林下植被和凋落物对寒温带森林生长季土壤CH4通量的影响

为进一步探究林下植被和凋落物管理对我国寒温带森林生长季土壤CH₄通量的影响,采用静态箱-气相色谱法对大兴安岭北部4种林型（白桦林、山杨林、樟子松林和兴安落叶松林）4种处理（自然状态、去除凋落物、去除林下植被以及去除林下植被和凋落物）的土壤CH₄通量排放特征进行观测研究。结果表明：该地区森林生长季土壤均表现为CH₄的汇,4种林型不同处理后土壤CH₄通量表现为单峰变化趋势,吸收峰值出现在7月或8月。自然状态4种林型土壤CH₄平均吸收通量表现为白桦林（-79.23±14.92）μg m^-2 h^-1>山杨林（-64.27±9.60）μg m^-2 h^-1>樟子松林（-62.54±15.48）μg m^-2 h^-1>兴安落叶松林（-48.73±12.26）μg m^-2 h^-1,兴安落叶松土壤CH₄平均吸收通量显著小于其他三种林型（P<0.05）。相比于自然状态,4种林型在去除凋落物后土壤CH₄吸收通量提高了2.12%-12.15%,但变化幅度均没有达到显著水平（P>0.05）。去除林下植被后4种林型CH₄吸收通量提高了0.84%-20.55%,且只有山杨林吸收增加达到显著水平（P<0.05）。同时去除林下植被和凋落物后,对白桦林和樟子松土壤CH₄通量影响不显著（P>0.05）,但对山杨林和兴安落叶松林影响显著（P<0.05）。总之,去除凋落物或林下植被均会提高土壤对CH₄吸收,去除林下植被对土壤CH₄通量的影响要大于去除凋落物的影响,但不同林型不同处理之间还存在差异。     

竹茶混交模式对表层土壤有机碳储量及组分的影响

为探究毛竹林下种植茶树对土壤有机碳储量与碳组分的影响,该研究以毛竹纯林、竹茶混交林和常绿阔叶林为研究对象,采集这3种林分类型的表层(0～10 cm)土壤,测定土壤有机碳(SOC)、碳组分、生物与非生物因素指标。结果表明:(1)竹茶混交林林下植物多样性相较于毛竹纯林显著降低,但其土壤有机碳密度(22.54±2.09)t·hm^-2、碳组分与毛竹纯林无显著差异(P>0.05)。竹茶混交林的矿物结合态有机碳(MOC)为(20.13±1.83)g·kg^-1,占总有机碳的92.66%。常绿阔叶林土壤有机碳密度比竹茶混交林和毛竹纯林高土壤有机碳密度分别高41.15%和41.00%(P<0.05)。(2)3种林分类型土壤微生物量碳(MBC)含量范围为0.58～3.08 g·kg^-1,土壤16S rRNA丰度范围为2.18×10¹⁰ ～5.65×10¹⁰copies·g^-1,固碳基因cbbL丰度范围为0.37×10⁸～ 1.10 ×10⁸ copies·g^-1,土壤微生物碳利用效率范围为0.03～0.28; 3种林分类型之间微生物相关指标不存在显著差异(P>0.05)。(3)3种林分类型SOC与土壤pH、砂粒含量和地上凋落物生物量呈显著负相关,与土壤黏粒含量、粉粒含量、总氮、C:N、总磷和铵态氮含量呈显著正相关(P<0.05)。(4)就不同碳组分而言,颗粒有机碳(POC)和MOC均与土壤pH、砂粒含量和根系生物量呈显著负相关,与土壤含水量、黏粒含量、粉粒含量、总氮、C:N、总磷和铵态氮含量呈显著正相关(P<0.05)。综上表明,竹茶混交改造会造成原生毛竹纯林林下植被多样性下降,但并未造成土壤碳储量下降; 而相较于常绿阔叶林,毛竹经营措施需要改进,以提升其碳汇效益。     

金属离子抑制细菌整合子捕获耐药基因盒的机制研究

【目的】探索金属离子对整合子捕获耐药基因盒的影响及其相关机制。【方法】在大肠埃希菌中构建一个1类整合子捕获耐药基因盒的体内模型。通过实时荧光定量PCR (real-time fluorescence quantitative PCR,RT-qPCR)测定不同浓度银(0.3、0.9、1.5 μg/mL的Ag⁺)和铜离子(5、50、100、150、210 μg/mL的Cu²⁺)干预后实验组和无金属离子干预对照组整合子整合频率,并用表型筛选法验证。利用质谱法测量细菌吸收的金属离子浓度,并进一步通过转录组测序方法分析银离子抑制细菌整合子捕获耐药基因盒的分子机制。【结果】qPCR和表型筛选法的结果表明,0.9 μg/mL银离子组整合频率为9.42×10^‒5 (6.49×10^‒5,1.44×10^‒4),1.5 μg/mL银离子组整合频率为7.29×10^‒5 (4.45×10^‒5,9.03×10^‒5),与对照组2.59×10^‒4 (2.24×10^‒4,3.33×10^‒4)相比整合频率明显降低,差异有统计学意义(P<0.001)。而不同浓度铜离子组与对照组没有明显差异。转录组测序方法对银离子作用前后大肠杆菌基因表达水平进行对比分析,通过GO功能和KEGG通路富集发现,差异表达基因与甲基半乳糖苷转运(methylgalactoside transport)、麦芽糖转运(maltose transport)、磷酸烯醇式丙酮酸-甘油磷酸转移酶活性(phosphoenolpyruvate-glycerone phosphotransferase activity)和甘油激酶活性(glycerone kinase activity)等功能有关以及涉及氨基酸糖和核苷酸糖代谢(amino sugar and nucleotide sugar metabolism)、鞭毛组装(flagellar assembly)、阳离子抗菌肽(CAMP)耐药性[cationicantimicrobial peptide (CAMP) resistance]、果糖和甘露糖代谢和磷酸糖转移酶系统[phosphotransferase system (PTS)]等代谢通路。通过蛋白互作网络筛选出前12个枢纽基因(ptsG、malE、lamB、lacZ、malK、basR、ais、ugd、nagE、metN、malQ和malF)。【结论】一定浓度银离子可以抑制整合子整合耐药基因盒。铜离子对整合频率影响不明显,因此不是所有具有杀菌性的金属离子都能对整合频率产生影响。银离子抑制细菌整合子捕获耐药基因盒可能是通过影响麦芽糖转运和碳分解代谢来调控。然而,碳分解代谢和麦芽糖转运过程很复杂,需要进一步研究。目前尚无细菌整合子转录组学相关研究,这为解决细菌耐药性问题提供了一个新的途径。     

圈养林麝粪样类固醇激素水平与麝香分泌的关系

基于非损伤取样和放射免疫法（RIA）,于2018年9月1日-10月15日期间对四川马尔康林麝繁育场的36头雄性圈养林麝（Moschus berezovskii）的粪样类固醇激素水平进行了检测,结合麝香分泌数据采集,探究了林麝的粪样睾酮和皮质醇水平与其麝香分泌的关系,结果表明：四川马尔康圈养林麝的麝香分泌（12.53±0.76）g（n=36）与粪样睾酮水平（106.03±12.34）ng/g（n=36）呈显著正相关（r=0.436,P < 0.01）,泌香较多雄麝（16.50±0.61）g（n=17）的睾酮水平（141.51±21.86）ng/g（n=17）显著地高于泌香较少（8.98±0.59）g（n=19）雄麝（74.27±7.93）ng/g（n=19）（t=-3.252,df=34, P < 0.01）,方程y=0.021x+10.270（R²=0.120,P < 0.05）可较好地拟合二者间关系。麝香分泌与皮质醇浓度（47.49±1.53）ng/g（n=36）相关不显著（r=0.078,P > 0.05）,泌香较多雄麝的皮质醇浓度（48.98±2.28）ng/g（n=17）略高于泌香较少雄麝（46.17±2.14）ng/g（n=19）,差异不显著（P > 0.05）,方程y=0.038x+10.730（R²=0.023,P > 0.05）可近似拟合皮质醇与泌香量间的关系。此外,雄麝睾酮水平对泌香量有显著影响（P < 0.05）,睾酮水平较高林麝泌香量（14.97±1.17）g（n=12）显著高于睾酮水平较低个体的泌香量（11.32±0.90）g（n=24）（F=4.79,P < 0.05）,而睾酮、皮质醇和年龄对麝香分泌的综合效应及变量间的交互作用均不显著（P > 0.05）。研究确定了圈养林麝粪样睾酮和皮质醇水平与麝香分泌量间的关系,可用于麝类驯养实践的麝香产量预测及品系选育。     

Phytoplankton primary production in a mesotrophic pond in sub-tropical Bangladesh

Annual gross primary productivity in mesotrophic Shahidullah Hall pond (Dhaka, Bangladesh) was 1383.35 g C m⁻² y⁻¹ (arithmetic mean). Daily primary productivity (between 1.6 and 6.8 g C m⁻² d⁻¹ was correlated with chlorophylla, day length and dissolved silica. Chlorophylla related significantly withk, incident light, SRP, alkalinity and conductivity. A negative correlation existed between biomass and rainfall. Productivity, biomass, conductivity, alkalinity, and SRP increased after mid-winter.k, I _k andZ _eu varied according seasonally.P _max related directly with temperature. Seasonal variation of ∝ ^B was 0.0049–0.0258 mg C (mg chla mmol PAR)⁻¹ m⁻². Q₁₀ was 2.12, community respiration 1334.99 g C m⁻² y⁻¹, and the underwater light climate 186.43μE m⁻² s⁻¹.     

Chromium-induced glucose uptake, superoxide anion production, and phagocytosis in cultured pulmonary alveolar macrophages of weanling pigs

The dose-dependent effects of chromium chloride (CrCl₃) and chromium picolinate (CrPic) were evaluated for their glucose uptake, superoxide anion (O ₂ ⁻ ) production, activity of glucose-6-phosphate dehydrogenase, and phagocytosis of incubated pulmonary alveolar macrophages in medium containing no or 5 × 10⁻⁸ M insulin. Glucose uptake was found to increase in cells treated with 20 μg/L CrCl₃. Incubation with 20 μg/L of CrPic enhanced glucose uptake and O ₂ ⁻ production in an insulin-dependent manner. However, the inclusion of CrPic to 100 μg/L in the medium absent of insulin also increased O ₂ ⁻ production. The activity of glucose-6-phosphate dehydrogenase was not affected by either the addition of Cr or insulin. The phagocytosis of Escherichia coli by macrophages was enhanced significantly (p<0.05) in medium containing 10–100 μg/L CrCl₃ or 20–100 μg/L CrPic in the presence of insulin. These results suggest that the addition of 10–20 μg/L CrCl₃ enhances directly the cellular activity of macrophages, whereas the effect of CrPic requires the cooperative action of insulin in enhancing their glucose uptake and phagocytosis.     

Monitoring of Changes in Selenium Concentration in Goat Milk During Short-Term Supplementation of Various Forms of Selenium

The goal of the experiment was to monitor the changes in the selenium concentration in goat milk during short-term oral supplementation of three different forms of selenium. The experiment involved 24 lactating goats of white shorthaired breed. Group C was the control; group S received selenium in the form of selenium-enriched yeast, group L in the form of lactate, and group B in the form of proteinate. Individual selenium preparations were administered individually orally in 250 μg Se dose per animal for 20 days. After the beginning of selenium supplementation, the selenium concentration in milk during the first 5 days grew gradually in group S. Between days 7 and 20 of Se supplementation, the mean Se concentrations in milk in groups were 12.53 ± 3.69 μg l⁻¹ (C), 25.90 ± 6.30 μg l⁻¹ (S), 13.14 ± 3.54 μg l⁻¹ (L), 11.70 ± 3.69 μg l⁻¹ (B). Differences between group S and other groups (C, B, L) were highly significant (p < 0.0001). Based on our results, selenium in the form of lactate and proteinate was excreted into the milk similarly, but selenium in the form of yeast, which contains high amount of selenomethionine, was excreted by milk in the highest amounts.     

代谢工程强化脱氮副球菌DYTN-1去除氮素污染物

【目的】脱氮副球菌(Paracoccus denitrificans)是一种环境友好的α-变形菌纲菌株,在有氧条件下也可进行反硝化过程,具有较好的脱氮能力。本研究以脱氮副球菌DYTN-1为底盘细胞,筛选氮素诱导型启动子用于强化硝化和反硝化途径,进而达到代谢工程强化脱氮副球菌DYTN-1去除氮素污染物的目的。【方法】通过接合转移的方法分别将过表达amoA、amoB、hao和nirS基因的重组质粒导入脱氮副球菌DYTN-1细胞中。经过荧光定量检测和氮素定量检测对脱氮副球菌DYTN-1的基因元件和氮去除能力进行表征。【结果】从基因组中挖掘了6个受NO₂^‒、NO₃^‒和NH₄⁺诱导的启动子,诱导差异为2‒26倍;且过表达nirS的菌株用2 g/L KNO₃处理24 h后培养基中NO₃^‒的残余量为野生型菌株的67%。同时过表达hao和nirS基因的菌株在用1 g/L NH₄Cl和2 g/L KNO₃处理12 h后,其NO₃^‒的剩余量仅为野生型菌株的50%,且最终总氮的降解效率达79.5%,剩余总氮仅为野生型菌株的一半。【结论】上述研究表明,利用筛选获得的启动子工具在P. denitrificans DYTN-1中进行代谢工程改造强化氮素污染物的去除具有可行性。     

模块化工程改造大肠杆菌生产L-色氨酸

L-色氨酸作为一种必需氨基酸,广泛应用于食品、饲料和医药等领域。目前,微生物法生产L-色氨酸存在转化率低等问题。为此,本研究通过敲除L-色氨酸操纵子阻遏蛋白(L-tryptophan operon repressor protein, trpR)、替换l-色氨酸弱化子(trpL)、引入抗反馈调节的aroG^fbr等,获得可积累11.80 g/L L-色氨酸的底盘菌株大肠杆菌(Escherichia coli)TRP3。在此基础上,将L-色氨酸合成途径分为中心代谢途径模块、莽草酸(shikimic acid, SA)途径至分支酸(chorismic acid, CHA)模块、分支酸至L-色氨酸模块,并借助启动子工程,通过平衡中心代谢途径模块、莽草酸途径至分支酸模块、分支酸至L-色氨酸模块,获得工程菌E.coli TRP9。在5 L发酵罐中,工程菌E.coli TRP9的L-色氨酸产量提升至36.08 g/L,糖酸转化率提升至18.55%,达到理论转化率的81.7%。本研究利用模块工程策略,构建了高产L-色氨酸生产菌株,为l-色氨酸的规模化生产奠定了良好的基础。     

β-半乳糖苷酶和阿拉伯糖异构酶共表达一步法催化乳糖到塔格糖

[目的] 构建一株以廉价原料乳糖为底物合成塔格糖的重组菌株,实现一步法高效生物合成稀有糖——塔格糖。[方法] 从Escherichia coli K-12基因组中,PCR扩增出阿拉伯糖异构酶araA和β-半乳糖苷酶lacZ基因,以SD-AS为连接子,利用pET28a-1载体串联表达于Escherichia coli BL21（DE3）,获得重组菌E.coli BL21/pET28a-araA-lacZ,对重组菌全细胞催化合成塔格糖的条件进行了工艺优化与放大研究。[结果] araA和lacZ基因在E.coli BL21中同时高效表达,在最优条件（pH 8.0、温度50℃、5 mmol/L Mn²⁺、添加0.5 mol/L硼酸和0.1% SDS）下,E.coli BL21/pET28a-araA-lacZ全细胞转化100 g/L乳糖,合成塔格糖最高产量达24.03±2.03 g/L,乳糖到塔格糖的摩尔转化率为45.67%,随着底物乳糖浓度的提高,塔格糖产量呈不同程度的提高,当投加500 g/L底物乳糖时,全细胞合成塔格糖产量最高达83.81±1.38 g/L。[结论] 通过2个关键靶酶的编码基因araA和lacZ在E.coli BL21细胞中进行共表达,实现了以重组菌全细胞为催化剂转化廉价底物乳糖,一步法高效合成稀有糖塔格糖,该研究为生物法制备低能量的功能性稀有糖奠定了较好的研究基础。     

提高微生物油脂生产能力的研究进展

微生物油脂是生物柴油生产领域具有广阔前景的新油脂资源。然而, 利用产油微生物进行油脂的工业化生产仍存在限氮条件下油脂生产强度不够高、对廉价高氮生物质原料的利用效率低等瓶颈问题。随着近年来发酵工程、生物信息学及分子生物学技术的发展, 国内外研究者利用不同策略优化微生物油脂的生产条件, 并对其油脂积累代谢途径进行改造, 旨在获得适用于工业化生产的产油性能优良的油脂菌。本综述总结了国内外利用生化工程、基因工程以及新兴的转录因子工程策略提高产油微生物油脂生产强度和扩大产油微生物廉价底物利用范围方面的研究进展, 并展望了基于组学研究、模块途径工程以及反向代谢工程的综合策略在理性改造产油微生物以提高其油脂发酵性能中的应用。     

酶的分子设计、改造与工程应用

酶工程的研究已经发展到分子水平 ,在体外通过基因工程、化学、物理等手段改造酶分子结构与功能 ,大幅提高了酶分子的进化效率和催化效率 ,生产有价值的非天然酶。对酶工程学若干“热点”和前沿课题的研究、应用进行了概述 ,分析了国际上酶工程研究及应用技术、手段、方法 ,包括体外分子进化、核酶和抗体酶的设计、酶分子的定向固定化技术、酶蛋白分子的化学修饰、融合酶、人工合成及模拟酶等技术 ,并展望了酶工程的技术进步和应用的新进展。     

Rational design of thiolase substrate specificity for metabolic engineering applications

Metabolic engineering efforts require enzymes that are both highly active and specific toward the synthesis of a desired output product to be commercially feasible. The 3‐hydroxyacid (3HA) pathway, also known as the reverse β‐oxidation or coenzyme‐A‐dependent chain‐elongation pathway, can allow for the synthesis of dozens of useful compounds of various chain lengths and functionalities. However, this pathway suffers from byproduct formation, which lowers the yields of the desired longer chain products, as well as increases downstream separation costs. The thiolase enzyme catalyzes the first reaction in this pathway, and its substrate specificity at each of its two catalytic steps sets the chain length and composition of the chemical scaffold upon which the other downstream enzymes act. However, there have been few attempts reported in the literature to rationally engineer thiolase substrate specificity. In this study, we present a model‐guided, rational design study of ordered substrate binding applied to two biosynthetic thiolases, with the goal of increasing the ratio of C6/C4 products formed by the 3HA pathway, 3‐hydroxy‐hexanoic acid and 3‐hydroxybutyric acid. We identify thiolase mutants that result in nearly 10‐fold increases in C6/C4 selectivity. Our findings can extend to other pathways that employ the thiolase for chain elongation, as well as expand our knowledge of sequence–structure–function relationship for this important class of enzymes.     

阿维菌素的生物合成与途径工程

阿维菌素是一种高效安全的大环内酯杀虫杀螨剂。本文介绍了阿维菌素生物合成的步骤及参与合成步骤的有关酶系统和基因簇。对阿维菌素 8个组分合成的遗传控制基因 ,特别是对其中B1a组分合成的遗传控制位点进行讨论分析 ,并介绍了利用途径工程改造阿维链霉菌生产合成单一高效组分B1a和提高活性组分产量的研究进展。     

Engineering of carboligase activity reaction in Candida glabrata for acetoin production

Utilization of Candida glabrata overproducing pyruvate is a promising strategy for high-level acetoin production. Based on the known regulatory and metabolic information, acetaldehyde and thiamine were fed to identify the key nodes of carboligase activity reaction (CAR) pathway and provide a direction for engineering C. glabrata. Accordingly, alcohol dehydrogenase, acetaldehyde dehydrogenase, pyruvate decarboxylase, and butanediol dehydrogenase were selected to be manipulated for strengthening the CAR pathway. Following the rational metabolic engineering, the engineered strain exhibited increased acetoin biosynthesis (2.24 g/L). In addition, through in silico simulation and redox balance analysis, NADH was identified as the key factor restricting higher acetoin production. Correspondingly, after introduction of NADH oxidase, the final acetoin production was further increased to 7.33 g/L. By combining the rational metabolic engineering and cofactor engineering, the acetoin-producing C. glabrata was improved stepwise, opening a novel pathway for rational development of microorganisms for bioproduction.     

生物法制备平台化合物乙偶姻的最新研究进展

乙偶姻(3-羟基-2-丁酮)作为一种应用广泛的食用香料和重要的平台化合物,具有广阔的工业应用前景。与传统的化学合成方法不同,高效、环保的乙偶姻生物制备方法,可以减轻资源和环境压力,促进我国低碳经济的发展。近来,生物法制备平台化学品乙偶姻取得了丰硕的研究成果。总结了最近几年国内外在该领域最新的研究热点及方向,简述了发酵法生产乙偶姻的优势菌株概况,重点综述了以糖类物质为底物生产乙偶姻的最新策略及研究成果、将微生物改造为生产手性乙偶姻的高效细胞炼制工厂以及将2,3-丁二醇或双乙酰作为发酵底物的研究趋势,并介绍了乙偶姻的分离纯化工艺。使用非致病性的安全菌株,高效率地利用廉价底物,并采用经济、简单、环保的分离纯化方式,从而生产具有高附加值的食品级或高手性纯度乙偶姻,是生物法制备乙偶姻产业化发展的可靠保障。     

组织工程的一般考虑

组织工程的最终目标是通过体外增减活细胞与其胞外环境相互作用而发育成具胡生物活性的组织或器官替代物,替换、修复组织或器官,或强化其生物学功能。本文主要讨论细胞移植和组织重建的影响因素和可能的方法。