牛DHX36解旋酶及其突变体底物结合与解旋活性研究

【目的】研究牛(Bos taurus)解旋酶DHX36(BtDHX36)及其突变体蛋白对含有G4结构底物结合和解旋活性的差异,为深入研究牛DHX36酶学特征和结构提供理论参考。【方法】制备底物16nt-ssDNA、Telomere G4和Telomere G4-16T,构建重组质粒pET15b-sumo-BtDHX36,通过Overlap PCR引入点突变,构建位于RSM区和OB域的突变重组质粒,分别将质粒导入大肠杆菌BL21(DE3)菌株中诱导表达;经过Ni-NTA和Hi Trap SP HP柱纯化得到目的蛋白。以解离平衡常数为判定指标,利用荧光各向异性法(FA)对酶蛋白与底物结合时的KCl浓度(0,50和100 mmol/L)、反应温度(25,30和37℃)、MgCl_2浓度(0,2和5 mmol/L)、pH(6.5,7.5和8.5)进行优化,确定BtDHX36的体外最佳底物结合条件,比较野生型BtDHX36及其突变体对含有G4结构底物结合活性的差异。以解旋比例和速率常数为判定指标,利用快速停流-荧光共振能量转移(FRET)技术,比较野生型BtDHX36及其突变体对G4结构的解旋活性差异。【结果】成功构建了野生型BtDHX36重组质粒pET15b-sumo-BtDHX36及其RSM区突变体BtDHX36~(R63AI65A)、BtDHX36~(Y69A)、BtDHX36~(K76AN77AK78A)和OB域突变体BtDHX36~(Y862A)的重组质粒,诱导表达纯化后得到了纯度大于95%的酶蛋白。确定了野生型BtDHX36的体外最佳底物结合条件为:KCl 50 mmol/L、MgCl_2 2 mmol/L、20 mmol/L Tris-HCl pH=7.5、反应温度37℃。各突变位点均可降低酶蛋白与Telomere G4的结合活性。OB域的Y862A突变和RSM区K76AN77AK78A突变对酶蛋白解旋Telomere G4-16T活性均无明显影响;RSM区的R63AI65A和Y69A突变对酶蛋白解旋Telomere G4-16T均有较大影响。【结论】获得了高纯度的野生型BtDHX36及突变体酶蛋白,明晰了其体外最佳底物结合条件;RSM区的R63AI65A和Y69A突变均可明显降低酶蛋白解旋Telomere G4-16T的活性。     

稻田土壤CH4排放及其关联微生物功能基因丰度对磺胺二甲嘧啶和铜污染的响应

为了研究畜禽粪便中磺胺类兽用抗生素磺胺二甲嘧啶(Sulfamethazine,SMZ)和典型重金属铜(Copper,Cu)复合污染对稻田土壤CH_4排放及其微生物相关功能基因丰度的影响,以猪粪为肥源,设置无污染、低、中、高4种SMZ和Cu不同污染水平的浓度,SMZ分别为0、0.1、1、5 mg·kg~(-1),Cu分别为0、100、500、1 500 mg·kg~(-1),两两组合共计16个处理,通过长时间室内模拟淹水培养实验,定期测定CH_4排放速率和相关土壤功能基因丰度,以期探索SMZ和Cu单一及复合污染影响CH_4排放和相关功能基因丰度的浓度效应及影响时长。结果表明:培养前期(0～12 d),不同浓度的SMZ和Cu未显著影响CH_4排放速率(P0.05);培养中后期(13～71 d),单一低浓度SMZ(0.1 mg·kg~(-1))处理对CH_4排放具有促进作用,高浓度SMZ(≥1 mg·kg~(-1))则反之,单一Cu污染及与不同浓度的SMZ复合污染处理均显著降低了CH_4排放速率(P0.05)。与对照(SMZ和Cu零添加)相比,除单一低浓度SMZ(0.1 mg·kg~(-1))处理的CH_4累积排放量增加了19.7%外,其他处理均显著降低了CH_4排放,尤其Cu和SMZ(≥1 mg·kg~(-1))复合污染处理显著降低了约77%(P0.05)。SMZ单一污染对16S rRNA-CH_4、mcr A和pmo A的基因丰度均有明显的浓度效应和输入时间效应,Cu单一污染则未有明显的浓度和时间效应,两者复合污染的联合作用比较复杂,主要取决于两污染物的起始浓度及SMZ输入土壤的时间。无论SMZ和Cu单一还是复合污染,中高浓度在培养的后期对三大基因丰度的影响均以降低为主,且pmo A基因丰度对Cu和SMZ单一及复合污染响应更敏感。研究表明,SMZ和Cu在一定程度上会降低稻田土壤CH_4排放及相关微生物基因丰度,有利于温室气体减排,但规模化养殖业畜禽粪便还田后导致抗生素和重金属高残留的环境风险仍不可忽视。     

农杆菌介导遗传转化获得转CP4基因籼稻的研究

  目的  建立籼稻‘中恢161’Oryza sativa subsp. indica ‘Zhonghui 161’农杆菌Agrobacterium tumefaciens介导的转化体系。  方法  以籼稻‘中恢161’的成熟胚为材料,设置了5个草甘膦质量浓度(100、200、300、400和500 mg·L?1)进行胚性愈伤组织的草甘膦敏感性试验。利用农杆菌介导法,将草甘膦抗性基因CP4-EPSPS导入‘中恢161’的胚性愈伤组织中,转化后的胚性愈伤组织分别在含有300、350和400 mg·L?1草甘膦的选择培养基上进行抗性筛选。抗性愈伤组织进一步分化、成苗。  结果  草甘膦质量浓度为300~400 mg·L?1时,愈伤组织褐化率约50%,具有很好的选择效果。经统计,300、350和400 mg·L?1草甘膦抗性筛选后,愈伤组织阳性率分别为40.16%、61.72%和84.04%,抗性愈伤组织的分化率为46.43%,成苗率为32.84%。共获得67株再生小苗,经PCR检测,43株成功转入CP4基因,再生植株阳性率为64.18%。  结论  建立了‘中恢161’农杆菌介导的转化体系。图5参20     

Some plants like it warmer: Increased growth of three selected invasive plant species in soils with a history of experimental warming

Soil warming can affect plant performance by increasing soil nutrient availability through accelerating microbial activity. Here, we test the effect of experimental soil warming on the growth of the three invasive plant species Trifolium pratense (legume), Phleum pratense (grass), and Plantago lanceolata (herb) in the temperate-boreal forest ecotone of Minnesota (USA). Plants were grown from seed mixtures in microcosms of soils with three different warming histories over four years: ambient, ambient +1.7 °C, and ambient +3.4 °C. Shoot biomass of P. pratense and P. lanceolata and plant community root biomass increased significantly in soils with +3.4 °C warming history, whereas T. pratense responded positively but not significantly. Soil microbial biomass and N concentration could not explain warming effects, although the latter correlated significantly with the shoot biomass of P. lanceolata. Our results indicate that soil with a warming history may benefit some invasive plants in the temperate-boreal ecotone with potential impacts on plant community composition. Future studies should investigate the impact of warming-induced differences in soil organisms and nutrients on plant invasion.     

杭州湾滨海湿地CH4排放通量的研究

2013年4—9月,利用静态明箱–气相色谱法对杭州湾裸滩湿地、海三棱藨草湿地、芦苇湿地和互花米草湿地CH4排放通量进行了原位观测,并利用室内厌氧培养法测定了0~30 cm深度的土壤CH4产生潜力。结果表明:整体而言,裸滩湿地表现为CH4的吸收源,CH4排放通量春季高、夏季低;海三棱藨草湿地、芦苇湿地和互花米草湿地表现为CH4的排放源,CH4排放通量均呈现夏季高、春秋季低的季节变化。平均CH4排放通量表现为:互花米草湿地(1.589 mg/(m2·h))芦苇湿地(0.722 mg/(m2·h))海三棱藨草湿地(0.218 mg/(m2·h))裸滩湿地(–0.068 mg/(m2·h)),互花米草湿地各月CH4排放通量均显著高于其他湿地。0~30 cm深度平均土壤CH4产生潜力表现为:互花米草湿地(0.050μg/(g·d)芦苇湿地(0.042μg/(g·d))裸滩湿地(0.030μg/(g·d)海三棱藨草湿地(0.027μg/(g·d)),互花米草湿地各土层CH4产生潜力显著高于其他湿地(除0~5 cm外)。裸滩湿地土壤CH4产生潜力没有明显的空间垂直变化趋势,CH4产生潜力最大值、最小值分别出现在10~20 cm和5~10 cm土层。其他3类湿地0~5 cm土层的CH4产生潜力最大,土壤CH4产生潜力整体上随着土壤深度的增加而减小;海三棱藨草湿地和芦苇湿地5~10 cm土层的CH4产生潜力最小,互花米草湿地20~30 cm土层的CH4产生潜力最小。土壤p H、有机碳和全氮含量对CH4排放通量有显著的影响。     

中红外光声光谱法测定土壤顶空氨气浓度

利用中红外光声光谱对土壤顶空氨气(NH3)浓度进行测定。结果表明,在动镜速率为0.16 cm/s,分辨率为4 cm–1时获得的NH3光谱噪音较少且信号强度高。利用选定的参数采集不同浓度NH3光谱,其中位于900~1 000 cm–1的特征吸收峰适于定量分析。分别选用该特征吸收的峰高、峰面积和第一主成分与NH3浓度进行回归建模,线性决定系数(R2)均达到0.9以上,表明三者都呈显著线性相关,中红外光声光谱可以用于快速测定土壤顶空中NH3浓度,这为土壤氮素循环研究提供了新手段。     

水稻根际和周围土壤中微生物功能基因丰度与碳、氮状况及其通量之间的关系

Rapid nitrogen（N） transformations and losses occur in the rice rhizosphere through root uptake and microbial activities. However,the relationships between rice roots and rhizosphere microbes for N utilization are still unclear. We analyzed different N forms（NH＋4,NO-3, and dissolved organic N）, microbial biomass N and C, dissolved organic C, CH4 and N2O emissions, and abundance of microbial functional genes in both rhizosphere and bulk soils after 37-d rice growth in a greenhouse pot experiment. Results showed that the dissolved organic C was significantly higher in the rhizosphere soil than in the non-rhizosphere bulk soil, but microbial biomass C showed no significant difference. The concentrations of NH＋4, dissolved organic N, and microbial biomass N in the rhizosphere soil were significantly lower than those of the bulk soil, whereas NO-3in the rhizosphere soil was comparable to that in the bulk soil. The CH4 and N2O fluxes from the rhizosphere soil were much higher than those from the bulk soil. Real-time polymerase chain reaction analysis showed that the abundance of seven selected genes, bacterial and archaeal 16 S rRNA genes, amoA genes of ammonia-oxidizing archaea and ammonia-oxidizing bacteria, nosZ gene, mcrA gene, and pmoA gene, was lower in the rhizosphere soil than in the bulk soil, which is contrary to the results of previous studies. The lower concentration of N in the rhizosphere soil indicated that the competition for N in the rhizosphere soil was very strong, thus having a negative effect on the numbers of microbes. We concluded that when N was limiting, the growth of rhizosphere microorganisms depended on their competitive abilities with rice roots for N.     

牛A型巴氏杆菌高、低毒力株LPS激活RAW264.7细胞TLR4信号通路的比较分析

本研究旨在比较牛A型多杀性巴氏杆菌高毒力株(PmCQ2)和低毒力株(PmCQ6)脂多糖(LPS)对小鼠巨噬细胞RAW264.7内TLR4信号通路的影响。选用体外培养RAW264.7细胞,经LPS刺激后检测细胞内TLR4信号激活及对TNF-α和IL-12p40表达的影响;并利用Western blot检测LPS对IκBα磷酸化及NF-κBp65活化的影响。结果显示,高毒力株LPS_PmCQ2和低毒力株LPS_PmCQ6分别刺激RAW264.7细胞后,均能显著提高TLR4的表达,并诱导细胞因子TNF-α和IL-12p40的分泌表达;LPS_PmCQ2和LPS_PmCQ6均能诱导IκBα磷酸化,促进NF-κBp65核转位,但二者没有显著差异(P>0.05)。本研究表明牛A型多杀性巴氏杆菌高、低毒力株LPS均能参与TLR4介导的小鼠巨噬细胞免疫应答,且二者对TLR4介导的IκBα-NF-κB信号通路的作用无显著差异,暗示牛A型多杀性巴氏杆菌对小鼠巨噬细胞的毒力与LPS无明显相关性,可能由其他毒力因子决定。