Highly efficient soluble expression and purification of recombinant human basic fibroblast growth factor (hbFGF) by fusion with a new collagen-like protein (Scl2) in Escherichia coli

Abstract

Human basic fibroblast growth factor (hbFGF) is involved in a wide range of biological activities that affect the growth, differentiation, and migration. Due to its wound healing effects and therapy, hbFGF has the potential as therapeutic agent. Therefore, large-scale production of biologically active recombinant hbFGF with low cost is highly desirable. However, the complex structure of hbFGF hinders its high-level expression as the soluble and functional form. In the present study, an efficient, cost-effective, and scalable method for producing recombinant hbFGF was developed. The modified collagen-like protein (Scl2-M) from Streptococcus pyogenes was used as the fusion tag for producing recombinant hbFGF for the first time. After optimization, the expression level of Scl2-M-hbFGF reached approximately 0.85?g/L in the shake flask and 7.7?g/L in a high cell-density fermenter using glycerol as a carbon source. Then, the recombinant Scl2-M-hbFGF was readily purified using one-step acid precipitation and the purified Scl2-M-hbFGF was digested with enterokinase. The digested mixture was further subject to ion-exchange chromatography, and the final high-purity (96%) hbFGF product was prepared by freeze-drying. The recovery rate of the whole purification process attained 55.0%. In addition, the biological activity of recombinant hbFGF was confirmed by using L929 and BALB/c3T3 fibroblasts. Overall, this method has the potential for large scale production of recombinant hbFGF.     

Assessing the response of forest productivity to climate extremes in Switzerland using model–data fusion

The response of forest productivity to climate extremes strongly depends on ambient environmental and site conditions. To better understand these relationships at a regional scale, we used nearly 800 observation years from 271 permanent long‐term forest monitoring plots across Switzerland, obtained between 1980 and 2017. We assimilated these data into the 3‐PG forest ecosystem model using Bayesian inference, reducing the bias of model predictions from 14% to 5% for forest stem carbon stocks and from 45% to 9% for stem carbon stock changes. We then estimated the productivity of forests dominated by Picea abies and Fagus sylvatica for the period of 1960–2018, and tested for productivity shifts in response to climate along elevational gradient and in extreme years. Simulated net primary productivity (NPP) decreased with elevation (2.86 ± 0.006 Mg C ha^?1 year^?1 km^?1 for P. abies and 0.93 ± 0.010 Mg C ha^?1 year^?1 km^?1 for F. sylvatica). During warm–dry extremes, simulated NPP for both species increased at higher and decreased at lower elevations, with reductions in NPP of more than 25% for up to 21% of the potential species distribution range in Switzerland. Reduced plant water availability had a stronger effect on NPP than temperature during warm‐dry extremes. Importantly, cold–dry extremes had negative impacts on regional forest NPP comparable to warm–dry extremes. Overall, our calibrated model suggests that the response of forest productivity to climate extremes is more complex than simple shift toward higher elevation. Such robust estimates of NPP are key for increasing our understanding of forests ecosystems carbon dynamics under climate extremes.     

Response of foundation macrophytes to near‐natural simulated marine heatwaves

Marine heatwaves have been observed worldwide and are expected to increase in both frequency and intensity due to climate change. Such events may cause ecosystem reconfigurations arising from species range contraction or redistribution, with ecological, economic and social implications. Macrophytes such as the brown seaweed Fucus vesiculosus and the seagrass Zostera marina are foundation species in many coastal ecosystems of the temperate northern hemisphere. Hence, their response to extreme events can potentially determine the fate of associated ecosystems. Macrophyte functioning is intimately linked to the maintenance of photosynthesis, growth and reproduction, and resistance against pathogens, epibionts and grazers. We investigated morphological, physiological, pathological and chemical defence responses of western Baltic Sea F. vesiculosus and Z. marina populations to simulated near‐natural marine heatwaves. Along with (a) the control, which constituted no heatwave but natural stochastic temperature variability (0HW), two treatments were applied: (b) two late‐spring heatwaves (June, July) followed by a summer heatwave (August; 3HW) and (c) a summer heatwave only (1HW). The 3HW treatment was applied to test whether preconditioning events can modulate the potential sensitivity to the summer heatwave. Despite the variety of responses measured in both species, only Z. marina growth was impaired by the accumulative heat stress imposed by the 3HW treatment. Photosynthetic rate, however, remained high after the last heatwave indicating potential for recovery. Only epibacterial abundance was significantly affected in F. vesiculosus. Hence both macrophytes, and in particular F. vesiculosus, seem to be fairly tolerant to short‐term marine heatwaves at least at the intensities applied in this experiment (up to 5°C above mean temperature over a period of 9 days). This may partly be due to the fact that F. vesiculosus grows in a highly variable environment, and may have a high phenotypic plasticity.     

雪灾干扰后林冠开阔度对黄心树幼苗更新的影响

幼苗更新是森林更新的关键过程,而林窗对于植物幼苗更新、生长及最终存活具有重要影响。极端气候事件会使森林在短时间内形成大量林窗,从而显著影响林内幼苗更新和存活。本研究以2015年雪灾后云南省哀牢山中山湿性常绿阔叶林优势种黄心树(Machilus bombycina)幼苗为对象,对194个1 m×1 m样方中的幼苗生长和死亡动态进行为期4年的监测,利用线性混合模型(LMM)和广义线性混合模型(GLMM),分析林冠开阔度对幼苗高度相对生长率、叶片相对增长率、新增率和死亡率的影响,研究了雪灾干扰后的林冠开阔度对植物幼苗更新的影响。结果表明:(1)林冠开阔度随时间推移逐渐变小(20.07%~9.97%),且速度由快到慢;(2)2015-2016年,已有幼苗高度相对生长率与林冠开阔度呈显著正相关(P<0.05),其余年份两者间无显著相关性;2016-2017年已有幼苗叶片相对增长率与林冠开阔度呈显著正相关(P<0.05),其余年份两者间无显著相关性;(3)林冠开阔度与幼苗新增率和死亡率均无显著相关性。本研究表明,雪灾后造成的林冠开阔度增加有利于黄心树幼苗生长。     

氮添加与降雨变化对红砂幼苗非结构性碳水化合物的影响

非结构性碳水化合物(NSC)在植物体内的含量及分配对植物生长和存活至关重要。开展氮沉降和降雨变化对幼苗NSC影响的研究,为揭示干旱导致幼苗死亡机理及预测气候变化背景下幼苗自然更新及培育提供依据。本研究以1年生红砂幼苗为对象,测定了不同降雨(降雨减少(W-)、自然降水(W)和降雨增多(W+))和氮添加(N0(0 g N·m^-2·a^-1)、N1(4.6 g N·m^-2·a^-1)、N2(9.2 g N·m^-2·a^-1)、N3(13.8 g N·m^-2·a^-1))条件下红砂幼苗各器官NSC及其组分含量。结果表明:红砂幼苗各器官NSC含量为28.8~71.8mg·g-1,叶片含量最高,茎含量最低。氮添加和降雨变化对红砂幼苗叶片和根系淀粉及总NSC含量有显著影响,而对茎无显著影响。各降雨条件下,氮添加均促进了红砂幼苗叶片淀粉和总NSC累积,在降雨增加30%的条件下氮的促进效应更显著,中高氮(N2和N3)叶片淀粉与总NSC含量显著高于低氮水平(N1和N0);在低氮降雨减少30%(N1W-)处理下,红砂叶片淀粉和NSC含量最小,而根系淀粉和NSC含量最大,即低氮干旱胁迫下红砂可通过NSC在不同器官的重新分配来适应胁迫环境。在自然降雨和降雨增加30%情况下,根系淀粉和NSC的含量随氮添加的增加而减小,且中高氮处理(N2和N3)显著低于对照(N0)。可见,叶片是红砂NSC的源,氮添加会促进红砂幼苗叶片NSC的累积,且这种促进效应与水分紧密相关,在降水增加情况下其效应更显著;而过量的氮添加会抑制根系NSC含量的积累,在低氮干旱胁迫下红砂也可通过叶片NSC向根系转移来适应逆境胁迫。     

极端干旱对草甸草原优势植物非结构性碳水化合物的影响

植物光合作用产生的非结构性碳水化合物(NSCs)水平可以反映植物和生态系统对环境变化的响应程度。近年来, 草原极端干旱事件的发生频率和持续时间增加趋势明显, 对生态系统结构和功能产生深远影响。该研究以内蒙古呼伦贝尔草甸草原为研究对象, 通过连续4年减少66%生长季降水量的控制实验来模拟极端干旱事件, 分析草原6种优势物种和植物功能群NSCs各组分对极端干旱的响应规律与机制。结果显示, 由于植物生物学、光合特性以及生理生态等特性的差异, 不同物种对干旱胁迫的响应具有明显差异。这表明草地植物NSCs组分及其利用策略对干旱胁迫的响应具有物种特异性, 从而导致其生物量的不同响应。将6种植物分为禾草和非禾草两类, 发现干旱显著增加了禾草的淀粉含量, 但对其可溶性糖含量无显著影响; 相反, 干旱显著增加了非禾草功能群的可溶性糖含量, 对其淀粉含量无显著影响, 表明不同功能群采取了不同的干旱应对策略。禾草选择将光合作用固定的能量进行储存以应对干旱胁迫, 其生物量对干旱响应不敏感; 而非禾草选择将能量以可溶性糖的形式直接供植物生长利用以及抵御干旱胁迫, 其生物量对干旱响应较为敏感。这一发现可为预测在全球气候变化背景下草甸草原生态系统结构与功能对极端干旱的响应提供科学参考。     

氮和水分添加对内蒙古荒漠草原放牧生态系统土壤呼吸的影响

土壤呼吸是生态系统碳循环的重要组成部分, 同时也是评价生态系统健康状况的重要指标, 对于评估退化草地恢复过程中生态系统功能具有重要意义。该研究在内蒙古四子王旗短花针茅(Stipa breviflora)荒漠草原长期放牧实验平台上进行, 该平台设置对照(CK)、轻度(LG)、中度(MG)和重度(HG) 4个放牧强度。通过在4个放牧处理区设置氮、水添加实验处理, 探讨不同放牧强度背景下, 氮、水补充对荒漠草原土壤呼吸过程的影响。结果表明: (1)历史放牧强度除2015年对土壤呼吸无显著影响, 2016和2017年都有显著影响, 放牧区3年平均土壤呼吸速率基本都高于对照区。此外, 氮和水分添加显著增加了MG区土壤呼吸速率, HG区氮、水同时添加对土壤呼吸速率有显著增加作用; (2)无论是历史放牧强度, 还是氮、水添加处理, 都没有改变荒漠草原生长季土壤呼吸速率的季节动态变化趋势, 土壤呼吸速率基本表现为单峰曲线模式, 峰值出现在水热同期的7月份; (3)不同年份生长季土壤呼吸速率对氮、水处理的响应并不相同, 氮添加至第3年产生显著影响。水分添加在平水年份(2015和2017年)对土壤呼吸产生显著影响, 但在丰水年份(2016年)无显著影响。氮、水共同添加分别在CK、LG和HG区3年平均土壤呼吸速率显著高于单独加水处理, 说明氮添加的有效性依赖于水分条件, 两者表现为协同作用; (4)不同处理下荒漠草原土壤呼吸的温度敏感性(Q₁₀)值介于1.13-2.41之间, 平均值为1.71。在无氮、水添加时, 放牧区的Q₁₀值都小于CK区, 总体表现为CK 大于 MG 大于 LG 大于 HG; 加水和氮水共同添加处理后, Q₁₀值都有明显增加, 其中NW处理下Q₁₀值都增加到2.0以上。上述结果说明在过去受不同放牧强度影响的荒漠草原在停止放牧后的恢复过程中, 土壤水分仍是影响土壤呼吸的主导环境因子, 外源氮添加只有在满足一定水分供给的基础上才起作用, 尤其是过去的重度放牧区土壤呼吸速率对氮、水补充的响应最为强烈。该研究结果可以为评估荒漠草原恢复过程中土壤呼吸速率受养分和水分影响提供基础资料和依据。     

西南山地流域NDVI变化特征及降水敏感性——以贵州沅江流域为例

归一化植被指数(Normalized Difference Vegetation Index,NDVI)是反映植被生长的重要指标,探究其时空变化特征,对生态保护、退耕还林和灾害防治具有重要意义。选取2000—2015年贵州沅江流域典型年份NDVI,采用重心分析、标准差椭圆、地理加权回归等方法,从干旱视角探究流域NDVI变化和降水敏感性。得出以下结论:(1)2000—2015年贵州沅江流域NDVI总体处于上升趋势,植被正在改善,期间西部植被改善最为明显,退耕还林还草取得较好成果。(2)2000—2015年贵州沅江流域干旱年与正常年NDVI空间变化趋势不一致,干旱程度对植被影响具有区域差异。(3)贵州沅江流域NDVI与降水呈负相关,空间敏感性自西向东递增,降水对流域西部喀斯特山区NDVI影响更明显。(4)2000—2015年贵州沅江流域NDVI时段变化与降水主要为负相关,降水越多NDVI时段变化敏感性越弱,退耕还林还草工程应重点放在负敏感区。研究结果可为退耕还林还草和干旱灾害防治提供科学参考。