地衣芽孢杆菌β-1,3-1,4葡聚糖酶基因的克隆及序列分析

β-葡聚糖是以β-1,3和β-1,4混合糖苷键连接形成的D-葡萄糖聚合物,主要存在于单子叶禾本科谷实中.有些微生物会产生降解β-葡聚糖的β-1,3-1,4-葡聚糖酶.将β-葡聚糖酶基因在食品级宿主菌中表达对动物饲料工业具有十分重要的应用价值.实验以地衣芽孢杆菌为材料,提取其总DNA,扩增其β-1,3-1,4葡聚糖酶基因序列,将该片段克隆到pMD 19-T载体进行测序,并将测序结果进行BLAST比对.发现其与GenBank中的两段基因序列具有较高的同源性.     

菜豆几丁质酶、烟草葡聚糖酶基因的分离克隆及双价表达载体的构建*

 几丁质酶基因（Chitinase,Chi）和葡聚糖酶基因（β-1,3-glucanase,Glu）具有协同抗真菌作用,根据GenBank号M13968,X53600分别设计引物,经RT PCR扩增,克隆菜豆Chi基因和烟草Glu基因, 将两个目的基因分别连上CaMV 35S启动子和终止子Nos,然后串联在一起共同组建于一个表达载体pBI121中,重组表达质粒经过限制性酶切分析鉴定,结果表明含有双价抗真菌基因的植物重组表达载体已构建成功。为非洲菊、香石竹等花卉抗真菌基因工程育种奠定了基础。     

木聚糖酶和葡聚糖酶对肉鸡生长性能、屠宰性能、胴体成分及血液指标的影响

文章旨在研究玉米-小麦-豆粕型日粮添加木聚糖酶和葡聚糖酶对1～42 d肉鸡生长性能、屠宰性能、胴体成分及血液指标的影响。试验选择健康、体重一致的1日龄Cobb肉仔鸡800只,随机分为2组,每组5个重复,每个重复80只。对照组饲喂玉米-小麦-豆粕型日粮,试验组饲喂基础日粮添加2000 U/g木聚糖酶+800 U/g葡聚糖酶,试验分为3个阶段,1～14 d,15～28 d和29～42 d。与对照组相比,酶制剂组显著提高了28～42 d肉鸡能量和蛋白质利用率(P <0.05),显著降低了平均日采食量、日增重和料重比(P <0.05)。对于试验全期,酶制剂组显著提高了1～42 d肉鸡能量和粗蛋白质利用率(P <0.05),同时显著降低了平均日采食量、日增重和料重比(P <0.05)。酶制剂组较对照组显著提高了肉鸡的屠宰率和腿肌率(P <0.05)。对照组与酶制剂组对肉鸡胴体干物质、脂肪和灰分含量的影响均无显著差异(P> 0.05)。日粮添加复合酶较对照组显著提高了28和42 d肉鸡血液T3的含量(P <0.05),显著降低了42 d肉鸡血液T4的含量(P <0.05),同时酶制剂组较对照组显著提高了42 d肉鸡血液葡萄糖的浓度(P <0.05)。与对照组相比,酶制剂组显著降低了28和42 d肉鸡血液尿酸含量(P <0.05),显著提高了14和42 d肉鸡血液甘油三酯含量(P <0.05)。综上所述,1～42 d肉鸡日粮添加2000 U/g木聚糖酶和800 U/g葡聚糖酶可以显著改善料肉比,提高屠宰性能,可改变血液中甲状腺激素和部分代谢物的浓度,但对胴体成分影响不大。     

Characterization of an Unknown Region Linked to the Glycoside Hydrolase Family 17 β-1,3-Glucanase of Vibrio vulnificus Reveals a Novel Glucan-Binding Domain

The glycoside hydrolase family 17 β-1,3-glucanase of Vibrio vulnificus (VvGH17) has two unknown regions in the N- and C-termini. Here, we characterized these domains by preparing mutant enzymes. VvGH17 demonstrated hydrolytic activity of β-(1→3)-glucan, mainly producing laminaribiose, but not of β-(1→3)/β-(1→4)-glucan. The C-terminal-truncated mutants (ΔC466 and ΔC441) showed decreased activity, approximately one-third of that of the WT, and ΔC415 lost almost all activity. An analysis using affinity gel containing laminarin or barley β-glucan revealed a shift in the mobility of the ΔC466, ΔC441, and ΔC415 mutants compared to the WT. Tryptophan residues showed a strong affinity for carbohydrates. Three of four point-mutations of the tryptophan in the C-terminus (W472A, W499A, and W542A) showed a reduction in binding ability to laminarin and barley β-glucan. The C-terminus was predicted to have a β-sandwich structure, and three tryptophan residues (Trp472, Trp499, and Trp542) constituted a putative substrate-binding cave. Linker and substrate-binding functions were assigned to the C-terminus. The N-terminal-truncated mutants also showed decreased activity. The WT formed a trimer, while the N-terminal truncations formed monomers, indicating that the N-terminus contributed to the multimeric form of VvGH17. The results of this study are useful for understanding the structure and the function of GH17 β-1,3-glucanases.     

Characterization of an Extracellular Serine Protease of Fusarium eumartii and its Action on Pathogenesis Related Proteins

Proteases have been proposed as part of the invasion strategies of some pathogenic fungi. In this work, a serine protease produced by the phytopathogenic fungus Fusarium solani f.sp. eumartii was purified and characterized. Purification of the enzyme was accomplished by gel filtration through a Superose 12 column, followed by hydrophobic interaction chromatography in Phenyl Superose and gel filtration chromatography through Superdex 75. Analysis of the purified enzyme by SDS/PAGE without heat treatment, revealed a single band, which corresponded to the proteolytic activity detected by zymogram. When this protein was subjected to denaturing conditions, two major polypeptides of approximately 30 and 33kDa were revealed. The N-terminal amino acid sequence of one of these polypeptides showed a high similarity with fungal mature serine proteases of the subtilisin family. This protease hydrolysed in vitro, specific polypeptides of potato intercellular washing fluids and cell walls. The protease was also able to degrade pathogenesis-related proteins from the intercellular washing fluids. The role of this serine protease as part of the fungal strategy to colonize potato tuber tissues is discussed.     

添加复合酶制剂对香蕉叶青贮品质的影响

试验旨在研究添加复合酶对香蕉叶青贮品质的影响。试验设对照组和3个复合酶组调制香蕉叶青贮,每个处理设3次重复,常温下贮存60d开封,测定青贮的发酵品质和化学成分。结果表明,3个复合酶组的pH和气体损失率极显著或显著降低（P〈0.01,P〈0.05）,干物质回收率和水溶性碳水化合物极显著升高和增加（P〈0.01）;在干物质回收率上,复合酶2组和复合酶3组极显著高于复合酶1组（P〈0.01）;在水溶性碳水化合物上,复合酶3组显著高于复合酶1组和复合酶2组（P〈0.05）;在中性洗涤纤维上,复合酶3组显著低于复合酶2组（P〈0.05）。试验结果提示,复合添加纤维素酶、葡聚糖酶及木聚糖酶能显著提高香蕉叶青贮品质,并且随复合添加水平升高青贮品质进一步提高。     

β—1,3—葡聚糖酶基因植物表达载体的构建

采用ＰＣＲ的方法,对严自大麦ｃＤＮＡ克隆的β－１,３－葡聚糖酶基因进行了扩增,在此基因的５’及３’端分别加入了克隆所需的ＸｂａＩ和ＳｓｔＩ限性酶切位点,定向克隆到经改造的质粒载体ｐＢｉｎｈ中,获得了含有β－１,３－葡聚糖酶基因的植物表达载体ｐＢｉｎｈ－Ｇｌｕ。     

Rice transformation with cell wall degrading enzyme genes from Trichoderma atroviride and its effect on plant growth and resistance to fungal pathogens

Three genes encoding for fungal cell wall degrading enzymes (CWDE), ech42, nag70 and gluc78 from the biocontrol fungus Trichoderma atroviride were inserted into the binary vector pCAMBIA1305.2 singly and in all possible combinations. The coding sequences were placed downstream of the rice actin promoter and all vectors were used to transform rice plants. A total of more than 1,800 independently regenerated plantlets in seven different populations (for each of the three genes and each…     

Glucanohydrolase enzyme activity in embryos of Scots,Corsican and lodgepole pines infected in vitro with Heterobasidion annosum

Embryos of Scots pine (Pinus sylvestris), Corsican pine (P. nigra var. maritima) and Lodgepole pine (P. contorta) were infected in vitro with Heterobasidion annosum and the activities of 1,3-β-glucanase, chitinase and β-glucosidase were monitored. Both the infected and control embryos of the three pine species showed the presence of the enzymes. Statistical analysis showed no significant increase (P > 0.05) in the levels of 1,3-β-glucanase in infected embryos for all the three pine species. However, there was a significant increase in levels of chitinase in infected embryos of P. contorta and P. nigra var. maritima and also in levels of β-glucosidase in infected embryos of all three of the pine species. The study has therefore revealed that constitutive levels of glucanohydrolase enzymes exist in the embryos of the three pine species and that the induction of chitinase and β-glucosidase was a factor in the response of the embryos to stimulation by H. annosum infection.     

转几丁质酶和葡聚糖酶双价基因棉花株系对黄萎病的抗性

 采用人工病圃、重病田及苗期室内鉴定相结合的方法,研究了转双价（Chi+Glu）基因的棉花株系61217-1对棉花黄萎病的抗性。结果表明,转基因棉花株系61217-1在人工病圃连续3年达抗病水平,病指极显著低于其受体中棉所24。2009年该转基因株系在河南安阳、江苏大丰及新疆石河子棉花黄萎病重病田的病指分别为18.7、18.1和16.7,均极显著低于受体中棉所24的病指。该转基因株系对来自河北、湖北、新疆的3个强致病力落叶型菌株均达抗病水平,病指分别为16.4、16.8和15.6,也极显著低于受体中棉所24的病指。表明转双价（Chi+Glu）基因的棉花株系61217-1具有优异稳定的抗黄萎病性。