嗜热子囊菌光孢变种cbh1基因的cDNA克隆及在毕赤酵母的高效表达

以嗜热子囊菌光孢变种(Thermoascus aurantiacus var. levisporus)总RNA为模板，通过RT-PCR克隆出外切纤维二糖水解酶基因cbh1片段，采用RACE方法获得全长cDNA克隆，其全长为1 710 bp，编码一种由457个氨基酸组成的单肽，推导的氨基酸序列中1～19位为信号肽序列，GenBank的登录号为AY840982。将该片段克隆到毕赤酵母(Pichia pastoris)分泌型表达载体pPIC9K上，获得表达重组质粒pPIC9K/cbh1，转化毕赤酵母GS115，所得重组子经PCR验证后进行诱导表达，筛选出一重组子GSp-15,经144 h诱导后，外切纤维二糖水解酶表达量为1.17 mg/mL，产酶活力为20.3 U/mL。     

Enzymatic Hydrolysis of Cellulose with Different Crystallinities Studied by Means of SEC-MALLS

The reactions of exo-cellulase （cellobiohydrolase, CBH） and endo-cellulase （endoglucanase, EG） were investigated by analyzing the insoluble residues of microcrystalline cellulose （MCC） and filter paper...     

Phenological Stage, Plant Biomass, and Drought Stress Affect Microbial Biomass and Enzyme Activities in the Rhizosphere of Enteropogon macrostachyus

Indigenous grasses have been effectively used to rehabilitate degraded African drylands. Despite their success, studies examining their effects on soil bioindicators such as microbial biomass carbon(C) and enzyme activities are scarce. This study elucidates the effects of drought stress and phenological stages of a typical indigenous African grass, Enteropogon macrostachyus, on microbial biomass and enzyme activities(β-glucosidase, cellobiohydrolase, and chitinase) in the rhizosphere soil. Enteropogon macrostachyus was grown under controlled conditions. Drought stress(partial watering) was simulated during the last 10 d of plant growth, and data were compared with those from optimum moisture conditions. The rhizosphere soil was sampled after 40 d(seedling stage), 70 d(elongation stage), and 80 d(simulated drought stress). A high root:shoot ratio at seedling stage compared with elongation and reproduction stages demonstrated that E. macrostachyus invested more on root biomass in early development, to maximise the uptake of nutrients and water. Microbial biomass and enzyme activities increased with root biomass during plant growth. Ten-day drought at reproduction stage increased the microbial biomass and enzyme activities, accompanying a decrease in binding affinity and catalytic efficiency. In conclusion, drought stress controls soil organic matter decomposition and nutrient mobilization, as well as the competition between plant and microorganisms for nutrient uptake.     

红壤丘陵区旱地和水旱轮作地土壤中纤维素降解功能微生物群落特征

为揭示农田土壤有机质中纤维素降解的微生物机制,依托红壤丘陵区长期定位试验,以两种土地利用方式(旱地和水旱轮作地)下两种施肥模式(化肥、秸秆还田配施化肥)的农田生态系统为研究对象,分析了表层土壤中纤维素含量、纤维二糖水解酶活性以及纤维素降解功能微生物丰度与群落结构的周年动态变化特征。结果表明:长期(13年)施肥后土壤中纤维素并未发生显著积累,且从周年动态变化来看,秸秆还田后旱地和水旱轮作地中纤维素分别在6个月和3个月内完全降解或被转化为其他形态;相关分析表明,纤维二糖水解酶活性与纤维素含量呈显著正相关,而真菌cbh I基因丰度与纤维二糖水解酶呈显著正相关(P0.01),因此功能基因cbh I可用于指示本研究供试土壤中降解纤维素的关键微生物群;聚类分析表明,旱地和水旱轮作地的纤维素降解微生物(含cbh I基因)互相分离,即与施肥相比,土地利用方式是引起土壤中纤维素降解微生物群落组成改变最主要的因素;克隆测序结果显示,两种土地利用方式下纤维素降解功能微生物均以伞菌和粪壳菌占绝对优势,分别占总克隆库的22.9%~39.5%(平均为34.7%)和17.7%~42.3%(平均为28.5%),其中秸秆还田后的纤维素降解过程可能由粪壳菌主导。研究结果阐明了红壤丘陵区旱地和水旱轮作地中秸秆还田后纤维素降解及其功能微生物群落的异同,为揭示农田土壤新鲜有机质中易分解组分(纤维素)的微生物转化机制提供了基础数据。