Effects of the severity and timing of basal leaf removal on the amino acids profiles of Sauvignon Blanc grapes and wines

The effects of the severity and timing of leaf removal(LR) on the amino acids of Sauvignon Blanc grapes and wines were studied during the 2017 growing season. High-performance liquid chromatography(HPLC) was used to analyze the amino acids profiles of grape berries and wines. The basal leaves were removed at three time points(40, 56 and 72 days after flowering, named LR40, LR56 and LR72, respectively) at two severity levels(one at which the first, third, and fifth basal leaves of each shoot were removed(50% level); and another at which the first six basal leaves were removed(100% level)). The results showed that leaf removal had little impact on total soluble solids(°Brix), titratable acidity, pH or berry weight. The LR72-50% treated grapes had higher berry weight, titratable acidity and °Brix than those of the other treatments. The highest concentrations of total amino acids and of total amino acids except proline were detected in LR72-50% treated grapes(2 952.58 and 2 764.36 mg L~(-1), respectively); the lowest were detected in LR72-100% treated grapes(2 172.82 and 2 038.71 mg L~(-1), respectively). LR72-50% treatment significantly promoted the synthesis of aspartic acid, serine, arginine, alanine, aminobutyric acid and proline at both severity levels for grapes, the concentrations of all of these amino acids were increased relative to the control concentrations. The LR72-50%, LR40-100% and LR72-100% treated wines had higher total amino acids concentrations and higher concentrations of some individual amino acids, such as arginine, alanine and serine, than did the control wines. Of all the amino acids studied, glycine, tyrosine, cysteine, methionine and lysine were not significantly influenced by the timing or severity basal defoliation in grapes and wines. The present study reveals the effects of the timing and severity of leaf removal on the amino acids profiles of grapes and wines.     

基于B/S的农业气象自动化观测系统国家级平台设计与应用

农业气象自动化观测系统国家级平台是依托中国气象局组织的农业气象自动化观测试点工作而设计开发,平台的搭建采用B/S架构实现多级用户、跨平台登录,采用XML及JSON等数据传输格式实现国家级、省级、台站级用户信息交互,实现农业气象观测XML数据传输状态的监控、台站端设备状态的评估以及数据内容的实时查询。     

Zerumbone attenuates MPP+-induced cytotoxicity in human neuroblastoma SH-SY5Y cells by inhibition of oxidative stress

AIM: To investigate the role of zerumbone (ZER) in 1-methyl-4-phenylpyridinium (MPP⁺)-induced cytotoxicity of human neuroblastoma SH-SY5Y cells. METHODS: Human neuroblastoma SH-SY5Y cells were cultured in vitro and the protective effect of ZER against MPP⁺-induced cytotoxicity was measured by CCK-8 assay. Flow cytometry was used to determine the apoptosis and reactive oxygen species (ROS). The expression of Parkinson disease protein 7 (PARK7) was knocked-down by using PARK7-specific short hairpin RNA (shRNA). The protein levels of PARK7, nuclear factor E2-related factor 2 (Nrf2) and heme oxygenase-1 (HO-1) were determined by Western blot. RESULTS: MMP⁺ remarkably reduced the cell viability in a dose-dependent and time-dependent manner. The SH-SY5Y cell injury model was established by treatment with MPP⁺ at 600 μmol/L for 24 h. ZER up-regulated the protein levels of PARK7 and Nrf2 (P<0.05), alleviated apoptosis (P<0.05), and reduced ROS production (P<0.05) in the SH-SY5Y cell injury model. Meanwhile, N-acetyl-L-cysteine (NAC) had the similar functions. Moreover, significant reductions in the protein levels of Nrf2 and HO-1 (P<0.05), and obvious increases in apoptosis (P<0.05) and ROS level (P<0.05) were demonstrated in PARK7-knockdown cells. CONCLUSION: ZER protects SH-SY5Y cells against MPP⁺-induced cytotoxi-city, which may be related to activation of PARK7/Nrf2/HO-1 pathway, and subsequent attenuation of oxidative stress and apoptosis.     

基于nanoUPLC-MS/MS的螺旋藻游离肽组成分析

为了研究螺旋藻中的多肽成分,以钝顶螺旋藻为原料,采用超滤法提取螺旋藻中的游离肽,利用nanoUPLC-MS/MS和PEAKS Studio分析谱图信息,结合NCBI数据库进行比对和从头测序分析,获得游离肽的结构组成和百分含量信息。结果表明,利用数据库比对法和从头测序法,分别得到可信的游离肽4 485个和20 597个,匹配到的蛋白质有1 036种。数据库比对结果中的游离肽主要为七肽到二十一肽,少量为二十一肽以上;从头测序结果中游离肽主要为二肽到十六肽,少量为十六肽以上。数据库比对结果中百分含量最高的游离肽是十肽,为15.95%;从头测序结果中百分含量最高的是五肽,达到24.09%。本研究结果为螺旋藻蛋白资源的进一步开发和利用提供了依据。     

超高效液相色谱-串联质谱法测定牛奶和奶粉中五种苯并咪唑类药物

为建立牛奶及奶粉中5种苯并咪唑类药物(阿苯达唑、芬苯达唑、奥芬达唑、噻苯达唑、苯硫氨酯)简便、准确的分析检测方法,以固相萃取为净化手段,采用Acquity UPLC^® BEH C₁₈(100 mm×2.1 mm,1.7 μm)色谱柱进行分离,电喷雾离子源(ESI⁺),多反应监测(MRM)模式检测。结果表明,5种苯并咪唑类药物在1~500 μg·L^-1范围内呈线性相关,相关系数(r)均大于0.996,加标平均回收率介于91.7%~97.8%之间,相对标准偏差(RSD)在1.4%~6.5%之间,基质效应均小于15%。5种药物的检出限均在0.1~3 μg·kg^-1范围内,定量限均在0.5~10 μg·kg^-1范围内。该方法操作简单、快速且灵敏度高,重现性好,能够用于牛奶及奶粉5种苯并咪唑类药物的同时测定。     

利用CRISPR/Cas9技术编辑BADH2-1/BADH2-2创制香米味道玉米新种质

【目的】香味是作物的重要食味品质之一。2-乙酰-1-吡咯啉（2-acetyl-1-pyrroline,2-AP）是主要香味物。BADH2是控制水稻等作物香味性状的关键基因,敲除该基因可以产生香味稻米。利用CRISPR/Cas9基因编辑技术在北京市农林科学院自育的玉米骨干亲本京724上敲除BADH2同源基因,以期获得有香米味道的玉米新种质材料。【方法】利用Ensembl数据库在线BLAST工具,将水稻OsBADH2蛋白序列在拟南芥、水稻和玉米蛋白序列数据库中进行序列比对,获得上述3个物种的BADH基因家族成员,并利用UniProt蛋白数据库中的结构域信息进行验证。进一步使用MEGA软件进行系统进化分析,获得玉米BADH2同源基因作为候选编辑靶标。基于CRISPR/Cas9基因编辑技术的原理,在候选基因的外显子处设计特异性靶点,并构建入CRISPR/Cas9基因编辑载体。再以玉米自交系京724为受体,利用农杆菌介导的遗传转化方法,通过磷酸甘露糖异构酶基因（phosphomannose isomerase,PMI）抗性筛选获得阳性转基因植株。转基因株系经测序明确其在靶基因中产生的突变类型。利用气相色谱质谱联用仪（gas chromatography-mass spectrometry,GC-MS）检测基因编辑株系T₁籽粒中香米主要香味物质2-AP的含量,以确认京724在基因编辑前后2-AP含量的变化。【结果】系统进化分析发现,玉米中存在2个BADH2同源基因,分别命名为ZmBADH2-1和ZmBADH2-2。ZmBADH2-1位于第4染色体,ZmBADH2-2位于第1染色体。2个基因均包含15个外显子和14个内含子,第4外显子间的核苷酸序列高度同源。在2个基因的第4外显子区域设计靶点并构建入CRISPR/Cas9基因编辑载体,通过遗传转化获得28株转基因株系。PCR扩增及测序分析结果显示,其中10株材料的2个ZmBADH2s在靶点区域均发生突变,突变基因型包括双等位突变和多等位突变,突变类型为不同数量的碱基缺失和插入。质谱检测结果显示玉米ZmBADH2双基因突变体籽粒中存在与香稻同样成分的2-AP。随机选取的4个T₁代基因编辑株系籽粒中,2-AP平均含量分别为438.29、404.63、348.65和161.82 μg·kg^-1,而未经过编辑的京724中未检测到2-AP。【结论】利用CRISPR/Cas9技术对玉米ZmBADH2-1和ZmBADH2-2同时进行定点敲除,创制出籽粒中具有香米味道的玉米骨干亲本新种质材料。     

Effects of bioflocs with different C/N ratios on growth,immunological parameters,antioxidants and culture water quality in Opsariichthys kaopingensis Dybowski

Biofloc technology (BFT) is a new ecological aquaculture technology that is intended not only to eradicate pollutants and elevate feed utilization but also to enhance immunity and antioxidant activity in aquatic animals. A 28‐day feeding trial was implemented to evaluate the effects of promoted bioflocs on the water quality, growth performance, immunological parameters and antioxidant status of Opsariichthys kaopingensis Dybowski juveniles in low exchange culture tanks. Three hundred sixty healthy O. kaopingensis juveniles (7.1 ± 0.02 g) were irregularly distributed among 12 tanks. Four C:N ratios in triplicate tanks were tried: C/N = 10.8:1 with a commercial diet (control), C/N = 15:1, C/N = 20:1 and C/N = 25:1. Anhydrous glucose (99.97%) was added to the commercial diet to maintain elevated carbon ratio in the three treatments. Uninterrupted 24‐hr aeration was supplied during the test by using an air‐stone connected to an air pump to develop the bioflocs in the water column. The results indicated that BFT significantly reduced total ammonia nitrogen (TAN), nitrite (NO2^?‐N), total phosphorus (TP) concentrations and water transparency (Secchi depth) in C/N 15, C/N 20 and C/N 25 (p < .05), whereas the chemical oxygen demand (COD) and nitrate nitrogen (NO3^?‐N) concentrations gradually increased over time. In addition, weight gain, specific growth rate (SGR), survival and protein efficiency ratio (PER) were significantly elevated in C/N 20, while food conversion rate (FCR) was considerably decreased (p < .05) in C/N 20, compared with the control. Significant increases in lysozyme (LSZ), glutamic pyruvic transaminase (GPT), glutamic oxaloacetic transaminase (GOT) and alkaline phosphatase (AKP) activities of O. kaopingensis were found in the C/N 20 group after a 28‐day feeding trial (p < .05). Comparing the antioxidant capacity of O. kaopingensis in gills, brains, kidneys, hepatopancreas, intestines and serum of juveniles from the four experimental groups, the activity of superoxide dismutase (SOD), total antioxidant capacity (T‐AOC), catalase (CAT) activity and glutathione peroxidase (GSH‐PX) activity of juveniles in the C/N 20 group were significant higher (p < .05), and the content of malondialdehyde (MDA) was considerably lower, than in the control. Overall, these findings suggest that BFT not only can enhance O. kaopingensis growth performance and strengthen antioxidant status but also can upgrade immune response, ensuring the sustainable development of aquaculture.     

ROCK promotes high glucose-induced cardiomyocyte apoptosis by inhi-biting PI3K/Akt signaling pathway

AIMTo investigate whether Rho-associated coiled-coil kinase (ROCK) is involved in high glucose-induced apoptosis of primary cardiomyocytes by regulating PI3K/Akt signaling pathway. METHODSPrimary Wistar rat cardiomyocytes were cultured and identified by α-sarcomeric actin (α-SCA) immunohistochemistry. Cardiomyocytes were treated with 5.5, 33 and 40 mmol/L glucose for 48 h. The cell viability was measured by MTT assay, and the mRNA expression of ROCK1 and ROCK2 in the cardiomyocytes was detected by RT-qPCR. Flow cytometry was used to analyze the apoptosis of the cardiomyocytes. The protein levels of ROCK1, ROCK2, cleaved caspase-3, Bcl-2, PI3K, Akt and p-Akt were determined by Western blot. In order to confirm the regulatory effect of ROCKs on PI3K/Akt signaling pathway, the cells were divided into control group (5.5 mmol/L glucose), high glucose group (33 mmol/L glucose) and high glucose+Y27632 (ROCK inhibitor) group. Western blot was used to detect the protein levels of ROCK1, ROCK2, PI3K, Akt and p-Akt. RESULTSAfter 48 h of high glucose exposure, the values of relative cell viability in 33 and 40 mmol/L glucose groups were (79.71±2.43)% and (68.41±7.49)%, respectively, both of which were significantly decreased compared with normal control group (P<0.05). After 48 h of high glucose exposure, the relative mRNA levels of ROCK1 and ROCK2 in 33 and 40 mmol/L glucose groups were significantly increased compared with normal control group (P<0.05). Compared with normal control group, the apoptotic rate in 33 and 40 mmol/L glucose groups was increased significantly (P<0.05). Compared with normal control group, the protein expression of ROCK1, ROCK2 and cleaved caspase-3 in 33 and 40 mmol/L glucose groups was increased (P<0.05), while the protein expression of Bcl-2 was decreased (P<0.05). No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed, while the protein level of p-Akt in 33 and 40 mmol/L glucose groups was decreased compared with normal control group (P<0.05). Compared with high glucose group, the expression of ROCK1 and ROCK2 was decreased in high glucose+Y27632 group. No significant difference in the protein levels of PI3K and Akt among the 3 groups was observed. Compared with normal control group, the protein level of p-Akt in high glucose group was decreased, and the protein level of p-Akt in high glucose+Y27632 group was increased significantly compared with high glucose group. CONCLUSION Under high glucose environment, ROCK may reduce the level of p-Akt by inhibiting the PI3K/Akt signaling pathway， thus promoting the apoptosis of cardiomyocytes.     

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。     

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。