CRISPR/cas系统及其在家禽上的应用研究进展

CRISPR/cas是一种获得性免疫防御系统,广泛存在于细菌和古细菌中。Strep tococcus pyogenes cas9(Spcas9)作为目前研究最多、最清楚的效应蛋白,因其酶活性高和靶点广而被应用于生命科学各个领域,但Spcas9分子量大、脱靶率高等缺点在一定程度上限制了其应用。随着CRISPR系统技术被深入开发,一些新效应蛋白被发现,本文就CRISPR/cas系统分类、原理、新效应蛋白发现及cas9(S.Pyogenes)多种变构体技术开发在及家禽上的应用进行综述,为相关领域的研究提供参考。     

Development of a semi-selective medium and an immunofluorescence colony-staining procedure for the detection of Clavibacter michiganensis subsp. sepedonicus in cattle manure slurry

Various compounds and basal media were tested for their suitability to create a semi-selective medium for isolation ofClavibacter michiganensis subsp.sepedonicus (Cms) from cattle manure slurry containing c. 10⁸ colony forming units (cfu) per ml.Plating efficiency of Cms in yeast glucose mineral medium (YGM) was 104% compared with yeast peptone glucose medium. Nalidixic acid, polymyxin B sulphate and the experimental disinfectant S-0208 inhibited colony growth of cattle slurry bacteria as compared with Cms in YGM. The optimal concentration of these inhibitors in combination was determined by modified agar diffusion tests and by pour plating in 24-well tissue culture plates. The semi-selective medium YGMI consisted of YGM supplemented with nalidixic acid (2 mg/l), polymyxin B sulphate (30 mg/l) and S-0208 (125 mg/l). Plating efficiency varied for Cms between 50.9 and 69.6%, for cattle slurry bacteria between 1.8 and 2.5% and for saprophytes from potato heel end extracts between 11.5 and 27.4%.Differentiation of Cms colonies from other colonies was based on their small and bluish colony morphology in pour plates and on immunofluorescence colony-staining (IFC). IFC of a pure culture of micro colonies of Cms in YGM was possible after one day incubation (colonies c. 5 cells). Green background fluorescence in the agar gels was prevented by addition of Tween 20 (0.1%) to the washing buffer and the use of 1% agar gels. IFC of macro colonies of Cms in YGMI, visible with 4x objective magnification, was possible after 4 days. The detection level of the target organism in artificially inoculated cattle slurry in YGMI based on colony morphology varied between 1.4×10³ and 2.3×10⁴ cfu per ml of cattle slurry. Miniaturized plating combined with IFC, using wells in tissue culture plates (=16 mm), proved suitable for detection, but was c. 30 times les sensitive. The recovery of Cms was negatively correlated with the number of saprophytic colonies in the agar plates (R ²=0.74).     

Effects of sodium ferulate on the nonenzymatic glycation and oxidation in kidneys of diabetic rats

AIM: To investigate the blocking effects of sodium ferulate (SF) on the nonenzymatic glycation and oxidation in kidneys of diabetic rats. METHODS: The diabetic rats induced by streptozotocin (STZ) were treated with SF (110 mg/kg) per day for 8 weeks. The renal weight/body weight, clearance rate of creatinine (Ccr), proteinuria, advanced glycation end products (AGEs) in renal cortex, and the malonyldialdehyde (MDA), fructosamine (FMN), antioxidant enzymes in serum and renal cortex were measured. The pathologic changes of kidneys were also observed. RESULTS: The levels of Ccr, proteinuria, FMN and AGEs in renal cortex, FMN in serum, and renal weight/body weight in diabetic control group were significantly higher than those in normal control group. In the diabetic control group, there was a highter level of MDA and a lower activity of superoxide dismutase (SOD) and catalase (CAT) in serum and renal cortex than those in normal control group. Except for FMN in renal cortex, the abnormalities were significantly ameliorated in the treatment group. The pathologic changes was significant in the diabetic control group, which was significantly ameliorated with the treatment of SF. CONCLUSIONS: SF protects the activity of antioxidant enzymes, clears away oxygen radicals and inhibits the deposit of AGEs in kindey, which may be the mechanisms of protecting effects of SF on kindey in diabetic rats.     

自动折叠喷洒臂的设计及运动学特性

为了解决当前卷盘式喷灌机所配套的喷灌车自动化水平较低的问题,设计了一种基于电动缸驱动的自动折叠喷洒臂,并在喷洒臂之间连接处设计了一种当喷洒臂完全展开时能够实现自密封的端面密封铰链.基于喷洒臂完全折叠时收拢于车架两侧,完全伸展时铰链左右铰合密封等特征建立各杆件参数约束方程,确定了喷洒臂的各杆件尺寸.通过矢量方程法对喷洒臂进行运动学分析,以电动杆推杆的转角、位移、速度、加速度为输入量,各杆件的角位移、角速度、角加速度为输出量,通过Simulink搭建喷洒臂的运动仿真框图并进行分析.结果表明:完全折叠时,喷管1与水平方向呈90°夹角;伸展过程中,各喷管运动平稳,且始终保持在水平直线方向;完全展开时,3个喷管转动90°,且各喷管铰接处完全闭合不发生水泄漏现象,说明设计具有一定的合理性.     

五轴数控加工叶片无干涉刀位轨迹的计算

根据微分几何特性，提出五坐标数控加工叶片时对叶片的工作面和背面的刀位轨迹计算分开予以讨论，对工作面而言，利用平头立铣刀加工时不存在干涉，而对背面而言，可能存在干涉。根据干涉的种类，分别对全局干涉和局部刀具干涉提出了快速判定依据，并对干涉点进行了修正。指出利用Iso-scallop法可获得最优的轨迹和蔗，并给出了实例。     

Effect of advanced glycation end products on inflammation in cultured cardiomyocytes

AIM: To investigate the effect of advanced glycation end products on inflammation in cultured cardiomyocytes.METHODS: Primary cardiomyocytes were isolated from Sprague-Dawley neonatal (1 to 2 days old) rats ventricles.The insulin resistant cardiomyocyte model was established.Neonatal rat ventricular myocytes were exposed to AGEs for 24 hours.TNF-α mRNA and PPAR-γ mRNA expressions were determined by RT-PCR.Activation of NF-κB in the cells was examined by using immunocytochemistry.The ultrastructure of the cells was detected by transmission electron microscope.RESULTS: The exprssion of TNF-α mRNA and the activation of NF-κB increased,the expression of PPAR-γ mRNA decreased compared with control group (P<0.05).The differences among different AGE-BSA groups were significant (P<0.05).The numbers of chondriosome and smooth endoplasmic reticulum increased.CONCLUSION: AGEs significantly increase TNF-α mRNA expression and NF-κB activation,and restrain the expression of PPAR-γ mRNA.These data suggest that AGEs play an important role in the onset of diabetic cardiomyopathy.     

路桥过渡段施工问题研究

在路桥过渡段施工时，会因基础不同、防水措施不足、施工质量等问题产生显著的沉降差异，从而引起桥头跳车现象。路桥过渡段的施工，应做好施工勘察设计工作、处理好软性地基、选好台后填筑材料、做好台背排水、压实填土，减少台后路基的沉降，提高路基的刚度，尽力实现路桥刚柔平稳过渡，达到防治桥台跳车的目的。     

利用地径建立林木材积估测模型的探讨

通过实测铁杉、红桦和湖北花楸的地径、胸径、树高和材积，我们建立了根据林木地径估测树木材积的数学模型，具有较高的相关系数，可以应用于森林资源管理和森林生态系统研究。     

PCR法克隆紫穗槐4-香豆酸:CoA连接酶全长cDNA

4 香豆酸 :CoA连接酶 (4CL)是木质素生物合成过程中重要的酶 .该文利用简并寡核苷酸PCR法结合cDNA末端快速扩增PCR法直接获得了紫穗槐 4CLAcDNA全长序列 ,避免了复杂的构建、筛选cDNA文库的过程 .先用简并PCR得到了 4CLA1片段 ,又据此片段设计反向嵌套引物 ,用RACE方法获得未知的 5′和 3′端序列 .所获得的全长 4CLAcDNA ,编码 5 40个氨基酸 .氨基酸序列同源性分析表明4CLA是典型的 4CL蛋白 ,含有预计的AMP binding位点、催化反应区和保守的Cys .     

Tensile strength of softwood butt end joints. Part 1: Effect of grain angle on adhesive bond strength

To study the effect of grain angle on the adhesive bond strength in wood, three-part Norway spruce wood specimens were bonded and tested in tension. The two axially orientated outer parts of the specimens were joined with the middle part by means of three adhesives typically used for load-bearing constructions, i.e. one-component polyurethane (PUR), melamine-urea-formaldehyde (MUF) and phenol-resorcinol-formaldehyde (PRF). The grain angle of the middle part was varied from 0° (end grain to end grain) to 90° (flat grain to end grain) in incremental steps of 10°. In general, PRF- and MUF-bonded samples exhibited highest tensile strength at end grain to end grain orientation of the three parts, while specimens bonded with PUR showed only 25% of the strength measured for PRF and MUF, respectively. At high grain angles (90°) all specimens showed similar strength values in the range of 30% of maximum strength of MUF- and PRF-bonded specimens. To explain the changing strength levels at different grain angle a composite failure criterion was applied.