茶树几丁质酶基因的克隆及其在干旱胁迫下的表达分析在干旱胁迫下的表达分析

以铁观音茶树叶片为材料,利用逆转录PCR及RACE法,克隆了茶树几丁质酶基因CsChi(GenBank登录号为KR078345).CsChi基因的cDNA全长为1 192 bp,包含972 bp的开放阅读框(ORF),编码323个氨基酸.生物信息学分析结果表明,CsChi蛋白的分子量为34.33 ku;理论等电点pI为8.44;原子组成为C1519H2285N413O464S18,总原子数为4 699;蛋白质结构分析显示该蛋白有6个蛋白的跨膜区域,属于跨膜蛋白;存在于细胞外;没有卷曲螺旋结构存在;CsChi基因编码的蛋白属于糖苷水解酶19家族,含有保守的ChtBD1结构域,与溶菌酶的保守结构域类似,可能兼具几丁质酶活性和溶菌酶活性,qPCR定量分析结果显示在不同干旱胁迫处理下茶树的CsChi基因的表达量,与对照组相比有所增加.推测CsChi基因在茶树干旱等逆境胁迫中起重要作用.     

斑节对虾Chitinase-2基因的克隆及其在蜕皮和幼体发育过程中的表达分析

研究以斑节对虾(Penaeus monodon)转录组获得的几丁质酶基因(Pm Chi)片段,运用RACE技术克隆了PmChi基因c DNA全长,命名为PmChi-2。PmChi-2基因c DNA全长2 050 bp,其中5'-非编码区(5'-UTR)144 bp、3'-UTR 319 bp和开放阅读框(ORF)1 587 bp,编码528个氨基酸。生物信息学分析显示,PmChi-2与其他甲壳动物Chi-2的相似性为78%~97%。实时荧光定量PCR结果显示,PmChi-2在蜕皮前期(D期)表皮中表达水平最高,在胃、鳃、腹神经节和眼柄中表达水平依次降低,在其他组织(肝胰腺、肠、肌肉、心脏)中几乎不表达。不同蜕皮阶段,PmChi-2在鳃、胃和表皮3种组织中的表达变化模式基本一致,均在蜕皮期(E期)表达量最低,而最高表达量在鳃中出现在蜕皮后期(A期),在胃和表皮中为D期。幼体不同发育阶段分析揭示PmChi-2 mRNA在幼体发育阶段的无节幼体到糠虾幼体第二期表达量维持在一个低水平,在糠虾幼体第三期PmChi-2 mRNA表达水平显著升高,在仔虾期又显著下降,推测PmChi-2 mRNA可能与斑节对虾幼体发育密切相关。研究结果表明PmChi-2基因可能在斑节对虾蜕皮以及幼体的变态发育中发挥重要作用,为深入研究斑节对虾几丁质酶发育调控提供了重要信息。     

产甲壳素酶菌株的筛选与诱变

以胶体甲壳素作为惟一碳源,从自然界微生物中筛选并诱导到一株产甲壳素酶活性较高的菌株,经16S r DNA鉴定为Aeromonas sp.。并以该菌株作为出发菌株,经过一次紫外诱变,酶活是初筛菌株的1.48倍。再经过二次紫外诱变或者紫外-Li Cl复合诱变,酶活分别是初筛菌株的3.16和3.87倍,最高酶活达到3.48 U/m L。最后通过连续传代培养,确定了菌株HMX-16经过紫外和复合诱变后突变性状稳定性良好。     

The activity and expression of chitinase in the equine lung and its activity in normal horses and animals with recurrent airway obstruction

Recurrent airway obstruction (RAO) is a chronic inflammatory condition in equine lung, which may share a common immunological basis with human asthma, in which dysregulated Th2 responses occur. Mammals express chitinases and chitinase-like proteins, two of which are active enzymes, chitotriosidase and acidic mammalian chitinase (AMCase). Both enzymes are upregulated in a range of inflammatory conditions, including asthma. We investigated the activity of chitinase in bronchoalveolar lavage fluid from horses with and without RAO in response to organic dust challenges. No significant differences were found in activity, although in one study RAO animals had elevated chitinase activity that fell short of statistical significance. The pH optimum and pH lability of the activity was consistent with the presence of chitotriosidase. RT-PCR amplification of the mRNA encoding chitotriosidase and AMCase in normal equine lung showed that chitotriosidase, but not AMCase, is expressed in trachea, bronchi, and peripheral lung tissue. The gene for chitotriosidase was identified from the Equus caballus (horse) genome 1.1 database and its similarity to the same genes from other species was determined. The results of this study indicate that the involvement of chitotriosidase in RAO is uncertain.     

根结线虫卵寄生真菌蜡蚧菌(Lecanicillium lecanii)产生的几丁质酶活性

卵寄生真菌产生的几丁质酶在卵孵化和寄生过程中发挥重要的作用,也是评价寄生线虫真菌生物防治潜力重要的生化指标之一。利用NAG法和pNP法分别测定蜡蚧菌(Lecanicillium lecanii)CFCC84957和CFCC80920菌系的几丁质降解酶系和外切酶活性,CFCC84957菌系接种4～6d含有0.2%胶体几丁质的培养滤液后,几丁质降解酶系到达活性高峰期,而CFCC80920菌系的培养滤液未检测到几丁质降解酶系。2个菌系的培养滤液从第2d开始均能检测到几丁质外切酶活性,CFCC84957和CFCC80920活性出现的最高峰分别在第12d和6d,但是CF-CC84957的活性值约是CFCC80920的4倍(分别为0.51μmol·h-1·ml-1、0.12μmol·h-1·ml-1)。含有0.01%糖基化几丁质的几丁质酶活性电泳检测到1条分子量为48.2kD的蛋白质谱带。     

南美白对虾几丁质酶的分离纯化

采用先“盐析”后“亲和吸附”的方法分离虾几丁质酶,纯化倍数为15.3倍,比活率73.3ImU·mg-1,产品得率50.6％;亲和分离胶为商品化的脱乙酰几丁质经再生后制备而成。方法简单易行适于放大生产。     

油菜几丁质酶的特点及其与抗菌核病的关系

通过测定甘蓝型油菜西农长角几丁质酶对底物的特异性等性质,结果表明,几丁质酶以脱乙酰化几丁质7B（乙酰化程度为30％）为底物时活性最高,而对8B,9B,10B（乙酰化程度为20％,10％,0％）和菌核菌细胞壁的活性较低。几丁质酶活性的最适宜反应温度为30℃-40℃中,并在25℃以下时活性大大降低,这与菌核菌生长和入侵的最适温度15℃-25℃不一致。西农长角的种子中几丁质酶的活性很强,发芽后逐渐下降,到第5天时开始提高。研究还发现,不同类型和抗性油菜材料的几丁质酶活性和对菌核菌的抗性间存在相关关系,相关系数达显著水平,这表明几丁质酶在提高油菜的抗菌核病能力起着重要的作用。     

Mycoparasitism of Gliocladium isolates in China Mycoparasitism of Gliocladium isolates in China

During an investigation of mycoparasitic fungi on sclerotia of Sclerotinia sclerotiorum in China, Gliocladium species was consistently encountered and isolated from natural soils collected over the country. Colonization frequencies on sclerotia of S. sclerotiorum by the fungi ranged from 40% to 100% when the sclerotia were introduced into soils and coincubated at 22-24 ℃ for 4 weeks. Identification showed that G. roseum, G. virens and G. catenulatum were the dominant species amon…     

植物几丁质酶及其应用研究进展

几丁质酶是高等植物普遍存在的一种重要的病程相关蛋白,近年来被广泛应用于植物抗病基因工程的研究.本文概述了国内外有关植物几丁质酶的研究情况,介绍了几丁质酶的特性、结构、分类、细胞学定位和功能及几丁质酶基因的克隆与其在植物基因工程中的应用.     

盾壳霉几丁质酶和葡聚糖酶酶动力学性质初步研究

盾壳霉几丁质酶和葡聚糖酶酶动力学性质研究表明:几丁质酶和葡聚糖酶的酶促反应,在本试验计量范围内,其反应速度与酶量和底物浓度均呈正相关,但两种酶促反应适宜的温度和pH值有较大差。几丁质酶降解几丁质的最适反应温度为50℃,最适pH值为8.0,葡聚糖酶降解葡聚糖的最适反应温度为60℃,最适pH值为3.0。