温度胁迫下沙葱萤叶甲小热激蛋白基因GdHsp20.6的克隆及表达分析

为明确小热激蛋白在沙葱萤叶甲Galeruca daurica应对温度胁迫中的作用，采用PCR方法克隆沙葱萤叶甲小热激蛋白基因Hsp20（GdHsp20.6）完整的开放阅读框（open reading frame，ORF）序列，应用在线软件对GdHsp20.6基因进行生物信息学分析，通过原核表达技术诱导表达及纯化其编码蛋白，并通过实时荧光定量PCR（real-time quantitative PCR，RT-qPCR）技术分析不同温度胁迫下GdHsp20.6基因的表达量。结果显示，GdHsp20.6基因ORF序列长度为543 bp，编码180个氨基酸，预测分子量为20.6 kD，无跨膜区和信号肽。GdHsp20.6氨基酸序列有高度保守的α-结构域。GdHsp20.6氨基酸序列与其它鞘翅目昆虫的Hsp20氨基酸序列有较高的一致性，其中与花绒寄甲Dastarcus helophoroides的Hsp20.99氨基酸序列的一致性最高，为63%。GdHsp20.6基因在大肠杆菌Escherichia coli BL21（DE3）细胞系中成功表达，经异丙基-β-D-硫代吡喃半乳糖苷（isopropyl-β-d-thiogalactoside，IPTG）诱导后GdHsp20.6蛋白成功表达，十二烷基硫酸钠-聚丙烯酰胺凝胶电泳（sodium dodecyl sulfate-polyacrylamide gel electrophoresis，SDS-PAGE）和Western-blot分析表明融合蛋白大小与预测大小一致，并纯化获得了纯度较高的目的蛋白GdHsp20.6。低温（-10~5℃）和高温（35~40℃）处理1 h以及处理后25℃恢复30 min均能诱导GdHsp20.6基因表达上调，并且0℃处理30~120 min也能诱导GdHsp20.6基因表达上调。表明GdHsp20.6基因在沙葱萤叶甲应对低温和高温胁迫中可能起着重要作用。     

Adaptation to Agenda 2000 (CAP reform) and optimisation of the farming system of French suckler cattle farms in the Charolais area: a model-based study

French suckler farmers need advice on the implications of the Agenda 2000 CAP reform for their farms and, in particular, on the incentives it offers for a more extensive mode of production. To support the dialogue between advisers and farmers, and thus help farmers with their decision-making, we constructed a linear programming (LP) model that optimises the farming system of the northern Massif Central Charolais suckler cattle farms, which may be either mixed (crop-livestock) or specialised (livestock). This model, called Opt'INRA, incorporated all of the production activities presently encountered in this zone, together with the constraints of the CAP premium attributions. We used it to study how, on the basis of their 1999 data, two farms, representing two situations frequently encountered in the Charolais area (a mixed crop-livestock farm and a specialised livestock farm), could best adapt to Agenda 2000.According to the model, for both of the farms studied, the economic impact of Agenda 2000 is relatively low, albeit negative. The adaptation of the system when possible does not lead to a significant increase in the gross margin of this farms. Agenda 2000 did not encourage farmers to extensify their farming system. On the other hand, this CAP reform discourages them from intensifying.     

四种钙结合蛋白基因在沙葱萤叶甲幼虫生长发育中的作用

为明确沙葱萤叶甲Galeruca daurica钙结合蛋白基因的功能,利用本实验室组装的沙葱萤叶甲转录组数据库鉴定钙结合蛋白基因序列,采用RNA干扰（RNA interference,RNAi）技术沉默沙葱萤叶甲3龄幼虫体内钙调蛋白（calmodulin,CaM）、钙网蛋白（calreticulin,CRT）、类钙磷蛋白（calcyphosine-like,CAPSL）和类肌钙蛋白C（troponin C-like,TnCl）共4种钙结合蛋白基因,观察幼虫发育历期、存活率、体重及蛹重的变化;应用实时荧光定量PCR测定干扰效率。结果表明,共筛选到41条编码钙结合蛋白的基因序列,选取4条具有完整开放阅读框的钙结合蛋白基因序列进行后续研究。4种钙结合蛋白基因的干扰效率从高到低依次为CAPSL（94.4%）、TnCl（76.2%）、CRT（70.5%）和CaM（54.5%）;干扰效率最高的时间分别为干扰后第4、2、6和2天。沉默CAPSL后,沙葱萤叶甲幼虫体重显著降低了21.0%~34.9%,存活率显著下降了53.5%,发育历期显著缩短了15.1%,蛹重显著降低了15.8%。沉默TnCl后,沙葱萤叶甲幼虫体重显著降低了10.5%~25.0%,存活率显著下降了19.1%,蛹重显著降低了11.0%,而对发育历期无显著影响。沉默CaM后,沙葱萤叶甲幼虫体重显著降低了5.9%~6.6%,存活率显著降低了8.7%,而幼虫发育历期和蛹重无显著变化。沉默CRT后,沙葱萤叶甲幼虫体重、蛹重、存活率和发育历期均无显著变化。表明CAPSL、TnCl和CaM在沙葱萤叶甲幼虫生长发育过程中发挥着重要作用,而CRT可能未参与沙葱萤叶甲幼虫生长发育的调控过程。     

Population attributable fractions of farm vector tick (Rhipicephalus appendiculatus) presence on Theileria parva infection seroprevalence under endemic instability

The primary objective of this study was to assess the impact of Rhipicephalus appendiculatus tick presence (exposure variable) on Theileria parva infection seroprevalence (outcome variable) in a group of cattle belonging to a farm using population attributable fractions (PAF). The analyses were based on a representative sample of 80 traditional smallholder mixed farms. The farms were selected by first stratifying the population administratively and implementing a multistage random sampling in Mbeere district in Kenya. The PAFs were estimated using the stratified, Bruzzi, and sequential partitioned PAF approaches. A secondary objective was, thus, to evaluate the impact of the approaches on the PAF estimates. The stratified and Bruzzi approaches estimated proportion of T. parva infection cases directly attributable to the exposure after controlling for confounding by agro-ecological zone (AEZ). The sequential partitioned PAF approach estimated a PAF associated with exposure after adjusting for any effect that the AEZ may have had by influencing the prevalence of the exposure. All analyses were carried out at the farm level where a farm was classified as infested if the tick was found on cattle on a farm, and infected if at least one animal on a farm was positive for T. parva antibodies. Variance estimation for PAFs was implemented using ‘delete-a-group’ jackknife re-sampling method. The stratified PAF (26.7% [95% CI: 9.0%, 44.4%]) and Bruzzi PAF (26.4% [95% CI: 9.6%, 43.2%]) were consistent in estimating a relatively low impact of farm vector tick presence with a relatively high level of uncertainty. The partitioned PAF (15.5% [95% CI: 1.5%, 29.6%]) suggested that part of the impacts estimated using the stratified PAF and Bruzzi approaches was driven by AEZ effects. Overall, the results suggested that under endemic instability in Mbeere district, (1) presence of R. appendiculatus was not a good indicator of T. parva infection occurrence on a farm; (2) ecological variation could play a role in determining infection impacts. This study provides a preliminary basis for evaluating the potential value and utility of estimating PAFs for variables amenable to control in tick-borne diseases (TBDs) epidemiological studies.     

沙葱萤叶甲气味结合蛋白GdauOBP20的基因克隆、 原核表达及其结合特性

【目的】 沙葱萤叶甲（Galeruca daurica）是近年来在内蒙古草原上暴发成灾的新害虫,本文旨在克隆沙葱萤叶甲气味结合蛋白基因GdauOBP20的cDNA全长序列,明确其重组蛋白与寄主植物挥发物的结合特性,为揭示沙葱萤叶甲嗅觉的分子机理打下基础。【方法】 基于沙葱萤叶甲转录组数据,利用RACE技术对沙葱萤叶甲气味结合蛋白基因GdauOBP20进行cDNA全长克隆;利用生物信息学软件预测分析其编码蛋白的理化特性和结构特征;通过原核表达系统表达目的蛋白,并使用Ni-NTA Agarose亲和层析柱进行重组蛋白纯化。最后采用荧光竞争结合的方法,以N-苯基-1-萘胺（N-phenyl-1-naphthylamine,1-NPN）为荧光探针,检测GdauOBP20重组蛋白与13种主要寄主挥发物的结合情况。【结果】 GdauOBP20的cDNA全长序列为567 bp（GenBank登录号MK250532）,其中5′末端非编码区长24 bp,3′末端非编码区长123 bp,具有ployA尾结构;开放阅读框（ORF）全长为420 bp,编码139个氨基酸。氨基酸序列中含有4个保守的半胱氨酸位点,属于Minus-C OBP亚家族。预测蛋白三级结构中含有6个α螺旋和两对由半胱氨酸形成的二硫键。成功构建了重组表达载体并获得了高纯度的重组蛋白。重组蛋白GdauOBP20与荧光探针1-NPN的结合常数为12.8 μmol·L ^-1,结合能力较好,可作为本试验的荧光报告子。在所测的13种主要寄主植物挥发物中,除与二烯丙基三硫醚无结合能力外,GdauOBP20重组蛋白与其他12种寄主植物挥发物均有不同程度的结合能力,其中与对二甲苯和环庚三烯的结合能力最强,解离常数分别为22.91和26.55 μmol·L ^-1,而与月桂烯结合能力最弱,解离常数为116.29 μmol·L ^-1。【结论】 沙葱萤叶甲GdauOBP20能与寄主植物的多种主要挥发性物质结合,推测其可能在沙葱萤叶甲对寄主植物的定位过程中发挥重要的作用。     

沙葱萤叶甲GdHsp70-2,GdHsp70-3的克隆鉴定与表达谱分析

为明确沙葱萤叶甲Galeruca daurica热激蛋白70 (Heat shock protein 70,Hsp70)基因的序列结构及其系统进化关系,本研究通过PCR技术克隆沙葱萤叶甲Hsp70基因cDNA全长序列与基因组序列,并进行生物信息学与表达谱分析。结果显示,克隆获得了沙葱萤叶甲2条Hsp70基因GdHsp70-2(GenBank登录号：MZ853083)和GdHsp70-3(Genbank登录号：OK585088),基因全长分别为2 410 bp和2 242 bp,各自编码657和646个氨基酸,均含有3个保守的HSP70家族特征序列,预测蛋白三维结构均由N-端ATPase功能域和C-端底物结合功能域所组成;系统发育分析表明GdHSP70-2,GdHSP70-3分别与松墨天牛(Monochamus alternatus)MaltHSC70-1、玉米根萤叶甲(Diabrotica virgifera virgifera) DvirHSP70-2的亲缘关系最近;基因组DNA克隆获得GdHsp70-2的两段内含子序列,GdHsp70-3则不含有内含子;表达谱分析结果表明GdHsp...     

沙葱萤叶甲的形态特征和生物学特性研究

采用野外调查和室内饲养相结合的方法,明确了沙葱萤叶甲(Galeruca daurica)的生活史和主要生物学特性,以期为该虫的预测预报和防治提供必要的基础。在锡林郭勒草原,沙葱萤叶甲1年发生1代,以卵在牛粪、石块及草丛下越冬,越冬卵最早于4月上旬开始孵化,4月下旬为孵化盛期。幼虫大量取食新鲜的沙葱(Allium mongolium)、多根葱(Allium polyrhizum)及野韭(Allium ramosum)等葱属植物。5月中旬老熟幼虫开始化蛹,6月上旬成虫开始羽化,随后进入越夏蛰伏期。8月下旬雌雄成虫开始交尾产卵。     

Effects of elevated atmospheric CO2 and N fertilization on abundance,diversity and C-isotopic signature of collembolan communities in arable soil

Rising atmospheric CO₂ concentrations are expected to have marked impacts on the carbon (C) turnover in agro-ecosystems through increased plant photosynthetic rates, leading to an enhanced biomass, and wider plant C/N ratios. Through increased carbon allocation below-ground, as well as through changed litter quality, CO₂ enrichment will indirectly affect soil faunal communities. In the present study we investigated how elevated atmospheric CO₂ and two different levels of N fertilization may affect abundance and diversity of collembolans, as important catalysts in decomposition processes, within an agro-ecosystem under winter wheat cultivation. The investigations were carried out in 2002 within a field experiment using the “Free Air CO₂ Enrichment” technique (FACE) at the Federal Agricultural Research Centre (Braunschweig, Germany). Stable C-isotopic analysis of collembolans, soil, and crops gave insight into C translocation. During our investigations δ¹³C values of all components analysed were significantly more negative under FACE compared to ambient air conditions. Stable C-isotopic signatures of collembolans were similar to those of soil under ambient air, but in between those of soil and roots under elevated CO₂ conditions. Our results revealed significant effects of both treatments (CO₂ enrichment and N fertilization) on density and species diversity of collembolans. Overall, collembolans were stimulated under elevated CO₂ conditions, showing an increased abundance of more than 50% (11 240 ind m⁻²) as well as a higher biodiversity (Shannon Weaver index = 2.5; evenness = 0.75) compared to ambient air conditions (7520 ind m⁻²; Shannon Weaver index = 2.2; evenness = 0.72). With regard to N supply, a decrease of about 20–30% under CO₂ enrichment and 45–55% under ambient air conditions in collembolan abundance with no alteration in diversity was recorded under reduced N fertilization. The observed impacts were species-specific.     

Yield and nitrogen utilization efficiency of the pseudocereals amaranth,quinoa, and buckwheat under differing nitrogen fertilization

In recent years, the cultivation of the pseudocereal species amaranth, quinoa, and buckwheat has gained rising attention. This study was undertaken to explore nitrogen (N) fertility requirements and nitrogen use efficiency of these species. For this purpose, a 2-year field experiment with N rates of 0, 80, and 120 kg N ha⁻¹ for amaranth and quinoa and 0, 30, and 60 kg N ha⁻¹ for buckwheat and two cultivars of each species was conducted.Grain yield of amaranth responded to N and ranged between 1986 and 2767 kg ha⁻¹. Nitrogen utilization efficiency (NUtE) ranged from 13.9 to 15.4 kg grain yield per kg above-ground plant N and decreased with increasing N rate. Higher grain yields and NUtEs seemed to be mainly inhibited by the low harvest index (0.22–0.23) of the investigated amaranth cultivars.Quinoa yielded between 1790 and 3495 kg grain ha⁻¹ and responded strongly to N fertilization. NUtE averaged 22.2 kg kg⁻¹ and did not decrease with increasing N rates.The grain yield of buckwheat did not respond to N fertilization and averaged 1425 kg ha⁻¹. N uptake increased only slightly with N fertilization. NUtE ranged from 16.1 to 20.0 kg kg⁻¹. Main problems occurring with the application of N to buckwheat were grain scattering and lodging.