在毕赤酵母中利用口蹄疫病毒(FMDV)2A实现dTomato和串联MagaininⅡ基因的共表达

抗菌肽(antibacterial peptides)是生物体内经诱导产生的一种具有生物活性的多肽,是天然免疫的重要组成部分.为了提高抗菌肽的表达量以及很方便地检测抗菌肽的表达情况,本研究利用口蹄疫病毒(Foot-and-mouth disease virus,FMDV) 2A将荧光蛋白dTomato基因和3个串联的MagaininⅡ基因融合到一个开放阅读框中(open reading frame,ORE),融合的基因被置于pPIC9K载体醇氧化酶基因(AOX1)启动子的下游,构建分泌型重组酵母表达载体pPIC9K-dTomato -2A-3M,将线性化的重组酵母表达载体通过电击法转入毕赤酵母(Pichia pastoris )GS115中,经G418筛选和PCR鉴定得到阳性转化子,然后将其转至摇瓶,在30℃、0.5％甲醇的条件下进行诱导表达,连续诱导3d.SDS-PAGE电泳图谱显示,在31 kD(dTomato)和9.5kD(3M)处有蛋白条带出现,在荧光显微镜下观察酵母表达上清发出红色荧光,对大肠杆菌(Escherichia coli)和金黄色葡萄球菌(Staphylococcus aureus)抑菌试验结果表明,重组抗菌肽串联的MagaininⅡ具有明显的抑菌效果.结果表明,由2A连接的融合基因(荧光蛋白dTomato和串联的MagaininⅡ)在毕赤酵母中成功的进行了表达,FMDV 2A在其C端剪切多聚蛋白,得到dTomato和串联的MagaininⅡ两个独立而有活性的蛋白,为小分子抗菌肽的表达提供一个高效的检测方法.     

猪生长抑素Ⅱ型受体基因(SSTR2)shRNA的构建与筛选

生长激素(GH)是调控动物生长的重要激素,GH分泌水平的高低主要受正性调控因子生长激素释放因子(GRF)和负性调控因子生长抑素(SS)的双向调节。SS通过生长抑素受体(somatostatin receptor,SSTR)发挥作用,减少GH的分泌水平。本研究设计了3条SSTR2的短发夹RNA(shRNA),构建猪(Sus scrofa)SSTR2真核表达质粒(pcDNA3.1(-)-SSTR2)和SSTR2慢病毒RNA干扰质粒(pshRNA-copGFP lentivector,LV-shRNA),并共转染中国仓鼠(Cricetulus griseus)卵巢细胞系(CHO),通过对转染细胞的荧光观察、Real-time PCR和酶联免疫法(ELISA)检测,结果表明,CHO转染细胞中猪SSTR2 mRNA表达水平不同程度的抑制,分别为90.4%、28.3%和86.3%,(P<0.05)。其中,LV-shRNA1表现出最高的沉默效率:转染效率在80%左右时,猪SSTR2mRNA水平沉默效率为90.4%,蛋白质水平降低了33.3%。本研究成功地筛选到了降低猪SSTR2基因表达的shRNA,为利用shRNA技术下调动物基因表达,构建转基因模型提供思路。     

三峡库区低碳农业发展模式与减排对策

在对三峡库区自然环境及生态农业发展存在问题分析的基础上,运用类比方法估算了三峡库区农业系统温室气体的排放量,结果显示2008年三峡库区耕地CO2,CH4,N2O的排放量分别为10,7,0.5万t/a。在此基础上进一步提出了山地综合立体开发模式、立体生态渔业养殖模式、水土流失综合防治模式、特色循环生态农业复合模式4种低碳农业发展模式及相应的减排对策。     

Response of Rice Seedlings to Copper Toxicity and Acidity

Metal toxicity and soil acidity affect plant growth. A hydroponic experiment was conducted to simulate effects of pH and copper concentration [Cu] on early growth and biomass production of high-yielding rice seedlings (Oryza sativa L. cv. ‘NIAB 6’). The rice seedlings were allowed to grow in Yoshida nutrient solution with 1 (control), 8, 16, and 32 μM Cu at buffered pH levels of 4.5, 5.0, and 5.5, respectively. Shoot and root growth, leaf chlorophyll content, and root lipid peroxidation and K⁺ leakage of 15-day-old rice seedlings were determined. Results show that [Cu] had significant adverse effects on rice seedlings. Shoot height and root length were reduced by 18%–60% and 7%–69%, respectively, compared with the control, and shoot and root weight decreased by 26%–56%, and 25%, respectively, in response to progressive increases in [Cu]. Similarly, leaf chlorophyll content decreased by 150%–245% compared with the control treatment. Rice seedling roots suffered from significant increases in lipid peroxidation followed by K⁺ leakage (> 8–23 times) in response to increasing [Cu]. Averaged across [Cu], seedling growth and biomass were greater at pH 5.5 than at other pH treatments. In some instances, increasing pH minimized the adverse effects of Cu at low concentrations on rice seedlings.     

Emission of dimethyl sulfide,carbonyl sulfide,and carbon bisulfide from paddy fields

Abstract

Emission rates of dimethyl sulficle (DMS), carbonyl sulfide (COS), and carbon disulfide (CS₂) to the atmosphere from paddy fields at Ryugasaki, Ibaraki Prefecture, Japan were measured by using the closed chamber method. DMS was emitted mainly through the rice plant and its emission rate was much higher than those of COS and CS₂. During a cropping period COS was slightly absorbed by the rice plant. Significant diurnal and seasonal variations of DMS iuxes were observed. The highest DMS iux was observed immediately after the heading day of rice plant. The annual DMS emission rate was in the order of mineral plot > straw plot > no-N plot, ranging from 4.5 to 6.9 mg S m^-2 yr^-1. The annual COS emission rate was in the order of straw plot > no-N plot > mineral plot, ranging from -0.2 to 1.8 mg S m^-2 yr^-1. The annual CS₂ emission rate was slightly higher in the straw plot, ranging from 0.9 to 2.0 mg S m^-2 yr^-1.     

不同磷肥用量对水稻土有机碳矿化和细菌群落多样性的影响

以水稻田田间定位试验为研究对象,利用三维荧光光谱技术(3D EEMs)和454测序技术,采集4个施磷水平(0、30、60、90 kg hm-2a-1)土壤,测定其有机碳矿化、溶解有机碳(DOC)组成和结构特征及细菌群落结构和丰度的变化。结果显示:水田施磷增加了土壤速效磷(Olsen-P),从而提高了土壤DOC含量,加速了有机碳的矿化速率和累积矿化量。3D EEMs结果表明,施磷分别显著增加了荧光指数和鲜度指数值1%~10%和3%~21%,而降低了腐殖化指数,且与土壤生化性质(Olsen-P、DOC和β-葡萄糖苷酶)具有显著相关性。说明施磷通过提高Olsen-P,促进了微生物源DOC的生成,同时降低了DOC的芳香化程度、分子量及腐殖化程度,从而提高了DOC生物可降解性。同时,施磷提高了细菌群落的丰度和多样性,特别是磷诱导了多种具有碳降解功能细菌的增加,从而加速了复杂有机碳的降解和甲烷氧化。此外,主成分分析表明稻田磷素施用量在30~60 kg hm-2a-1时对土壤有机碳矿化及细菌群落多样性的提高作用最为明显。因此,适度施磷能显著提高涉碳降解微生物的活性,从而提高DOC的生物可降解性,加速有机碳的矿化速率,促进稻田土壤有机碳循环。     

过量空气系数与废气再循环率耦合对汽油机性能的影响

为了提高汽油机部分负荷下的燃油经济性,该文通过改变废气再循环率(EGR,exhaust gas recirculation)和过量空气系数,在一台增压进气道喷射汽油机上进行试验,研究了过量空气系数与EGR耦合对发动机部分负荷下燃油经济性和排放的影响。结果表明:随着EGR率和过量空气系数α的增加,燃烧循环变动率变大,燃烧持续期变长,且EGR是影响两者的主要因素;泵气损失显著减少,有效燃油消耗率显著降低,与EGR率=0和过量空气系数α=1条件下相比,EGR率=20%和过量空气系数α=1.1条件下的有效燃油消耗率降低了8.37%;随着EGR率的增加,THC(total hydrocarbon)排放增加,但NOx排放显著降低,高达约80%。因此,将三效催化转化器和EGR技术相结合,燃烧稀混合气,可以在满足欧Ⅴ排放法规的同时,大幅提高汽油机部分负荷下的燃油经济性。     

拟南芥DA1-Related Protein 2基因参与调控植物对盐胁迫的响应

土壤盐渍化是目前农业生产面临的主要问题之一,同时种子萌发转绿作为植物幼苗形态建成的基础对盐胁迫最为敏感。本研究以Col-0、Ler野生型拟南芥和osr1短根突变体拟南芥为试验材料,通过图位克隆方法得到调控根生长的DAR2(DA1-Related Protein 2)基因。本研究利用RT-PCR方法,发现生长10 d的Col-0幼苗在200 mmol·L 1氯化钠条件下处理6 h和12 h,DAR2基因受盐胁迫诱导;200 mmol·L 1氯化钠处理后,组织化学染色结果显示,萌发1 d的根尖韧皮部和3 d的叶片pDAR2::GUS的表达上升,进一步表明DAR2基因受到盐胁迫的诱导。统计不同MS培养基[0(CK)、100 mmol·L 1氯化钠、150 mmol·L 1氯化钠、200 mmol·L 1氯化钠、150 mmol·L 1氯化钾、200 mmol·L 1甘露醇]上Col-0和dar2-3的萌发率和转绿率发现:随着氯化钠浓度的逐渐增大,突变体无论萌发还是转绿时间明显比野生型晚。在150 mmol·L 1氯化钾和200 mmol·L 1甘露醇培养条件下,萌发和转绿的时间也比野生型要晚。这些结果表明突变体在萌发和转绿期对盐胁迫的敏感性比野生型明显增强,进一步证明了突变体对盐胁迫的敏感性并不是对离子的特异响应。这些研究结果为深入了解逆境胁迫下植物早期生长发育的可塑性调控机制奠定了基础,同时也为通过生物技术改良作物抗逆性提供了理论依据。     

Effect of mineral nitrogen fertilizer forms on N2O emissions from arable soils in winter wheat production

Nitrogen fertilizers are supposed to be a major source of nitrous oxide (N₂O) emissions from arable soils. The objective of this study was to compare the effect of N forms on N₂O emissions from arable fields cropped with winter wheat (Triticum aestivum L.). In three field trials in North‐West Germany (two trials in 2011/2012, one trial in 2012/2013), direct N₂O emissions during a one‐year measurement period, starting after application of either urea, ammonium sulfate (AS) or calcium ammonium nitrate (CAN), were compared at an application rate of 220 kg N ha^?1. During the growth season (March to August) of winter wheat, N₂O emission rates were significantly higher in all three field experiments and in all treatments receiving N fertilizer than from the non‐fertilized treatments (control). At two of the three sites, cumulative N₂O emissions from N fertilizer decreased in the order of urea > AS > CAN, with emissions ranging from 522–617 g N ha^?1 (0.24–0.28% of applied fertilizer) for urea, 368–554 g N ha^?1 (0.17–0.25%) for AS, and 242–264 g N ha^?1 (0.11–0.12%) for CAN during March to August. These results suggest that mineral nitrogen forms can differ in N₂O emissions during the growth period of winter wheat. Strong variations in the seasonal dynamics of N₂O emissions between sites were observed which could partly be related to weather events (e.g., precipitation). Between harvest and the following spring (post‐harvest period) no significant differences in N₂O emissions between fertilized and non‐fertilized treatments were detected on two of three fields. Only on one site post‐harvest emissions from the AS treatment were significantly higher than all other fertilizer forms as well as compared to the control treatment. The cumulative one‐year emissions varied depending on fertilizer form across the three field sites from 0.05% to 0.51% with one exception at one field site (AS: 0.94%). The calculated overall fertilizer induced emission averaged for the three fields was 0.38% which was only about 1/3 of the IPCC default value of 1.0%.     

等高绿篱--坡地农业复合系统土壤N2O排放特征

N2O是一种重要的温室气体, 具有很强的温室效应。当前全球变化条件下, 人类活动和农业生产行为产生的N₂O排放增加是当前倍受关注的问题。本研究于2008年11月-2009年10月, 利用静态箱 气相色谱技术对亚热带地区紫穗槐(Amorpha fruticosa L.)绿篱枝叶还田条件下冬小麦 夏玉米轮作田土壤N₂O排放通量进行原位监测, 观测紫穗槐枝叶移出(AR)、翻施(AI)、表施(AC)及作物单作(CK)4种处理下整个生长季土壤N₂O的排放量, 对等高绿篱 坡地农业复合生态系统土壤N₂O排放通量变化及其影响机制进行研究。结果表明, 整个冬小麦 夏玉米轮作期, 4个处理土壤N₂O排放通量呈现出相似的季节变化特征, AR、AI、AC、CK处理全生长季的排放总量为127.62 mg·m^-2、209.66 mg·m^-2、208.73 mg·m^-2、77.52 mg·m^-2。作物不同生育阶段N₂O日均排放通量在冬小麦季表现为: 开花-成熟期>拔节-开花期>出苗-拔节期; 在夏玉米季表现为: 拔节-抽雄期>播种-拔节期>抽雄-成熟期。本试验综合评估了等高绿篱 坡地农业复合生态系统土壤N2O排放通量变化及其影响机制。研究显示, 土壤N₂O排放通量在冬小麦季与土壤温度相关性显著, 在夏玉米季与土壤水分相关性显著。在复合生态系统中紫穗槐复合种植及枝叶还田显著促进土壤N2O排放, 翻施处理产生的N2O量大于表施处理。