum sensing)系统中的信号分子 ,它参与诱导调控许多植物病原菌致病基因的表达。苏云金芽胞杆菌的AiiA蛋白能降解这类AHLs分子 ,进而可减弱病原菌致病基因表达产生的病害。苏云金芽胞杆菌杀虫晶体蛋白基因cry3Aa的启动子是一种不依赖芽胞形成的启动子 ,它相对于其它cry类基因的启动子有启动基因转录时间早 ,转录时间长的优点。通过重叠延伸PCR ,用杀虫晶体蛋白基因cry3Aa启动子替换编码AiiA蛋白的基因aiiA自身的启动子 ,构建了融合基因pro3A aiiA。将融合基因装入穿梭载体pHT3 0 4的BamHI SphI位点 ,得到重组质粒pBMB686并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株BMB686的AiiA蛋白表达量在各个生长时期均高于对照菌株 ,对AHLs分子的降解活性和对胡萝卜软腐欧文氏菌感染马铃薯产生病害的抑制能力也明显优于对照菌株     

苏云金芽胞杆菌杀线虫伴胞晶体蛋白基因cry6Aa的克隆与表达

通过对晶体蛋白N-末端氨基酸测序,设计简并探针,从对根结线虫高毒力苏云金芽胞杆菌YBT-1518菌株中克隆到1个含有杀线虫晶体蛋白基因的片段。序列测定表明该序列含有两个ORF(orf1和orf2),其中orf1与基因cry6Aa1同源性为98%,已在GenBank上登录(Acc.NO.AF499736),并被命名为cry6Aa2。将克隆的该片段克隆到穿梭载体pHT304上,并转化苏云金芽胞杆菌无晶体突变株BMB171,重组菌株可形成米粒状伴胞晶体。生物测定表明,表达的毒素蛋白对北方根结线虫的LC50为9.47μg/mL,毒力与出发菌株(10.74μg/mL)相当。     

Characteristics of the membranes of the pellicle of the ciliatePseudomicrothorax dubius

Summary The membranes of the pellicle of the ciliatePseudomicrothorax dubius are investigated using thin section electron microscopy and freeze-fracture replicas. The plasma membrane is covered by a surface coat and is connected to the outer alveolar membrane by short, sometimes branched, bridges. The inner alveolar membrane is coated on both sides. The epiplasm lies in intimate contact with the cytoplasmic surface of this membrane, and there is a corresponding deposit on the other surface. This deposit is regularly striated.The epiplasmic layer and the alveoli are interrupted at sites of cytotic activity,e.g., the attachment sites of trichocysts, the cytoproct, and the parasomal sacs. The striated deposit ends where the epiplasm ends, indicating a direct relationship between these two epimembranous layers.There is a deposit along the sides of the first part of the tip of the trichocysts, and in this region the trichocyst membrane is free of intramembranous particles.The membrane of the parasomal sacs has a coat on both surfaces. That on the extraplasmic surface is similar to the surface coat of the plasma membrane. The origin of the cytoplasmic coat is unknown. The cytotic activity of these sacs is indicated by their highly irregular profiles.     

The early development of the tail and the transformation of the shape of the nucleus of the spermatid of the domestic fowl,Gallus gallus

Summary The differentiation of the spermatid, especially in reference to the formation of the flagellum, and transformation of the shape of the nucleus was investigated in the domestic fowl.In the early stage of the spermatid, a prominent Golgi apparatus appears around the centrioles. The Golgi vesicles then surround the axial-filament complex which develops from the distal centriole. These vesicles fuse to form continuous membrane at the earliest stage of flagellar formation, and in the succeeding stage Golgi lamellae are attached to the plasma membrane of the developing flagellum. From these observations, it is assumed that Golgi apparatus may be a source of the membrane system of the flagellum.The microtubules distributed around the nucleus form the circular manchette. The anterior region of the nucleus with the manchette is cylindrical in shape and the posterior region without it remains irregular in shape. When the circular manchette has been completed, the whole nucleus acquires a slender cylindrical shape. The circular manchette then changes into the longitudinal manchette. The nuclei of spermatids without a longitudinal manchette are abnormal in shape. In view of these observations it is assumed that the nuclear shaping of the spermatid may be accomplished by circular manchette and the maintenance of shape of the elongated nucleus by longitudinal manchette.The authors wish to thank Mr. Takayuki Mori for his helpful suggestions and technical advices     

Characterization of the composition of the aqueous humor and the vitreous body of the eye of the frog Rana temporaria L

This study aimed to analyze the aqueous humor (AH) and the vitreous body (VB) of the eye of the adult frog Rana temporaria L. as a representative species of amphibians, which lead a semi-terrestrial life. The presence of collagen, albumin, uric acid and electron donors was shown in both media; however, there are slight differences in their concentrations. To determine collagen, a spectral-fluorescent probe, cyanine dye, was used. The presence of collagen in AH of the frog was found at the first time. The total content of electron donors (ascorbic and uric acids, tryptophan, and tyrosine) in VB and HA was roughly estimated at ~ 1.5 × 10^− 4 mol/L. Both VB and AH absorb light in similar UV regions. The total protein and albumin contents in AH were found to be somewhat higher than those in VB. The uric acid content was at an equally low level in both intraocular media. It is supposed that the similarity of VB and AH compositions shown in this work is due to some exchange between VB and AH contents in the course of accommodation. The role of intraocular fluids in physiological functions of the eye and in protecting the retina against UV light is discussed.     

Observations on the permeability of the choriocapillaris of the eye

Summary The choriocapillaris is a fenestrated capillary bed located posterior to the retinal pigment epithelium. It serves as the main source of supply to the photoreceptors, retinal pigment epithelium, and other cells of the outer retina. The permeability of these capillaries to intravenously injected ferritin (MW — approx. 480,000; mol. diam. 11 nm) was examined in the mouse, rabbit, and guinea pig, each of which is characterized by a different type of retinal vascularization. In all three species, the bulk of the ferritin remained in the capillary lumina, where it appeared to be blocked at the level of the diaphragmed fenestrae. Some ferritin was present in endothelial cell vacuoles. The results confirm previous work on the rat choriocapillaris and indicate that the barrier function of the choriocapillary endothelium is present even among species in which the retinal circulation differs significantly.Supported by NIH grant EY03418