不同钙磷配比及浓度对多花黑麦草生长特性的影响

为揭示多花黑麦草（Lolium multiflorum Lamk）对不同钙磷环境的适应机制，设置3个不同钙磷比（1∶1、2∶1、5∶1），每个钙磷比下设置两种钙磷浓度，以不添加钙和磷的营养液处理为对照，探讨不同钙磷水平下多花黑麦草形态和生理指标的变化；并利用隶属函数综合评价法来筛选适宜多花黑麦草生长的钙磷配比及浓度。结果表明：①Ca/P比为1∶1时，随Ca2+、P浓度的增加，株高、叶宽、光合色素和脯氨酸（proline, Pro）含量均呈下降趋势，丙二醛（malondialdehyde, MDA）和超氧化物歧化酶（superoxide dismutase, SOD）活性呈升高趋势；②Ca/P比为2∶1时，随Ca2+、P的增加，株高、叶宽存在一定的下降，叶绿素含量、MDA含量和SOD活性差异不显著，类胡萝卜素和Pro含量升高；③Ca/P比为5∶1时，随Ca2+、P浓度的增加，株高、叶宽、光合色素含量升高，MDA含量下降，Pro含量和SOD活性变化不明显；④P浓度相同时，随着Ca2+浓度的增加，叶绿素、类胡萝卜素含量和SOD活性呈降低趋势；⑤通过隶属函数综合评价，多花黑麦草适宜生长的Ca、P浓度分别为50和10 mg·kg-1。以上结果为多花黑麦草的栽培种植和钙磷响应机理研究提供理论依据。     

Seaweed polysaccharide mitigates intestinal barrier dysfunction induced by enterotoxigenic Escherichia coli through NF-κB pathway suppression in porcine intestinal epithelial cells

This study aimed to investigate the protective effects and underlying mechanism of seaweed polysaccharide (SWP) on intestinal epithelial barrier dysfunction induced by E. coli in an IPEC-J2 model. A preliminary study was done to screen optimum SWP concentrations by cell viability, cytotoxicity, apoptosis and proliferation evaluation. The regular study was conducted to evaluate the protective effects of SWP against E. coli challenge via the analysis of transepithelial electrical resistance (TEER), tight junction proteins, NF-κB signalling pathway, proinflammatory cytokines and the E. coli adhesion and invasion. Our results show that 4 h E. coli challenge down-regulated tight junction proteins expression, decreased TEER, activated NF-κB signalling pathway and increased proinflammatory response, which indicates that the E. coli infection model was well-established. Pre-treatment with 240 μg/ml SWP for 24 h alleviated the 4 h E. coli -induced intestinal epithelial barrier dysfunction, as evidenced by the up-regulated expression of Occludin, Claudin-1 and ZO-1 at both mRNA and protein level and the increased TEER of IPEC-J2 cells. Pre-incubation with 240 μg/ml SWP for 24 h inhibited the activation of the NF-κB signalling pathway by 4 h E. coli challenge, including the decreased mRNA expression of TLR-4, MyD88, IκBα, p-65, as well as the reduced ratio of protein expression of p-p65/p65. Also, pre-treatment with 240 μg/ml SWP for 24 h decreased proinflammatory response (IL-6 and TNF-α) induced by 4 h E. coli challenge and decreased the E. coli adhesion and invasion. In conclusion, SWP mitigated intestinal barrier dysfunction caused by E. coli through NF-κB pathway in IPEC-J2 cells and 240 μg/ml SWP exhibited better effect. Our results also provide a fundamental basis for SWP in reducing post-weaning diarrhoea of weaned piglets, especially under E. coli -infected or in-feed antibiotic-free conditions.     

土壤微生物生物地理学：国内进展与国际前沿

土壤微生物生物地理学是研究土壤中微生物空间分布格局及其随时间变化的一门科学,是土壤微生物学和微生物生态学等领域的研究前沿。近年来,尽管土壤微生物生物地理学研究取得了巨大进展,目前仍面临诸多难题与挑战。本文简要回顾了土壤微生物生物地理学的发展历程,重点介绍近年来我国在森林、草地和农田生态系统中土壤微生物生物地理学研究的主要进展。同时进一步阐述了目前土壤微生物生物地理学研究的国际前沿方向,包括微生物群落空间分布及其驱动机制、群落构建过程与共存网络、微生物地理分布与生态系统功能的关联以及预测微生物群落对全球变化的响应。最后,对土壤微生物生物地理学未来的研究方向进行了展望,强调了清晰的微生物物种定义、微生物群落的时间动态、多组学与合成生物学技术以及高精度的预测模型在土壤微生物生物地理学研究中的重要性。     

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.     

利用CRISPR/Cas9系统敲除葡萄中VviEDR2提高对白粉病的抗性

利用CRISPR/Cas9系统定点编辑葡萄白粉病感病基因VviEDR2（Enhanced disease resistance 2），在VviEDR2的DUF1336结构域设计靶位点VviEDR2-T1，构建CRISPR/Cas9敲除载体，通过农杆菌介导法转化‘无核白’葡萄胚性愈伤组织。对PCR阳性植株进行靶位点扩增测序，结果表明，共有8个转基因植株在靶位点处发生不同类型的双等位基因突变，编辑效率为32%；突变体植株生长势较弱，叶片较小，茎秆丛生、细弱。进一步对突变体植株进行抗病检测，结果表明，接种葡萄白粉菌（Erysiphe necator Schw.）5 d后，突变体植株叶片上白粉菌孢子仅能萌发出少量较短初级菌丝，表皮细胞产生大量明显的H2O2，而野生型叶片中白粉菌萌发出大量初级菌丝、次级菌丝和吸器，无明显H2O2产生。这些结果表明，可以利用CRISPR/Cas9技术编辑葡萄感病基因VviEDR2，提高葡萄白粉菌抗性。     

中国板栗238份品种（系）叶片形态、解剖结构及其抗旱性评价

采用多功能图像分析法、冷冻切片法和指甲油印记法，对238份板栗品种（系）叶片的形态、解剖结构及气孔特征进行了观察测量，运用变异系数、主成分分析对33项指标进行了筛选，后运用隶属函数法进行抗旱性综合评价。结果表明：（1）238份板栗品种（系）33项指标中除上表皮角质层厚度、上表皮细胞宽度、第一层栅栏组织细胞密度、第一层栅栏组织细胞宽、下表皮细胞宽度和下表皮角质层厚等6项指标的变异系数小于10%，各品种系（间）差异较小外，其余27项指标的变异系数均大于10%，在各品种（系）间差异较大；经主成分分析从27项指标中筛选出10项作为238份板栗品种（系）抗旱性综合评价的典型指标。（2）利用隶属函数分析将238份品种（系）划分为4类：第Ⅰ类包括31个品种（系），平均隶属值为0.60 ~ 0.80，抗旱性极强；第Ⅱ类包括48个品种（系），平均隶属值为0.50 ~ 0.59，抗旱性强；第Ⅲ类包括84个品种（系），平均隶属值为0.30 ~ 0.49，抗旱性中；第Ⅳ类包括75个品种（系），平均隶属值为0.20 ~ 0.29，抗旱性弱。     

依据显微结构及光合特性探讨蝴蝶兰花芽分化的时期

以蝴蝶兰‘大辣椒’为试验材料，对花芽分化进程及期间光合特性和碳水化合物、可溶性蛋白及激素含量的变化进行研究。结果表明：花芽长度为0、2、4、8、16和24 cm时，分别处于花芽分化初始期、花序原基分化期、花原基分化期、萼片原基分化期和花瓣原基分化期（16和24 cm）。蝴蝶兰叶片的净CO2吸收速率在花芽发育前期（0 ~ 4 cm）没有显著变化，花芽8 cm时显著降低。花芽中的碳水化合物和可溶性蛋白的含量显著高于叶片，碳水化合物在花芽长度为4 cm时达到稳定水平，可溶性蛋白含量在花芽8 cm时达到叶片与花芽的平衡；赤霉素（GA）的含量在花芽2 cm时达到最大值，生长素（IAA）含量在花芽4 cm时显著升高，玉米素（ZT）含量在花芽8 cm时显著降低，而ABA含量在花芽发育的过程中并没有显著变化。由此可知，当蝴蝶兰花芽开始分化萼片原基（8 cm）时，光合生理及生化物质基本达到一个相对稳定的水平，此阶段的蝴蝶兰花芽已彻底完成成花分化。     

枣AP2/ERF转录因子鉴定及其响应枣疯病植原体表达分析

利用生物信息学方法，从植物转录因子数据库和NCBI数据库中分别得到175和164个候选的枣AP2/ERF转录因子序列，使用DNAMAN软件进行序列比对、去除重复序列，采用SMART软件预测蛋白结构域发现，枣基因组中包含有145个AP2/ERF基因，其中ERF、AP2、RAV亚家族分别含有116个、23个、5个，另有1个独立基因。预测枣AP2/ERF转录因子氨基酸数量在111 ~ 692之间，分子量在12 446.87 ~ 76 154.10之间，pI在4.31 ~ 10.11之间。鉴定出的145个AP2/ERF转录因子，105个分别定位到12条染色体上，40个未能定位。在此基础上，利用嫁接病芽方法将枣疯病植原体转至健康枣植株，通过转录组测序和qRT-PCR手段，分析了AP2/ERF转录因子对枣植原体侵染的响应，在植原体侵染枣的6个不同时期，枣AP2/ERF表达数量和表达量均不相同，共有48个差异表达基因，其中ZjAP2*9、ZjERF49和ZjERF91是响应枣疯病植原体最为重要的AP2/ERF转录因子。     

大花君子兰叶绿体基因组及其特征

采用Illumina MiSeq测序平台对大花君子兰（Clivia miniata）叶片总DNA进行测序，通过组装获得了其叶绿体基因组（cpDNA）全长序列（158 114 bp）。对其cpDNA注释得到135个基因，包含87个蛋白编码基因、40个tRNA基因和8个rRNA基因。采用生物信息学方法对获得的cpDNA进行简单序列重复（SSR）分析和密码子偏好性分析。结果显示：①大花君子兰cpDNA中共有61个SSR位点，其中单核苷酸、二核苷酸、三核苷酸、四核苷酸、五核苷酸和六核苷酸重复数分别为38、9、2、8、3和1个，多数SSR分布在基因间隔区；②大花君子兰cpDNA密码子偏爱以A或U（T）结尾，亮氨酸使用频率最高，半胱氨酸使用频率最低。基于24种植物的cpDNA全长和23种植物的叶绿体ycf2基因序列进行系统发育分析，结果显示大花君子兰与石蒜科植物在同一分支，显示最近的亲缘关系，支持大花君子兰属于石蒜科。基于叶绿体ycf2的系统发育分析结果与基于cpDNA全长的系统发育分析研究结果大部分相同，支持ycf2基因可以代替cpDNA全长用于植物系统发育分析。