表达载体pCAGGS显著增强禽流感DNA疫苗的免疫保护效果

【目的】将A/Goose/GuangDong/1/96(H5N1)[GD/1/96(H5N1)]的HA基因插入鸡β-actin启动子高效真核表达载体pCAGGS，构建了DNA疫苗质粒pCAGGHA5，以提高H5亚型禽流感DNA疫苗的表达水平和免疫保护效果。【方法】将pCAGGHA5和表达GD/1/96(H5N1)HA基因的质粒pCIHA5通过间接免疫荧光法和Western-blot分析检测转染293T细胞后瞬时表达的HA抗原蛋白，随之将pCAGGHA5及pCIHA5分别以100 ?g和10 ?g剂量一次免疫3周龄SPF鸡，4周后用100 LD50的HPAIV GD/1/96(H5N1)鼻腔途径进行攻击。【结果】间接免疫荧光法和Western-blot分析表明2种表达质粒均可正确表达H5亚型HA抗原蛋白，载体pCAGGS表达水平显著高于载体pCI；免疫SPF鸡后，100?g pCAGGHA5可形成5/5的免疫保护，100?g pCIHA5可形成2/4的免疫保护，10?g pCAGGHA5可形成对免疫鸡5/5的免疫保护，而10?g pCIHA5 则基本不能形成免疫保护，pCAGGHA5诱导的HI抗体水平远远高于pCIHA5。【结论】鸡β-actin启动子表达载体pCAGGS可显著提高HA基因体外表达水平和H5亚型禽流感DNA疫苗诱导的保护性抗体免疫反应水平，增强免疫保护效果。     

BABA诱导烟草幼苗抵御干旱胁迫的研究

β-氨基丁酸(BABA)是一种高效的植物诱抗剂,能够增强植物对多种胁迫的抵御能力。通过施加外源BABA来减轻干旱胁迫对烟草幼苗的毒害,并从渗透调节物质,抗氧化酶系统来探讨其作用机理。结果表明,在干旱胁迫下,5 mmol·L-1 BABA可以有效地改善烟草幼苗生长状况,提高其相对含水量及存活率。BABA预处理不仅显著提高干旱胁迫下烟草幼苗渗透调节物质可溶性糖、可溶性蛋白和脯氨酸含量,还增强烟草幼苗超氧化物酶(SOD)和过氧化物酶(POD)活性。此外,BABA预处理提高干旱胁迫下烟草幼苗过氧化氢(H2O2)含量,降低丙二醛(MDA)含量。综上,BABA诱导烟草幼苗抵御干旱胁迫是诱导植物渗透调节物质累积及抗氧化酶活性调节的综合结果。     

硅钨酸负载介孔分子筛SBA-15的制备及催化性能研究

[目的]探讨负载型介孔分子筛SiW12/SBA-15的制备及催化性能。[方法]采用水热合成法,以P123为模板剂制备介孔材料SBA-15,由硅钨酸浸渍进一步合成介孔催化剂SiW12/SBA-15。通过IR、TG、SEM和TEM进行表征,并且将负载型介孔分子筛SiW12/SBA-15用于催化α-蒎烯的环氧化反应。研究硅钨酸负载量、催化剂加入量、过氧乙酸与原料摩尔比及SBA-15的焙烧温度对环氧化产物得率的影响。[结果]将硅钨酸负载到SBA-15上并未破坏介孔分子筛的结构,硅钨酸仍保持着Keggin的基本结构。在550℃下焙烧SBA-15,负载量为30%,催化剂用量为2%,CH3COOOH与α-蒎烯的摩尔比为1.2∶1时,α-蒎烯的转化率为99.4%,产物2,3-环氧蒎烷的得率为90.45%。[结论]通过负载法能够制备负载型介孔分子筛SiW12/SBA-15。该催化剂对α-蒎烯的环氧化反应有良好的催化效果。     

NLRP3/ASC/caspase-1/IL-1β/IL-18轴在大鼠脓毒症急性肾损伤中的作用

[目的]观察盲肠结扎穿孔(Cecal ligation and puncture,CLP)所致的脓毒症诱导急性肾损伤(AKI)后大鼠不同时间点的血清及肾组织中NLRP3/ASC/caspase-1/IL-1β/IL-18的表达情况。[方法]建立大鼠CLP诱导的脓毒症AKI模型,并于手术后不同时间点用ELISA试剂盒方法测定血清尿素氮(BUN)、血清肌酐(Scr)水平及IL-1β/IL-18的表达量;采用Western blotting法检测肾脏组织NLRP3/ASC/caspase-1/IL-1β/IL-18蛋白水平,并对肾脏组织进行HE染色病理分析。[结果]CLP诱导的脓毒症大鼠血清BUN和Scr水平显著增高,肾小管坏死也显著增加,均于CLP术后24 h达到高峰,与假手术组大鼠相比差异显著(P0.05或P0.01);大鼠肾脏组织中NLRP3/ASC/caspase-1/IL-1β/IL-18及血清IL-1β/IL-18水平也随着损伤程度的加重表现出升高趋势,与假手术组相比差异均达到显著水平。[结论]大鼠脓毒症AKI后肾组织或血清中NLRP3/ASC/caspase-1/IL-1β/IL-18蛋白水平表达增加,表明NLRP3炎症小体的激活可能对脓毒症AKI及其病理改变有促进作用。     

深绿木霉SS003产胞壁降解酶发酵条件的响应面优化

【目的】优化深绿木霉（Trichoderma atroviride）SS003菌株在真菌细胞壁诱导下产β-葡聚糖酶和几丁质酶的培养条件,为其生防机理研究奠定基础。【方法】设置不同培养温度（24、26、28、30、32、34℃）、培养时间（24、48、72、96、120、144、168 h）和初始接种量（3~9块菌块）分别进行单因素诱导SS003菌株产胞壁降解酶发酵条件优化试验;根据单因素优化结果,利用Design-Expert 8.05b中的Box-Behnken中心组合设计原理,对培养温度、培养时间、初始接种量对SS003菌株产酶培养条件进行响应面分析优化。【结果】SS003菌株在诱导培养下产β-葡聚糖酶的最优培养条件为：培养时间73.70 h、培养温度27.82℃、初始接种量5.19块菌块;几丁质酶的最优培养条件为：培养时间82.91 h、培养温度27.70℃、初始接种量5.03块菌块。【结论】响应面法可用于深绿木霉SS003菌株产胞壁降解酶优化条件的筛选,能有效提高深绿木霉SS003产β-葡聚糖酶和几丁质酶的产酶量。     

An ABAGE-Like Metabolite of Botrytis cinerea Isolate BC4 Inhibited the Growth of Hypocotyls and Roots of Amaranthus retroflexus Seedlings

A metabolite, which had an inhibitory effect on plant growth, was isolated from cultural filtrate of Botrytis cinerea isolate BC4 by column chromatography on silica gel and preparative HPLC. Its structure was determined from HPLC-ESI MS, GC, IR, ^1H NMR and ^13C NMR spectral data, as well as chemical hydrolysis. The results showed that this metabolite was quite similar to abscisic acid-β-D-glucopyranosyl ester（ABAGE） in structure. The inhibitory effect of the ABAGE-like metabolite on plant growth was investigated using a weed Amaranthus retroflexus L. as a bioassay material. The results showed that it inhibited hypocotyls and roots growth of A. retroflexus （Amaranthus retroflexus L.） seedlings when its concentration was over 0.5 μM. The concentrations for 50% inhibition of hypocotyls and roots growth of A. retroflexus seedlings were 2.8 and 1.4 μM, respectively.     

不同类型枇杷果实着色期间果肉类胡萝卜素含量的变化

 【目的】分析不同类型枇杷果实着色过程中果肉类胡萝卜素的变化特点，探讨果肉外观颜色差异形成的原因。【方法】以‘大红袍’、‘青种’分别代表红沙和白沙两类不同枇杷品种，自果实开始着色起，分期采样，进行枇杷果肉类胡萝卜素提取、纯化和HPLC分析。【结果】果实进入着色期后，果肉叶黄质含量迅速下降，至果实成熟时下降平缓，而α-胡萝卜素、β-胡萝卜素、β-隐黄质和玉米黄素成分均呈上升趋势，尤其在采收前7～14 d上升迅速。果实成熟后，‘大红袍’果肉的类胡萝卜素总量为‘青种’的２倍以上；两品种果肉叶黄素类色素含量接近，但类胡萝卜素含量差异较大，‘大红袍’ 的胡萝卜素含量是 ‘青种’的4倍左右。【结论】胡萝卜素积累的多少，尤其是β-胡萝卜素含量的高低是造成‘青种’和‘大红袍’果肉类胡萝卜素总量积累差异的主要原因，也是影响这两个品种果肉色泽深浅的主要因素。     

黑曲霉突变株ZM-8产纤维素酶条件的研究

经筛选的黑曲霉突变株ZM-8是一株高产纤维素酶菌株,其β-葡萄糖苷酶活性特别高.采用正交试验对其产酶条件进行了优化,结果表明其最适培养基配方为:氮源(NH4)3PO4,含氮量0.6%,含水量300%;玉米秸秆粉与麸皮质量比为4∶1,接种量1∶30.最佳培养条件为:培养温度为35℃,培养时间为72 h,初始pH值为6.5.在优化后的培养条件下测定纤维素FPU酶、C1酶、CMC酶和β-葡萄糖苷酶的活力分别为5.25、0.48、19.60U/mL和42.86 U/mL,约为优化前各酶活的3.0倍、1.8倍、2.5倍和2.1倍.     

美国红鱼和大黄鱼β-珠蛋白基因的克隆和遗传进化分析

根据已发表的其他脊椎动物β-珠蛋白基因序列设计并合成1对特异性引物,通过RT-PCR方法首次克隆到美国红鱼和大黄鱼β-珠蛋白cDNA,其开放读码框均由447个碱基组成,编码148个氨基酸的多肽,预测的相对分子量分别为16 333和16 148 Da,两者核苷酸序列和推导的氨基酸序列同源性分别为90.4%与81.2%.将推导的美国红鱼和大黄鱼β-珠蛋白氨基酸序列与其他鱼类及人β-珠蛋白氨基酸序列进行同源性比较,其同源性在35.5%～70.3%,它们与同属真口鱼纲的真鲷、黄尾鲱鱼、鲤鱼、虹鳟等的同源性较高,而与肉鳍鱼纲的肺鱼及板鳃纲的鲨鱼的同源性较低;二级结构预测表明,美国红鱼和大黄鱼β-珠蛋白存在类似哺乳动物的8个α-螺旋区,在近端与远端与血红素结合的组氨酸高度保守.     

Molecular characterization of benzimidazole-resistant B. cinerea field isolates with reduced or enhanced sensitivity to zoxamide and diethofencarb

Sensitivity profiles of Botrytis cinerea field isolates to zoxamide and the molecular basis of the resistance mechanism involved in cross-resistance relationships between benzamides, benzimidazoles and N-phenylcarbamates were investigated. B. cinerea isolates collected from southern, central and northern Greece were characterized based on their sensitivity to zoxamide, the benzimidazole carbendazim and the N-phenylcarbamate diethofencarb. Isolates exhibiting baseline sensitivity to carbendazim and zoxamide but no sensitivity to diethofencarb were considered wild type (S phenotype) and accounted for 44% of the total strains sampled. Thirty-three percent of the isolates had increased sensitivity (HS phenotype) to zoxamide and diethofencarb and were highly resistant to carbendazim compared to S isolates. Eight percent of the sample was highly resistant (HR phenotype) to all anti-tubulin agents studied. The rest of the isolates were moderately resistant to zoxamide (MR phenotype) and equally sensitive to benzimidazoles and N-phenylcarbamates compared to isolates of the S phenotype. Fungitoxicity tests with botrycides belonging to other chemical classes revealed no cross-resistance relationships between zoxamide and the phenylpyrrole fludioxonil, the dicarboximide iprodione, the hydroxyanilide fenhexamid, the anilinopyrimidine cyprodinil, the carboxamide boscalid and the strobilurin-type fungicide pyraclostrobin. Study of fitness characteristics did not show any significant difference between zoxamide resistant and sensitive isolates with respect to the parameters tested. PCR-RFLP analysis of a part of the β-tubulin gene sequence detected mutations in position 198 for both HS and HR zoxamide-sensitivity phenotypes. DNA sequence analysis of the B. cinerea β-tubulin gene revealed two previously described benzimidazole-resistance-conferring mutations. The first one was the glutamic acid (GAG) to alanine (GCG) change at position 198 (E198A), which was identified in all HS isolates. The second mutation (E198K) was a GAG-to-AAG substitution resulting in the replacement of glutamic acid with lysine present in all B. cinerea isolates highly resistant to all three anti-tubulin classes of fungicides. A number of mutations in other positions of the β-tubulin gene were detected in the moderately zoxamide-resistance phenotype.