Biochar addition mitigates nitrogen loss induced by straw incorporation and nitrogen fertilizer application

Biochar has been shown to be potentially beneficial for enhancing yields and soil properties, and diminishing nitrogen (N) losses. However, it remains unclear how biochar regulates soil carbon (C) and N to mitigate N losses induced by straw mixing with N fertilizer in dryland soils. Therefore, we investigated the effects of straw mixing (S₁), S₁ with biochar (SB) and no straw inputs (S₀), and routine urea application rates (N₁) and 70% of routine rates (N_0.7) on yields and N losses, and identify the relationship between N losses and soil C and N compounds. Results showed that N_0.7 and N₁ were suitable for the maize and wheat seasons, respectively, contributing to mitigating N losses without reducing crop yields. Moreover, in the maize season, N_0.7-SB significantly mitigated the straw-induced NH₃-N and N₂O-N emissions by 106% and 81%, respectively. In the wheat season, N₁-SB reduced the straw-induced NH₃-N and N₂O-N emissions by 35% and 66%, respectively. In addition, N_0.7-SB sharply reduced soil inorganic N (SIN) storage in the maize season. Furthermore, the NH₃-N and N₂O-N emission rates were negatively correlated with dissolved organic carbon/SIN content (0–20 cm) (DOC/SIN_0-20). N losses (N₂O-N and NH₃-N emissions and SIN storage) were positively correlated with SIN_0-20, but negatively correlated with soil organic carbon / SIN_0-20 (SOC/ SIN_0-20). This study provides further evidence that biochar with an appropriate N application rate decreased SIN_0-20 and increased DOC/SIN_0-20, thus reducing SIN storage and the straw-induced gaseous N emissions without decreasing crop yields.     

纳米氧化锌和纳米二氧化钛对尾草履虫急性毒性效应的比较研究

[目的][方法] 本研究以原生动物尾草履虫（Paramecium caudatum）为实验对象, 对目前广泛应用的两种纳米材料，纳米二氧化钛（TiO2）和纳米氧化锌（ZnO）的24小时急性毒性进行了比较研究，分析了草履虫在两种纳米材料胁迫下抗氧化酶活性的改变，并结合两种纳米材料的超微形态，探讨了纳米材料毒性效应的机制，为两种纳米材料的环境毒性监测与评价提供依据。[结果]结果显示：对尾草履虫的24小时半数效应浓度(24h-EC50)分别为51.580mg·L-1（TiO2）、0.444mg·L-1（ZnO）；最低有影响浓度(24h-LOEC)分别为1.712mg·L-1（TiO2）、0.352mg·L-1（ZnO）；以EC50浓度24小时胁迫尾草履虫，获得抗氧化酶活性改变为：超氧化物歧化酶(SOD)，过氧化氢酶(CAT)和过氧化物酶(POD)活性与对照组相比有不同程度的改变，经成对检验分析，两种纳米材料对尾草履虫的POD酶表现出较为明显的抑制作用。[结论]上述结果表明：纳米氧化锌对草履虫的24小时急性毒性大于纳米二氧化钛，这与纳米氧化锌对草履虫抗氧化酶的抑制作用更强，以及纳米氧化锌(30nm)的超微形态相较于纳米二氧化钛(25nm)更加纤细和尖锐有关；本研究获得的24小时急性毒性指标可用于监测和评价两种纳米材料的环境毒性，并为纳米材料的环境容量和生态安全阈值确定提供参考。     

内源H2O2对水稻种子萌发的影响

通过用不同浓度的烟酰胺腺嘌呤二核苷酸磷酸（nicotinamide adenine dinucleotide phosphate, NADPH）合成酶抑制剂碘二苯（diphenylene iodonium, DPI）及过氧化氢（hydrogen peroxide, H₂O₂）清除剂二甲基硫脲（dimethylthiourea, DMTU）分别培养水稻（Oryza sativa L.）种子,研究内源H₂O₂对种子萌发过程胚根、胚芽和胚根根尖活力等的影响。结果表明,DPI及DMTU培养的水稻种子其胚根生长和胚芽生长均受到抑制,尤其是DPI对胚根生长的抑制作用更为显著。DPI和DMTU对水稻种子萌发的影响均呈现出浓度效应,即浓度越高,抑制作用越强。其中,DPI对水稻种子萌发的抑制作用比DMTU的更为明显。此外,胚根根尖的超氧阴离子（superoxide anion, O ^2-）和H₂O₂含量随DMTU浓度增大而减少,根尖细胞受损也越严重。由此推测,内源H₂O₂可能参与调控水稻种子萌发过程。     

CRISPR/Cas9介导的多基因编辑系统在大豆中的应用

自CRISPR/Cas9（clustered regularly interspaced short palindromic repeats/CRISPR-associated protein 9）基因 组编辑技术发现以来，迅速在作物中得到广泛应用。但是，CRISPR/Cas9多基因编辑系统在大豆中的研究尚待开 发。本文利用CRISPR/Cas9介导的多基因编辑系统，分别构建了两个载体，一个载体含6个靶点，编辑7个大豆基因 （4个Glycine max ASYMMETRIC LEAVES1（GmAS1）同源基因和3个GmAS2 同源基因），另一个载体含8个靶点，编辑 11个G. max AGAMOUS 家族同源基因（4个GmAG 同源基因，2个G. max SEEDSTICK（GmSTK）同源基因和5个G. max SHATTERPROOF1（GmSHP1/2）同源基因）。大豆遗传转化后，经表型鉴定和靶点检测发现，CRISPR/Cas9介导的多 基因编辑系统在大豆中成功实现了多基因编辑。当3个GmAS1 同源基因和3个GmAS2 同源基因同时突变时，导致 大豆叶片向远轴面弯曲、皱缩且叶柄变短的表型。当2个GmSHP1 同源基因和2个GmSTK 同源基因同时突变时，导 致豆荚停止发育的不育表型。     

CRISPR/Cas9技术敲除酿酒酵母gpd2基因对产2,3-丁二醇的影响

旨在利用CRISPR/Cas9基因编辑技术敲除酿酒酵母甘油-3-磷酸脱氢酶基因(gpd2),探究其对2,3-丁二醇产量的影响。根据酿酒酵母(Saccharomyces cerevisiae)W5甘油-3-磷酸脱氢酶基因(gpd2)设计供体片段及gRNA片段,将gRNA片段与可表达Cas9蛋白的敲除载体相连,之后将重组质粒及供体DNA片段转化到S. cerevisiae W5细胞中,根据表型筛选及PCR验证获得gpd2基因缺失菌株。结果表明目的基因gpd2敲除成功,基因缺失菌株与原始菌株经发酵实验相比,甘油产量下降22.01%,乙醇产量提高24.65%,2,3-丁二醇产量下降10.60%。gpd2基因的敲除并没有提高2,3-丁二醇的产量,原因可能是逐渐积累的NADH会优先被细胞内大量的乙醇脱氢酶所氧化,作用于乙醇的产生,而不是优先作用于2,3-丁二醇的合成。本实验构建了适用于酿酒酵母的基因敲除系统,该系统对进一步探究酿酒酵母其他代谢产物与2,3-丁二醇合成之间的关系具有实际的借鉴意义。     

不同质地黑土净氮转化速率和温室气体排放规律研究

为探讨黑龙江省半干旱地区不同质地黑土的净氮转化速率和温室气体排放规律,以壤砂土和粉壤土为研究对象开展室内培养试验,对土壤净硝化速率和净矿化速率、N2O和CO2排放速率与累积排放量进行研究。结果表明:7d培养期间壤砂土的平均净矿化速率和CO2平均排放速率分别为0.49mgN kg-1 d-1和0.30mgCO2-C kg-1 h-1,显著低于粉壤土的平均净矿化速率(1.37 mgN kg-1 d-1)和CO2平均排放速率(0.47mgCO2-C kg-1 h-1)。壤砂土的平均净硝化速率和N2O平均排放速率分别为1.65mgN kg-1 d-1和212.6ngN2O-N kg-1 h-1,显著低于粉壤土的5.02mgN kg-1 d-1和521.3ngN2O-N kg-1 h-1。壤砂土和粉壤土的N2O排放比率分别为0.081%~0.301%和0.210%~0.254%。研究表明,土壤质地显著影响土壤净氮转化速率和温室气体排放,壤砂土较低的pH、有机碳和水溶性有机碳含量是导致其净硝化速率、净矿化速率以及N2O、CO2排放速率显著低于粉壤土的主要原因。     

围垦对滨海稻田土壤N2O还原潜力的影响

稻田湿地生态系统的N₂O还原消耗潜力对缓解大气温室气体效应具有重要意义，而滨海自然湿地围垦改造成稻田后耕层土壤的N₂O还原速率及其微生物机制却鲜有报道。选取崇明岛光滩湿地为对照（WK0），比较研究不同围垦年限（19、27、51、86 a）的围垦区稻田耕作层土壤N₂O还原速率演替规律及其微生物数量变异特征。结果表明，土壤总有机碳含量（TOC）随围垦年限增长而显著增加，而土壤pH值、SO₄^2-浓度和EC值则均随围垦年限增长而呈逐渐下降趋势。土壤N₂O还原速率随围垦年限增长而显著增加，其中围垦86 a稻田土壤达到25.5 μg N₂O·g^-1·d^-1，与光滩湿地相比增加了58.4%。定量PCR结果发现，功能基因nosZ Ⅰ和nosZ Ⅱ拷贝数也随着围垦年限增长而显著增加，其中围垦86 a的稻田土壤功能基因分别为1.72×10⁸ copies·g^-1和4.36×10⁸ copies· g^-1，比光滩湿地稻田高出一个数量级。相关性分析发现土壤N₂O还原速率与功能基因nosZ Ⅰ拷贝数呈显著正相关，而功能基因nosZ Ⅱ拷贝数随围垦年限的增加率远高于功能基因nosZ Ⅰ；N₂O还原速率、功能基因nosZ Ⅰ、nosZ Ⅱ拷贝数与3个土壤理化指标（pH、EC、SO₄^2-）均呈负相关。因此，围垦造田促进了滨海湿地土壤N₂O还原过程，而功能基因nosZ Ⅰ数量的大幅增加是N₂O还原速率增加的重要原因。     

Effects of calpain-2 and autophagy-related protein 5 on hepatocyte apoptosis induced by endoplasmic reticulum stress

AIMTo investigate the effects of calpain-2 and autophagy-related protein 5 (Atg5) on apoptosis of BRL-3A rat normal liver cells during endoplasmic reticulum stress (ERS) induced by dithiothreitol (DTT). METH?ODS: BRL-3A cells were treated with DTT at 2.0 mmol/L for 0, 6, 12 and 24 h to induce ERS. Real-time cell analysis (RTCA) was used to measure the effect of DTT on BRL-3A cell proliferation. Apoptosis and cell cycle distribution were analyzed by flow cytometry. The mRNA expression of calpain-2 and Atg5 was detected by real-time PCR. The protein levels of calpain-2, Atg5, Atg7, Atg12 and microtubule-associated protein 1 light chain 3 (LC3) were determined by Western blot. The interaction between calpain-2 and Atg5 was investigated by co-immunoprecipitation (Co-IP). RESULTSThe proliferation of BRL-3A cells treated with DTT was significantly inhibited. The apoptosis of BRL-3A cells was significantly increased after DTT treatment for 6, 12 and 24 h as compared with 0 h group (P<0.05). The cell cycle was arrested in G₁ phase after DTT treatment (P<0.05). After DTT treatment for 6, 12 and 24 h, the mRNA expression of calpain-2 and Atg5 in the BRL-3A cells was significantly increased as compared with 0 h group (P<0.05). The protein levels of calpain-2, Atg12 and Atg7 in the cells treated with DTT for 6, 12 and 24 h were significantly higher than those in 0 h group, and the ratio of LC3-II/LC3-I was also significantly higher than that in 0 h group, while Atg5 expression was significantly lower than that in 0 h group (P<0.05). The results of Co-IP found that the anti-calpain-2 antibody precipitated Atg5 protein from the cell lysates, and the anti-Atg5 antibody also precipitated calpain-2 from the cell lysates, which confirmed the interaction between calpain-2 and Atg5. CONCLUSION Calpain-2 may participate in ERS-induced hepatocyte apoptosis by interacting with Atg5.     

Roles of protein phosphatase 4 in down-regulation of eNOS Ser633 phosphorylation induced by palmitic acid in human umbilical vein endotheli?al cells

AIMTo investigate the roles of protein phosphatase 4 (PP4) in down-regulation of endothelial nitric oxide synthase (eNOS) Ser633 phosphorylation induced by palmitic acid (PA). METHODSHuman umbilical vein endothelial cells (HUVECs) were treated with PA at 25 μmol/L, 50 μmol/L, 100 μmol/L and 200μmol/L for 36 h, or treated with PA at 100 μmol/L for 12 h, 24 h, 36 h and 48 h. Protein phosphatase 2A (PP2A) family inhibitor fostriecin (FST, 20 nmol/L) or okadaic acid (OA, 5 nmol/L) was selected to pretreat the HUVECs for 30 min. Protein phosphatase 4 catalytic subunit (PP4c) siRNA or protein phosphatase 2A catalytic subunit (PP2Ac) siRNA was transfected into the HUVECs. The protein expression levels of of eNOS, PP4c and PP2Ac, as well as the level of eNOS Ser633 phosphorylation, were detected by Western blot. The intracellular nitric oxide (NO) content was measured by DAF-FM DA. RESULTS(1) Compared with control group, the levels of eNOS Ser633 phosphorylation were decreased in PA groups in which the HUVECs were treated with 25 μmol/L, 50 μmol/L, 100 μmol/L and 200 μmol/L PA for 36 h (P<0.05) and 100 μmol/L PA for 24 h, 36 h and 48 h (P<0.05). No significant difference in the level of total eNOS protein expression among all the groups was observed. (2) Compared with control group, both FST and OA pretreatment reversed the reduction of eNOS Ser633 phosphorylation (P<0.05) and the decrease in intracellular NO content (P<0.05) induced by PA. No significant difference in the level of total eNOS protein expression among all the groups was observed. (3) Compared with si-Control group, the PP4c protein expression was significantly reduced (P<0.05), while the level of eNOS Ser633 phosphorylation was significantly increased in si-PP4c group (P<0.05). Although the levels of PP2Ac protein expression declined significantly (P<0.05), the level of eNOS Ser633 phosphorylation remained unchanged in si-PP2Ac group. No significant differencein the level of total eNOS protein expression among all the groups was found. CONCLUSION PA significantly reduces the level of eNOS Ser633 phosphorylation and the content of NO in the HUVECs, which may be due to PA inducing the activation of the PP2A family member PP4 rather than PP2A.     

MCP-1/CCR2 signaling pathway mediates alcohol-induced angiogenesis in breast cancer

AIM To investigate the role of monocyte chemoattractant protein-1 (MCP-1) and its receptor CC chemokine receptor 2 (CCR2) in ethanol-promoted breast cancer angiogenesis and the underlying mechanism. METH?ODS: A mouse model of transplanted breast tumor with moderate alcohol consumption was established. The correlations between the expression of MCP-1/CCR2 and the expression of angiogenesis markers [platelet endothelial cell adhesion molecule-1 (PECAM-1) and vascular endothelial growth factor (VEGF)] in tumor tissues were examined by immunohistochemistry. In vitro, a 3D tumor-endothelial co-culture system was established to observe tumor angiogenesis and the role of MCP-1/CCR2 signaling pathway in alcohol-mediated angiogenesis. The cell migration ability was detected to clarify whether MCP-1/CCR2 enhanced cell mobility to form new vessels. RESULTS MCP-1 and CCR2 were both highly expressed in the breast tumor tissues of tumor-bearing mice consuming alcohol, and their expression levels were consistent with the angiogenic markers PECAM-1 and VEGF (P<0.05). The interaction between mouse breast cancer E0771 cells and endothelial cells was observed to promote angiogenesis in the 3D tumor-endothelial co-culture system with or without alcohol stimulation. MCP-1 promoted this kind of tumor angiogenesis, while CCR2 antagonist effectively inhibited the tumor angiogenesis and especially blocked alcohol-induced angiogenesis. Activation of MCP-1/CCR2 signaling pathway enhanced the migration ability of endothelial cells. CONCLUSION The MCP-1/CCR2 signaling pathway plays an important role in promoting the angiogenesis of breast cancer stimulated by alcohol. The mechanism might be that MCP-1 improves the migration of endothelial cells and then promotes angiogenesis.