斑石鲷TGF-β1基因克隆和表达分析

虹彩病毒是造成斑石鲷(Oplegnathus punctatus)工厂化养殖中大规模死亡的主要病原,其感染力极强,感染后的斑石鲷死亡率高,严重影响了斑石鲷养殖产业发展。转化生长因子TGF-β1是一种重要的免疫调节因子,在病毒免疫应答中发挥重要作用。为研究TGF-β1在斑石鲷被虹彩病毒感染过程中发挥的作用,运用RACE和实时荧光定量qRT-PCR技术对TGF-β1进行了基因克隆,并对其进行在不同组织、不同时间点相对表达量的差异分析。结果显示,斑石鲷TGF-β1基因c DNA序列全长为3157 bp,5’非编码区长712 bp,3’非编码区长1278 bp,开放性阅读框长1167 bp,编码388个氨基酸,基因组包含6个外显子和5个内含子。同源分析发现,TGF-β1和鱼类相似度较高,与半滑舌鳎(Cynoglossus semilaevis)的同源性最高,为76.67%。TGF-β1在斑石鲷健康组织(肝脏、脾脏、肾脏、头肾、心脏、鳃、胃、肠和皮肤)中均有表达,在头肾、肠、肝脏和皮肤组织中表达量较高,而在脾脏和肾脏组织表达量较低。为进一步研究TGF-β1在病毒感染过程中相对表达量的变化,对健康斑石鲷注射虹彩病毒进行刺激,随后比较了TGF-β1在脾脏、肝脏、肾脏、头肾4个不同组织、不同时间点的相对表达量的差异,在头肾、脾脏和肝脏中,病毒刺激后TGF-β1的表达量均出现升高,但在脾脏和肝脏中,峰值出现在刺激后第4天,而在头肾中峰值出现在感染后的第10天。在肾脏中,病毒刺激后的TGF-β1的表达呈现下降趋势,0 d表达量最高,4、7 d依次降低,7 d降至最低,10 d有所恢复。以上研究表明,TGF-β1可能响应了虹彩病毒对机体的刺激,可能在对病毒免疫应答中发挥作用。而病毒感染后不同组织中TGF-β1相对表达量的差异,则值得进一步研究。     

Assessment of phosphorus bioavailability in cultivated Andisols from a long-term fertilization field experiment using chemical extractions and soil enzyme activities

Various chemical extraction methods have been used to evaluate soil phosphorus (P) availability in different ways, and therefore inconsistent results are often obtained. This study examined the usefulness of the resource allocation model for extracellular enzymes for evaluating P availability in soils from a more biological perspective. Potential P availability was evaluated using the Truog and Bray-2 tests, the Hedley sequential extraction procedure, and enzyme activity assessments in cultivated Andisols from a 70-year-old fertilization experiment. Both the ratio of acid phosphatase to β-D-glucosidase activities and the ratio of alkaline phosphatase to β-D-glucosidase activities showed significant negative correlations with potentially available inorganic P, suggesting that microorganisms preferentially expended resources in the form of phosphatase production rather than β-D-glucosidase production to acquire P. Additionally, crop P content had a significant negative relationship to the ratio of alkaline phosphatase to β-D-glucosidase activities. These results suggest that the resource allocation model for extracellular enzymes is useful for evaluating P availability in Andisols.     

Cadmium accumulation and distribution in cucumber plant

An experiment developed in soilless culture was used to study the cadmium (Cd) accumulation, and distribution of Cd in cucumber (Cucumis sativus var. peonero‐mixfl) plant. Four treatments were established (0, 5, 10, and 20 mg Cd⁺² L^‐1). Uptake, and transport of Cd were increased with time, and Cd concentration in the nutrient solution. Fruit accumulation of Cd varied from 16 to 92 mg kg^‐1 depending on the treatments. The fresh weight, and dry matter accumulation of cucumber plant organs (roots, stem, leaves, and fruits) was affected by cadmium treatment. A decrease of the total, a, and b chlorophyll increasing Cd concentration in nutrient solution, and time of experiment were observed. The incidence of this metal on the content of chlorophyll b seem to be faster than chlorophyll a. Cucumber plant could be a feasible plant for pollution experiments due to their high sensibility, and transport efficiency.     

Ramifications of crop residue loading for soil microbial community composition,activity and nutrient supply

Variable results have been reported on the effects of crop residue loads on soil microbial properties. We investigated changes in soil bacterial composition, β-glucosidase enzyme activity and nutrient bioavailability in response to wheat residue loading. The treatments included three levels of above-ground wheat residues (removed, retained or supplemented), with or without fertilizer N. Bacteroidetes, Firmicutes and Verrucomicrobia (the first two are copiotrophs) were less abundant where residues were removed than where residues were retained or supplemented, but the reverse was true for Actinobacteria, Cyanobacteria, Chloroflexi and Nitrospirae (all oligotrophs, although some Actinobacteria can be copiotrophic). Actinobacteria were also less abundant where fertilizer N was applied, and the abundances of their genera (including Arthrobacter and Mycobacterium) increased where residues were removed, confirming that they were oligotrophic in this study. β-diversity showed similar differences in the bacterial community structures because of residue management, but α-diversity was not affected by residue management or N fertilizer. β-glucosidase enzyme activities increased as C inputs increased with residue manipulation and N fertilizer. The enzyme activities increased with increasing residue loading in the 0–15 cm soil depth, but decreased with soil depth. Soil K supply increased with increasing residue loading, but nitrate-N supply was highest with residue retention. These results demonstrate remarkable resilience of soil microbial functioning under a wide range of crop residue inputs, without adverse effects on enzyme activity attributable to inorganic N fertilizer. The increasing β-glucosidase activity with increasing residue loading probably explains why crop residue return does not always increase soil C stocks.     

2种辅料对苹果蠹蛾性信息素含量测定的影响

[目的]研究淀粉、β-环糊精(β-CD)2种辅料对苹果蠹蛾性信息素含量测定的影响.[方法]采用气相色谱法,检测淀粉、β-CD以及分别采用淀粉、β-CD制成的粉剂和颗粒剂中苹果蠹蛾性信息素的含量.[结果]混合物中苹果蠹蛾性信息素的提取回收率为99.64％～ 100.02％;粉剂和颗粒剂中苹果蠹蛾性信息素的平均回收率为99.95％ ～ 100.00％.[结论]β-CD和淀粉均不影响苹果蠹蛾性信息素的含量测定,相容性较好,可进一步进行剂型研究.     

控制pH值和溶解氧对地衣芽孢杆菌HDYM-04分批发酵产β-甘露聚糖酶的影响

为了探究地衣芽孢杆菌产β-甘露聚糖酶的能力,探究在发酵过程中pH值和溶氧水平对产β-甘露聚糖酶的影响。以地衣芽孢杆菌(Bacillus licheniformis) HDYM-04为试验菌株,通过3,5-二硝基水杨酸比色法（DNS法）测定酶活力。结果表明,控制pH值至7.0直至发酵结束,在发酵的初始阶段(12~20 h)效果明显,酶活力水平均处于3200 U/mL以上,同时菌体密度达到5.5以上。其次,控制溶氧在25%左右时,保持菌体的生长状态,酶活力高峰持续时间较长（6~20 h保持在3000 U/mL左右）。因此控制pH值7.0,溶氧在25%左右时,在发酵的初始阶段,有良好的产酶能力。     

β-葡聚糖及其在猪营养中的应用研究

β-葡聚糖是广泛分布于真菌、细菌和植物中的一种功能性多糖,具有调节机体免疫、抗感染、调节血糖等多种生物活性和功能,在饲料逐步禁用抗生素的前提下,日益引起人们的关注。文章在介绍β-葡聚糖来源、结构和理化性质的基础上,重点论述了该多糖激活免疫细胞、调控与免疫反应相关的细胞信号传递、保护和修复胰岛β细胞、改善胰岛素抵抗和糖代谢等机理,以及β-葡聚糖在猪生产中的应用,并对其研究方向和应用前景做了展望。     

Extraction and purification of an endo-1,4-β-xylanase from wheat malt

Wheat and wheat malt are known to be high quality raw materials in the beer brewing process. The size and structure of wheat arabinoxylans (AXs) and their enzymatic degradation products have a profound impact on beer properties, such as viscosity, turbidity, filtration rate and foaming properties. AXs are of great benefit to human health due to their anti-tumor properties and potential roles in the immune system. Endo-1,4-β-xylanase is a key enzyme for degradation of AXs and production of small molecule AXs, thereby altering the properties of AXs, such as solubility, viscosity, and turbidity. In this study, extraction and purification of an endo-1,4-β-xylanase from wheat malt was described. The results showed that, after 40%–60% ammonium sulfate precipitation, Q-Sepharose Fast Flow anion exchange chromatography, Phenyl-Sepharose 6 Fast Flow Hydrophobic chromatography, and twice sephacryl S-100 HR gel filtration, a final endo-1,4-β-xylanase was obtained. The purified wheat malt endo-1,4-β-xylanase showed a single band on SDS-PAGE with a molecular weight of 27.8 kDa, which had a purification fold of 12.08 and a specific activity of 4.47 μ/mg.     

鸡β2-微球蛋白的基因克隆与原核表达

本研究通过RT-PCR从正常鸡外周血淋巴细胞总RNA中扩增chβ2m 基因全长片段，然后将其克隆至原核表达载体pET-32a（＋），转化大肠杆菌BL21（DE3），用IPTG对其进行诱导表达，利用HiTrap亲和柱对表达产物进行纯化。结果显示，采用RT-PCR扩增出chβ2m基因全长约360 bp，编码120个氨基酸，与基因库公布的相一致，同源性为100%；经0.8 mM IPTG诱导表达后，SDS-PAGE分析发现chβ2m融合蛋白主要以可溶性形式存在于细菌裂解上清；利用His标签纯化该蛋白， SDS-PAGE分析仅见约34 kDa大小的chβ2m融合蛋白条带；Western-blot分析表明，纯化后的chβ2m融合蛋白与特异单克隆抗体反应呈现特异性的条带。以上结果证实，本研究成功表达并获得了纯化的可溶性chβ2m融合蛋白，为进一步对chβ2m结构及其功能研究奠定基础。     

Muscovy duck retinoic acid-induced gene I (MdRIG-I) functions in innate immunity against H9N2 avian influenza viruses (AIV) infections

Retinoic acid inducible gene I (RIG-I) is a cytosolic pattern recognition receptor that senses pathogen-associated molecular patterns (PAMPs). Muscovy duck (Cairina moschata) is a large duck different from other species of ducks, and is more susceptible to some microbial pathogens. In this study, the Muscovy duck RIG-I gene (MdRIG-I) was identified. Quantitative RT-PCR showed that MdRIG-I mRNA was widely expressed in different tissues, especially in those with mucosa. RIG-I null DF-1 cells transfected with DNA constructs encoding MdRIG-I or CARDs domain can activate IRF-3 and NF-κB to up-regulated activity of IFN-β promoter. The components of the signaling pathway downstream of RIG-I in mammalian cells including IRF-3, NF-κB, IFN-β and the IFN-stimulated genes Mx-1, PKR and MDA5 were significantly up-regulated in CARDs-overexpressing-DF-1 cells. Implicating RIG-I in the antiviral response to an infection in vivo, we found that RIG-I expression in brain, spleen, lung and bursa were up-regulated in ducks challenged with H9N2 avian influenza virus (AIV), whose six internal genes were closely related to the H7N9 and H10N8 AIV. In vitro, DF-1 cells transfected with MdRIG-I plasmid can respond significantly to H9N2 AIV, evident through enhancement of IFN-β promoter activity and decreased virus titer. Altogether, these results indicated that MdRIG-I is a novel member of RLR gene family, engaging in the early stage of antiviral innate immunity.