Substrate-induced changes in microbial community-level physiological profiles and their application to discriminate soil microbial communities

The addition of simple substrates could affect the microbial respiration in soils. This substrate-induced respiration is widely used to estimate the soil microbial biomass, but little attention has been paid to its influence on the changes of community-level physiological profiles. In this study, the process of microbial communities responding to the added substrate using sole-carbon-source utilization （BIOLOG） was investigated. BIOLOG is biased toward fast-growing bacteria; this advantage was taken to detect the prompt response of the active microbial communities to the added substrate. Four soil samples from agricultural fields adjacent to heavy metal mines were amended with L-arginine, citric acid, or D-glucose. Substrate amendments could, generally, not only increase the metabolic activity of the microbial communities, but also change the metabolic diverse patterns compared with no-substrate control. By tracking the process, it was found that the variance between substrate-induced treatment and control fluctuated greatly during the incubation course, and the influences of these three substrates were different. In addition, the application of these induced changes to discriminate soil microbial communities was tested. The distance among all samples was greatly increased, which further showed the functional variance among microbial communities in soils. This can be very useful in the discrimination of microbial communities even with high similarity.

Transcriptomic and physiological analyses identifying Lanzhou lily(Lilium davidii var.unicolor)drought adaptation strategies

Drought stress is the main limiting plant growth factor in arid and semiarid regions.The Lanzhou lily(Lilium davidii var.unicolor)is the only sweet-tasting lily grown in these regions of China that offers highly edible,medicinal,health,and ornamental value.The Tresor lily is an ornamental flower known for its strong resistance.Plants were grown under three different drought intensity treatments,namely,being watered at intervals of 5,15,and 25 d(either throughout the study or during specific growth stages).We measured the biomass,leaf area,photosynthetic response,chlorophyll content(SPAD value),and osmoregulation of both the Lanzhou lily and the Tresor lily(Lilium‘Tresor’).Additionally,we employed RNA sequencing(RNA-Seq)and qRT-PCR to investigate transcriptomic changes of the Lanzhou lily in response to drought stress.Results showed that under drought stress,the decreasing rate in the Lanzhou lily bulb weight was lower than the corresponding Tresor lily bulb rate;the net photosynthetic rate,transpiration rate,and stomatal conductance of the Lanzhou lily were all higher compared to the Tresor lily;osmoregulation constituents,such as glucose,fructose,sucrose,trehalose,and soluble sugar,in the Lanzhou lily were comparatively higher;PYL,NCED,and ERS genes were significantly expressed in the Lanzhou lily.Under moderate drought,the biosynthesis of flavonoids,circadian rhythms,and the tryptophan metabolism pathway of the Lanzhou lily were all significant.Under severe drought stress,fatty acid elongation,photosynthetic antenna protein,plant hormone signal transduction,flavone and flavonol biosynthesis,and the carotenoid biosynthesis pathway were all significant.The Lanzhou lily adapted to drought stress by coordinating its organs and the unique role of its bulb,regulating photosynthesis,increasing osmolyte content,activating circadian rhythms,signal transduction,fatty acid elongation metabolism,and phenylalanine and flavonoid metabolic pathways,which may collectively be the main adaptation strategy and mechanisms used by the Lanzhou lily under drought stress.

Physiological and genome-wide gene expression analyses of cold-induced leaf rolling at the seedling stage in rice(Oryza sativa L.)

Leaf rolling and discoloration are two chilling-injury symptoms that are widely used as indicators for the evaluation of cold tolerance at the seedling stage in rice. However, the difference in cold-response mechanisms underlying these two traits remains unknown. In the present study, a cold-tolerant rice cultivar, Lijiangxintuanheigu, and a cold-sensitive cultivar, Sanhuangzhan-2, were subjected to low-temperature treatments and physiolog-ical and genome-wide gene expression analyses were conducted. Leaf rolling occurred at temperatures lower than 11℃, whereas discoloration appeared at moderately low temperatures such as 13℃. Chlorophyll contents in both cultivars were significantly decreased at 13℃, but not altered at 11℃. In contrast, the relative water content and relative electrolyte leakage of both cultivars decreased significantly at 11℃, but did not change at 13℃. Expression of genes associated with calcium signaling and abscisic acid (ABA) degradation was significantly altered at 11℃ in comparison with 25℃ and 13℃. Numerous genes in the DREB, MYB, bZIP, NAC, Zinc finger, bHLH, and WRKY gene families were differentially expressed. Many aquaporin genes and the key genes in trehalose and starch synthesis were down regulated at 11℃ in comparison with 25℃ and 13℃. These results suggest that the two chilling injury symptoms are temperature-specific and are controlled by different mechanisms. Cold-induced leaf rolling is associated with calcium and ABA signaling pathways and is regulated by multiple transcriptional regulators. The suppression of aquaporin genes and reduced accumulation of soluble sugars under cold stress results in a reduction in cellular water potential and consequently leaf rolling.

Soil Microbial Community-Level Physiological Profiling as Related to Carbon and Nitrogen Availability Under Different Land Uses

The goal of this work was to assess soil microbial respiration, determined by the assay of community-level physiological profiling in an oxygen-sensitive microplate （O2-CLPP）, in response to endogenous C and several individual C substrates in the soils with different organic C contents （as a function of soil type and management practice）. We also used the O2-CLPP to determine the respiratory response of these soils to endogenous C and amended C substrates with N addition. A respiratory quotient （RQ） was calculated based on the ratio of the response to endogenous soil C vs. each C-only substrate, and was related to total organic carbon （TOC）. For assessing N availability for microbial activity, the effect of N supplementation on soil respiration, expressed as Nr^tio, was calculated based on the response of several substrates to N addition relative to the response without N. Soils clustered in 4 groups after a principal component analysis （PCA）, based on TOC and their respiratory responses to substrates and endogenous C. These groups reflected differences among soils in their geographic origin, land use and C content. Calculated RQ values were significantly lower in natural forest soils than in managed soils for most C-only substrates. TOC was negatively correlated with RQ （r = -0.65）, indicating that the soils with higher organic matter content increased respiratory efficiency. The N addition in the assay in the absence of C amendment （i. e., only endogenous soil C present） had no effect on microbial respiration in any soil, indicating that these soils were not intrinsically N-limited, but substrate-dependent variation in Nr^tio within soil groups was observed.

Effect of the Type of Vitis vinifera Cultivation in the Cenophenoresistome and Metabolic Profiling （CLPP） of Edaphic Bacterial Communities

In the present work, bacterial soil communities of different grapevine exploitation samples are studied in order to elucidate the possible influence of different agrarian management techniques （use of fertilizers, irrigation with river water） may have on the rhizospheric microbiome of Vitis vinifera plants. Therefore, it is postulated the Cenophenoresistome as a novel methodology to evaluate complex communities＇ global resistance against different antibiotics, by using and adjusting a serial of techniques traditionally applied to evaluate a monospecific population＇s resistance against antibiotics （Vitek, ATB and disk diffusion methods）. Likewise, the metabolic profile （CLPP： comunity level physiological profile） of bacterial communities is studied by Biolog ECO. In relation to the functional structure of the bacterial communities, it is observed that the metabolic profile （diversity, kinetics and CLPP） of unexploited soils differs from soils under anthropic influence. It is discussed the causes of resistance in the human clinic antibiotic treatment based on the agrarian management, especially with the contamination transmitted by irrigation water, which could be associated with changes in edaphic communities. The results obtained in the present study through two different approaches （Cenophenoresistome and metabolic profiles） are consistent with each other, suggesting that both methods can be good bioindicators of the state of humankind-altered soils that host natural ecosystems. Likewise, the concept of Cenophenoresistome is proposed as a bioindicator of soil response to alteration processes, as well as a possible predictor of its evolution in edaphic remediation processes.

ACCELERATED DISCOVERY AND PROFILING OF PHYSIOLOGICALLY ACTIVE COMPONENTS IN COMPLEX SAMPLES BY HPTLC-EDA-HRMS

On the one hand,the separation of thousands of compounds in a complex extract is thrilling,but may be still be separated unsatisfactorily.Hence,the question arises where to stop in high-sophisticated separation science?Which technical effort is economically justifiable in routine?On the other hand,the separation itself does not imply an effect-directed answer to questions such

Effect of Different Vegetation Types on the Rhizosphere Soil Microbial Community Structure in the Loess Plateau of China

The Loess Plateau in China is one of the most eroded areas in the world. Accordingly, vegetation restoration has been implemented in this area over the past two decades to remedy the soil degradation problem. Understanding the microbial community structure is essential for the sustainability of ecosystems and for the reclamation of degraded arable land. This study aimed to determine the effect of different vegetation types on microbial processes and community structure in rhizosphere soils in the Loess Plateau. The six vegetation types were as follows：two natural grassland （Artemisia capillaries and Heteropappus altaicus）, two artificial grassland （Astragalus adsurgens and Panicum virgatum）, and two artificial shrubland （Caragana korshinskii and Hippophae rhamnoides） species. The microbial community structure and functional diversity were examined by analyzing the phospholipid fatty acids （PLFAs） and community-level physiological profiles. The results showed that rhizosphere soil sampled from the H. altaicus and A. capillaries plots had the highest values of microbial biomass C, average well color development of carbon resources, Gram-negative （G-） bacterial PLFA, bacterial PLFA, total PLFA, Shannon richness, and Shannon evenness, as well as the lowest metabolic quotient. Soil sampled from the H. rhamnoides plots had the highest metabolic quotient and Gram-positive （G＋） bacterial PLFA, and soil sampled from the A. adsurgens and A. capillaries plots had the highest fungal PLFA and fungal：bacterial PLFA ratio. Correlation analysis indicated a signiifcant positive relationship among the microbial biomass C, G- bacterial PLFA, bacterial PLFA, and total PLFA. In conclusion, plant species under arid climatic conditions signiifcantly affected the microbial community structure in rhizosphere soil. Among the studied plants, natural grassland species generated the most favorable microbial conditions.

燕麦盐胁迫响应基因的差异表达与生理响应的关系

以耐盐燕麦品种VAO-9为材料,通过Illumina测序与数字基因表达谱技术对300mmol/L NaCl处理前后的叶片cDNA文库进行RNA-Seq与DGE分析,同时测定0(CK)、100、200、250、300mmol/L NaCl胁迫下VAO-9幼苗叶片的相对电导率、丙二醛含量和脯氨酸含量,探讨燕麦盐胁迫响应基因的差异表达与生理响应的关系。结果表明:(1)RNA-Seq分析得到Unigenes 65 801条,其基因表达呈现高度的不均一性和冗余性;若差异基因表达谱鉴定分析以log2Ratio≥2且FDR值≤0.001为选择标准,则发现上调和下调表达基因在胁迫0.5h时分别有306和64个,在胁迫3h时分别有639和290个,胁迫24h时分别有1 488和882个。(2)KEGG代谢分析显示,有23 652条Unigenes比对到KEGG中的128条代谢途径,包括与逆境胁迫相关的植物激素信号转导途径、ABC转运蛋白途径、肌醇磷酸代谢途径、渗透调节途径等。(3)在300mmol/L NaCl处理下燕麦叶片的相对电导率、丙二醛含量、脯氨酸含量等生理指标的变化与相关差异表达基因的变化趋势基本一致,说明基因差异表达量与生理反应密切相关。研究认为,在相同的栽培及胁迫处理条件下,可根据植物盐响应生理指标的变化判断耐盐基因的表达情况。

大兴安岭森林沼泽类型与火干扰对土壤微生物群落影响

[目的]研究森林沼泽演替与火干扰条件下土壤微生物结构与多样性变化,为进一步揭示土壤微生物群落在森林沼泽保护与恢复中的作用提供依据。[方法]采用磷脂脂肪酸法与BIOLOG方法,研究大兴安岭南瓮河国家自然保护区内主要森林沼泽类型(兴安落叶松-狭叶杜香-藓类沼泽、兴安落叶松-兴安杜鹃-藓类沼泽、兴安落叶松+白桦-苔草沼泽)与2006年受不同火强度干扰沼泽(重度火烧的兴安落叶松-兴安杜鹃-藓类沼泽和中度火烧的兴安落叶松+白桦-苔草沼泽)土壤微生物群落特征,探讨沼泽主要发育阶段与火干扰强度对土壤微生物群落的影响。[结果]研究区域土壤微生物群落以16:00(16.29±5.62 nmol·g-1)、甲烷氧化菌(18:1ω8t)(9.89±8.61 nmol·g-1)与16:1ω7c(9.79±3.24 nmol·g-1)的微生物为优势种群。土壤微生物总PLFAs含量、革兰氏阳性菌(G+)中a15:0、i16:0、i17:0、革兰氏阴性菌(G-)中的cy19:0、真菌中的18:2ω6c、甲烷氧化菌(18:1ω8t)与森林沼泽发育阶段、火干扰明显相关(p<0.05)。一般饱和直链脂肪酸/单烯饱和脂肪酸比(Sat/Mon)偏低,其比值随沼泽发育呈现增加趋势,受到火干扰后明显增加(p<0.05);真菌/细菌比(F/B)与革兰氏阳性菌/革兰氏阴性菌(G+/G-)未随着沼泽发育呈现出规律性变化,其比值受火干扰后明显发生改变(p<0.05)。土壤细菌与真菌对6类碳源的利用能力明显不同(p<0.001),其中土壤细菌对α-D-Lactose与L-Threonine利用存在差异性(Fα-D-Lactose=2.87 p=0.080,FL-Threonine=3.00 p=0.078),土壤真菌对D-Mannitol、D-glucosaminic Acid利用存在差异性(FTween 80=2.75,p=0.088,FD-Mannitol=3.53 p=0.047,FD-glucosaminic Acid=4.67 p=0.022),但沼泽类型与火干扰未对土壤微生物功能多样性产生影响(p>0.05)。[结论]土壤微生物量与沼泽发育阶段相关;沼泽发育与火干扰改变土壤微生物群落结构。土壤细菌与真菌对碳源利用方面具有选择性。

多模态数据赋能教师画像:从简笔画走向全息画像

教师数字画像是实现精准诊断、及时干预和个性化服务的基础,然而国内学者少有关注,特别是多模态数据赋能全息化描绘教师画像的潜力没有得到很好的发掘。本研究从多学科角度系统解析了多模态的内涵,即多模态是表征不同生理、心理或行为反应的不同形态数据。以此为基础,本研究构建了以生理数据为主要成分的多模态数据全息临摹教师画像的机理及画像模型,模型包括关注发展期望勾勒的愿景层、关注研修动态描绘的学习层、关注本体特征刻画的属性层三个层面。之后,研究构建了基于全息画像的个性化机制(包括适性的动力机制和个人路径生成机制)以及机制得以运转的多模态学习分析方略(包括涵盖去伪存真、多维共描、多面临摹三个层面的多模态数据融合分析方略以及涉及精准教学、失败中学、个性化学习三种学习理念的适应性精准决策方略)。希望这一研究成果能对教师全息画像的临摹提供可行的方案,对以此为基础的智慧研修系统与个性化研修服务的构建提供有价值的参考。

老化油泥处理过程中的微生物群落变化特征

通过在油田现场构建2个中试规模的生物堆(秸秆添加堆和对照堆)对老化油泥进行处理,运用BIOLOGTM系统对油泥中微生物群落水平的生理特性进行分析,测定其微生物代谢活性、代谢多样性及代谢特征差异性,以研究堆体各层次间微生物群落代谢特征的变化规律.结果表明,与对照堆相比,秸秆添加堆的多项生理特性指标均得到显著提升,其中微生物代谢多样性指数变化范围由1.64～3.02升至2.83～3.29,并且表现出时间上的稳定性和空间层次间的均质性;在微生物碳源代谢特征的差异性分析中,对照堆各层次随时间推移表现出向其外层顺次变化的规律;油泥中总异养菌及石油烃降解菌的数量与环境温度关联性较强,石油烃降解菌的数量在试验末期取得峰值,达4.15×107CFU/g.

荒漠草原不同植被类型土壤微生物群落功能多样性

利用Biolog EcoplateTM技术通过对荒漠草原生态系统中沙米、白沙蒿、柠条、沙冬青和人工乔木林5种植被类型土壤微生物群落功能多样性分析,以探讨不同植被类型对土壤微生物群落代谢功能多样性的影响。结果表明,5种植被类型土壤微生物群落代谢活性有显著的差异(P<0.01),5种植被类型土壤微生物群落利用碳源的种类也存在差异,氨基酸类、羧酸类和糖类是其主要的碳源利用类型。在Biolog ECO板培养基接种培养96 h后,5种植被类型土壤微生物群落多样性显示,物种丰富度指数(R)和均匀度指数(EH)差异显著,Shannon指数(H′)差异不显著。

广西柑橘溃疡病菌菌系分化研究

从广西北海、桂林地区11个柑橘品种溃疡病材料上分离得到13株致病菌,进行菌系分化研究表明:在所测试的35项生理特征中有25项(柠檬酸盐、丙二酸盐、36℃生长、耐盐性(1～3%)、吐温20、吐温80、水杨素、卫茅醇、葡萄糖、乳糖、纤维二糖、苯丙氨酸、棉子糖、松三糖、侧金盏花醇、菊糖、核糖、甘露醇、阿拉伯糖、海藻糖、蜜二糖、山梨糖、七叶苷、酒石酸盐、明胶)完全相同,10项(醋酸盐、果糖、枸橼酸盐、鼠李糖、木糖、蔗糖、麦芽糖、半乳糖、淀粉、甘露糖)存在差异;16项生化特征测试中有12项(细胞色素C氧化酶、过氧化氢酶、甲基红试验、乙酰甲基甲醇试验、水解七叶苷、果聚糖产生、硝酸盐还原、脲酶、苯丙氨酸脱氨酶、精氨酸双水解酶、卵磷脂酶、明胶水解)完全相同,4项(水解淀粉、吲哚产生、硫化氢产生、石蕊牛奶)存在差异;3种噬菌体鉴定试验能够区分出13个菌株菌系分化现象的存在,且分为6种敏感型,而菌株细胞脂肪酸成分分析则不能区分菌系分化情况。

马铃薯连作栽培对土壤化感物质及微生物群落的影响

土壤微生物是土壤生态系统的重要组成部分,长期连作严重影响了作物产量和品质,改变了土壤生态系统过程。试验以不同连作年限马铃薯和倒茬地根际土壤为材料,采用气相色谱和质谱联用技术以及群落水平生理活性(CLPPs)和磷脂脂肪酸(PLFAs)生物标记法,对不同连作年限马铃薯根际土壤化学物质及微生物群落结构、功能进行研究。结果表明,连作马铃薯根际土壤乙酸乙酯浸提的酸、中、碱性相中都有质量分数高达50%左右的邻苯二甲酸二丁异酯和少量的顺式-14-二十九烯;和倒茬相比,长期连作根际土壤酯类、烯烃类和苯类物质都有明显的积累。随连作年限的延长,微生物群落利用碳源的能力呈现先增加后降低的趋势,连作4 a时最强;而连作10 a时微生物群落对主要碳源的利用能力总体较弱,以碳水化合物为碳源基质的微生物类群代谢能力显著降低;以碳水化合物、羧酸类化合物和氨基酸为代谢基质的微生物类群组成明显受到抑制。主要磷脂脂肪酸中,以16∶1、16∶0、18∶2ω6t和18∶1ω9c表征的PLFA含量较高,不同类型的PLFA含量随连作时间的增加呈现单峰型变化趋势,连作4 a时土壤微生物生物量最高;随着连作年限的增加,细菌/微生物群落生物量比值升高,而真菌/微生物群落生物量比值下降;倒茬种植明显改善了土壤主要微生物群落生物量及结构组成。长期连作产生的邻苯二甲酸二丁异酯及顺式-14-二十九烯积累,可能是导致马铃薯连作障碍的主要化感物质;这些化学物质显著改变了土壤微生物群落结构与功能,连作4 a可能是马铃薯连作栽培障碍的临界点。

FIZZ1对大鼠主动脉内皮细胞血管新生的促进作用及其机制探讨

目的探讨抵抗素样分子α(FIZZ1)对大鼠主动脉内皮细胞血管新生的影响及其机制。方法采用贴壁法培养Wistar大鼠主动脉内皮细胞,将实验分为4组:A组(对照组)、B组(5×10–9mol/L FIZZ1组)、C组(1×10–8mol/LFIZZ1组)、D组(2×10–8mol/L FIZZ1组)。采用Matrigel小管形成实验评价不同浓度的FIZZ1对大鼠主动脉内皮细胞体外血管新生的影响。采用全基因表达谱检测FIZZ1对主动脉内皮细胞信号通路活性的影响,筛选出活性有显著差异的信号通路。结果 FIZZ1刺激后,B、C、D组新生血管数目(分别为22.6±2.9、28.5±3.3、36.9±5.0个/HP)均明显高于对照组(19.7±2.6个/HP,P<0.01),且呈剂量依赖性。全基因表达谱检测提示,FIZZ1刺激后有22条信号通路的活性出现明显改变,其中活性上调信号通路12条,如调节细胞肌动蛋白骨架通路(rno04810)等,活性下调信号通路10条,如上皮细胞细菌侵入通路(rno05100)等。结论 FIZZ1可促进大鼠主动脉内皮细胞血管新生,其机制可能与信号通路调节细胞肌动蛋白骨架通路(rno04810)活性上调有关。

复垦矿区土壤有机碳和微生物活性变化及其解析

以适当比例混合煤矸石、粉煤灰和活性污泥并种植黑麦草构建植物-矿区复合基质体系,测试复合基质有机碳及酶活性变化,利用群落水平生理结构(CLPP)对复合基质微生物功能多样性进行解析,分析了矿区复合基质中有机碳动态变化及其与酶活性、微生物多样性关系。结果表明:植物-矿区复合基质体系3种基质中有机碳随复垦年限增长而增加,且在第3年煤矸石+粉煤灰+污泥(F+G+S)区有机碳达到最大,分别是煤矸石+粉煤灰(F+G)区和对照区的1.1和1.2倍。3个复垦区域复合基质中蔗糖酶、脱氢酶、酸性磷酸酶酶活性随复垦时间增长而增加。微生物多样性CLPP解析结果表明,微生物种类在复垦1年时最多,微生物优势度和均一性随复垦时间逐渐变大;同时3个区域基质微生物多样性与营养元素相关性不大,可能有机碳可获得性低成为微生物生长的限制因子。

Metabolic diversity and seasonal variation of soil microbial communities in natural forested wetlands

This study explores the effects of vegetation and season on soil microorganisms and enzymatic activity of different wetlands in a temperate climate.Microbial carbon metabolism diversity was assessed using community-level physiological profiles(CLPP)with 31 different carbon substrates.CLPP indicated that significant interactions occur during carbon substrate metabolism of the microorganisms.Furthermore,the different types of vegetation present in the wetland ecosystem combined with the seasonal effects to influence microbial carbon metabolism and enzymatic activity.The most significant differences occurred to carbohydrates,carboxylic acids,and amino acids.The Mantel test confirmed positive correlations between soil enzymatic activities and microbial carbon metabolism.Soil microorganisms in Betula ovalifolia and Carex schmidtii wetlands used carbon substrates more efficiently in summer than those in other forested wetlands during other periods.Enzymatic activities also showed a similar trend as microbial carbon metabolism.The results demonstrate that microbial carbon metabolism patterns can be used as biological indicators in wetland ecological alterations due to vegetation type or to seasonal factors.

不同品系高羊茅应答高温胁迫的初级代谢产物分析

为探讨高温对高羊茅初级代谢的影响,对耐热性不同的两个高羊茅基因型PI 578718(耐热)和PI 234881(热敏感),进行40℃/35℃(昼/夜各12h)模拟高温胁迫21d,分别于不同时间段取样测定生理以及代谢产物的变化。结果表明,在高温胁迫下,草坪质量、叶绿素含量、叶片相对含水量、根系活力持续下降,电解质渗漏加剧,与耐热型基因型相比,热敏感基因型各生理指标变化更明显。通过GC-MS分析,检测并鉴定了25种初级代谢产物,主要包括有机酸和脂肪酸(9种)、氨基酸(9种)、糖类和糖醇(7种),其中氨基酸(如脯氨酸、缬氨酸、谷氨酸)、糖类(如蔗糖)和糖醇(如肌醇)在叶片中大量积累,而有机酸和脂肪酸类(如柠檬酸、棕榈酸)在根系中大量积累。高温胁迫下叶片和根系中代谢产物如缬氨酸、谷氨酸、脯氨酸、蔗糖、肌醇、柠檬酸、棕榈酸含量的升高或维持,可能是耐热基因型PI 578718与热敏感基因型PI 234881之间耐热性差异的主要原因,而且柠檬酸作为抗氧化剂并参与呼吸作用的三羧酸循环途径,它的高积累,可能更有助于提高高羊茅对高温胁迫的耐性和适应性。

Degradation of chlorpyrifos in laboratory soil and its impact on soil microbial functional diversity

Degradation of chlorpyrifos at different concentrations in soil and its impact on soil microbial functional diversity were investigated under laboratory condition. The degradation half-live of chlorpyrifos at levels of 4, 8, and 12 mg/kg in soil were calculated to be 14.3, 16.7, and 18.0 d, respectively. The Biolog study showed that the average well color development （AWCD） in soils was significantly （P 〈 0.05） inhibited by chlorpyrifos within the first two weeks and thereafter recovered to a similar level as the control. A similar variation in the diversity indices （Simpson index lID and McIntosh index U） was observed, but no significant difference among the values of the Shannon-Wiener index H＇ was found in chlorpyrifos-treated soils. With an increasing chlorpyrifos concentration, the half-life of chlorpyrifos was significantly （P ≤ 0.05） extended and its inhibitory effect on soil microorganisms was aggravated. It is concluded that chlorpyrifos residues in soil had a temporary or short-term inhibitory effect on soil microbial functional diversity.

赤拟谷盗章鱼胺受体3(TcOctβR3)cDNA克隆、表达及功能

【目的】章鱼胺信号系统在调节昆虫行为和生理过程中具有至关重要的作用。赤拟谷盗(Tribolium castaneum)作为一种模式昆虫,被广泛用于解析昆虫生长发育及生理等调控机制的研究工作。本研究以赤拟谷盗为对象,旨在明确章鱼胺受体在调节赤拟谷盗行为和生理方面的功能。【方法】根据Gen Bank登录的相关序列信息(XP_008198078),利用RT-PCR技术克隆赤拟谷盗章鱼胺受体基因Tc OctβR3的c DNA序列。利用在线生物信息学分析软件预测该基因的开放阅读框、编码的氨基酸序列以及跨膜结构域等信息,基于邻接法构建该基因与其他昆虫相关序列的系统发育树,明确系统进化关系。分别提取赤拟谷盗各发育阶段(卵、幼虫、蛹和成虫)、不同组织(中枢神经系统、脂肪体、中肠、后肠、马氏管、精巢和卵巢)以及饥饿胁迫后的RNA,以赤拟谷盗核糖体蛋白S3(Tc RPS3)为内参基因,采用实时定量PCR技术分析该基因在赤拟谷盗不同发育阶段、不同组织以及在饥饿胁迫下的表达模式。运用哺乳动物异源表达系统在人胚胎肾细胞HEK293中瞬时表达Tc OctβR3,进而利用第二信使c AMP含量测定技术分析Tc OctβR3与配体的结合能力。最后,通过体外合成赤拟谷盗Tc OctβR3的双链RNA,利用RNA干扰以及轨迹球行为分析等技术探究该基因的生理功能。【结果】序列分析结果表明,赤拟谷盗Tc OctβR3开放阅读框全长1 305 bp,编码434个氨基酸,序列中含有G蛋白偶联受体典型的7个跨膜结构域。基于邻接法构建的系统发育树表明,该基因编码的蛋白质与小蜂甲(Aethina tumida)的OctβR3亲缘关系最近。实时定量PCR分析结果表明,Tc OctβR3在赤拟谷盗各发育阶段均有表达,尤其在低龄幼虫期转录水平最高,而在其他发育阶段表达量无显著差异;在赤拟谷盗不同组织中,Tc OctβR3在中枢神经系统的表达量显著高于其他组织;赤拟谷盗幼虫在经饥饿处理24 h的过程中,Tc OctβR3的表达量呈先下降后上升的趋势,且在处理6 h的表达量最低,为对照的0.47倍,在16 h表达量最高,为对照的1.80倍,最后恢复到正常水平。通过HEK293细胞异源表达Tc OctβR3后,c AMP测定结果表明章鱼胺(OA)呈浓度依赖性地激活Tc OctβR3,其EC50为8.68×10-7 mol·L-1,萘甲唑啉(NA)的激动活性最高,其有效中浓度EC50为8.56×10-8 mol·L-1。4种供试生物胺激动剂活性强弱为:萘甲唑啉>酪胺(TA)>章鱼胺>多巴胺(DA)。进一步采用RNA干扰技术的分析结果表明,注射ds RNA能有效抑制Tc OctβR3的表达,沉默效率高达61.5%,但干扰该基因表达后不会影响赤拟谷盗成虫的爬行速度和产卵量。【结论】Tc OctβR3在赤拟谷盗中枢神经系统可能发挥重要作用,能调节幼虫对饥饿胁迫的响应。明确了Tc OctβR3的分子生物学特性,可为将来以其为靶标筛选高效激动剂和抑制剂提供理论依据。