布鲁氏菌LPS对小鼠巨噬细胞中NLRP3炎症小体转录水平的影响

为研究布鲁氏菌LPS对巨噬细胞中NLRP3炎症小体的影响,本试验提取布鲁氏菌2308、RB51和△WbkA的LPS,以不同浓度与小鼠巨噬细胞相互作用,荧光定量PCR检测其对NLRP3、ASC、Caspase1、IL1-β、IL18转录水平的影响。结果显示RB51LPS和△WbkA LPS上调NLRP3炎症小体相关基因的转录水平,且呈浓度依赖性,而浓度对2308LPS调节NLRP3炎症小体相关基因转录的作用不大;且同一浓度下,RB51LPS和2308LPS比△WbkA LPS更好的调节Caspase1、IL1-β、IL18的转录水平。     

西安蔬菜主产区土壤硝态氮累积现状与蔬菜产品、浅层地下水氮素污染调查研究

以西安郊区主要周边蔬菜产区为研究地点,通过分析蔬菜鲜样、菜田土壤2 m垂直分层土样、浅层地下水的水样,研究蔬菜及菜田生态系统氮肥污染现状.结果表明,按我国的地下水硝酸盐含量标准,在选点采样中1.5～3 m深的地下水硝酸盐含量超标,菜田土层土质疏松与浇水次数较多的菜田硝酸盐向下淋洗现象明显,温室较露地2 m深土层硝态氮与铵态氮分布浓度高,菜田2 m深土层中常有硝态氮与铵态氮的聚集峰,不同深度地下水的菜田韭菜硝酸盐含量差异明显.     

Shikonin inhibits neuronal apoptosis induced by OGD through targeting PI3K/Akt signaling pathway

AIM: To explore the effect of shikonin on rat primary cortical neurons in oxygen-glucose deprivation (OGD)-induced injury model.METHODS: The neurons were pretreated with shikonin at different concentrations (0.02, 0.2, 2 and 20 μmol/L) followed by treatment with OGD. Lactate dehydrogenase (LDH) release assay and fluorescein diacetate/propidium iodide (FDA/PI) double staining were used to detect neuronal viability and apoptosis, and then the optimal concentration of shikonin was determined. LY294002 (PI3K/Akt signaling pathway inhibitor, 1 μmol/L) was added before the addition of shikonin, and the protein level of p-Akt (Ser473) in the neurons was determined by Wes-tern blot. LDH release assay and FDA/PI double staining were also used to detect neuronal viability and apoptosis.RESULTS: A certain concentration (0.2~20 μmol/L) of shikonin increased the viability of impaired neurons (P<0.05) and the protein level of p-Akt (Ser473) in the neurons (P<0.05). The effect of shikonin on neuronal p-Akt (Ser473) levels and the cell death were blocked by LY294002 (P<0.05).CONCLUSION: A certain concentration of shikonin reduces OGD-induced apoptosis of rat primary cortical neurons by activating PI3K/Akt signaling pathway.     

桔小实蝇14-3-3基因的克隆和表达谱分析

【目的】克隆桔小实蝇14-3-3蛋白的基因（Bactrocera dorsalis 14-3-3,Bdor 14-3-3）,研究Bdor 14-3-3 mRNA在不同组织和不同发育时期的表达情况,探索其是否参与了桔小实蝇的发育过程。【方法】采用反转录聚合酶链式反应(RT-PCR)和快速扩增cDNA末端(RACE)技术克隆Bdor 14-3-3;采用实时荧光定量PCR（real-time PCR）方法,研究Bdor 14-3-3 mRNA在桔小实蝇不同组织及不同发育时期的相对表达量。【结果】克隆获得了Bdor 14-3-3基因,其编码区长度为747 bp,编码248个氨基酸残基。氨基酸序列一致性分析表明,在昆虫纲中,桔小实蝇与刺舌蝇14-3-3蛋白的序列一致性最高,为98.8%,与豌豆蚜的序列一致性最低,为85.4%。实时荧光定量PCR分析表明,Bdor 14-3-3 mRNA在桔小实蝇不同组织和发育时期都有表达;在雌虫头（去除触角）和翅中的相对表达量均较高,分别是触角的1.39和1.44倍;在雄虫胸和后足中的相对表达量均较高,分别是前足的1.28 和1.23倍。在蛹的早期发育过程中Bdor 14-3-3 mRNA表达量逐渐升高,到7 d蛹期相对表达量达到最高峰,是10 d蛹的4.91倍。【结论】克隆获得了Bdor 14-3-3基因,其表达的14-3-3蛋白可能参与了桔小实蝇的变态发育过程,尤其在蛹的发育过程中Bdor 14-3-3可能发挥着重要作用。     

氮肥后移对土壤氮素供应和冬小麦氮素吸收利用的影响

采用田间试验研究了氮肥后移对土壤氮素供应和冬小麦氮素吸收利用的影响。结果表明,与农民习惯施氮（N 300 kg/hm2,基肥和拔节肥各占1/2）比较,氮肥后移处理（N210kg/hm2,基肥、拔节肥和孕穗肥各占1/3）在不降低小麦产量的同时,大大提高了氮肥利用率,且全生育期氮素表观损失极低。过量施用氮肥(N 300 kg/hm2)明显提高了60 cm以下土层硝态氮含量,增加了其向地下水淋溶迁移的风险。氮肥后移可提高小麦成熟期0-20cm土层硝态氮积累量,降低其在20-100cm土层的积累。基于冬小麦不同生育阶段的氮素吸收量而进行氮肥后移是可行的,氮肥后移可节省氮肥30%,是较为理想的施氮方式。     

温室土壤硝态氮积累的温度、水分、施氮量耦合效应

为了探讨温室作物生产水肥管理和土壤温度对土壤硝态氮累积的影响，本文选取使用5年的温室土壤样品进行培养试验，研究温度、水分、施氮量及其耦合效应对温室土壤硝化作用和硝态氮累积的影响。试验结果表明：温室土壤硝态氮累积量可用“S”曲线进行定量描述，硝化过程中最大硝化速率、延迟期和最大可能累积量是参数土壤温度，含水率和NH₄-N含量的函数；通过正交回归分析得出影响最大硝化速率的因素主次顺序依次为温度、含水率、温度与含水率的交互作用、水肥的耦合作用以及施氮量；影响延迟期的因素主次顺序依次为土壤水分、土壤温度、施氮量以及水肥耦合作用；最大可能累积量与温度、含水率及施氮量呈指数关系，其中施氮量影响最大，土壤温度次之，而这3个因素之间的交互作用对最大可能累积量没有明显的影响。利用回归模型，可为不同温度环境及水肥条件下硝态氮累积量及氮利用的有效性预测提供依据。     

亚洲璃眼蜱唾液腺一新功能基因的克隆与测序

HaB1是亚洲璃眼蜱雌成蜱唾液腺差异表达基因文库中的一个片段,根据其序列设计引物HaB1-GSP1和HaB1-GSP2,以唾液腺总RNA为模板,RACE法扩增获得HaB1的未知3′-末端。测定该末端序列,进行序列拼接,设计全长引物5-′CCAGTCCGAAGGAAGGGCG-3′和5-′TCTC-CGGGCACGTGAAGTGTC-3′。以cDNA第一链为模板,扩增获得基因全长。经核查表明,该基因为一新基因(登录号AY803896)。     

2%加收米水剂防治香蕉叶鞘腐烂病的田间药效试验

比较了A(2%加收米AS500倍单剂)、B(2%加收米AS500倍配用25%敌力脱EC1000倍)和C(2%加收米AS500倍配用25%势克EC2000倍)三种使用方法对香蕉叶鞘腐烂病的防治药效。结果表明:对香蕉叶鞘腐烂病的防治药效排列是:A相似文献   

飞蝗表皮蛋白LmKnk3-5′的抗体制备及组织定位

【目的】LmKnickkopf3-5′(LmKnk3-5′)是飞蝗（Locusta migratoria）蜕皮发育中重要的表皮蛋白。本文旨在制备LmKnk3-5′特异抗体并对其进行组织定位,为LmKnk3-5′生物学功能研究提供蛋白水平的证据,同时为进一步深入探究LmKnk3-5′与其他表皮蛋白在飞蝗表皮形成过程中的协同作用打下基础。【方法】首先比对飞蝗Knickkopf(Knk)家族4个基因LmKnk、LmKnk2、LmKnk3-FL和LmKnk3-5′的氨基酸序列,选取LmKnk3-5′的3段特异抗原序列R1、R2和R3,设计包含BamH I、Hind III酶切位点的引物,以LmKnk3-5′全长cDNA为模板,PCR获得LmKnk3-5′的特异抗原片段;然后将特异抗原片段与pET-32a经双酶切、T4连接酶连接、测序验证、成功构建重组质粒后,转入BL21（DE3）表达菌株中,加入IPTG（终浓度0.5 mmol·L^-1）低温（16℃）诱导重组蛋白表达,SDS-PAGE检测重组蛋白表达情况;接着扩大培养可在菌体裂解液的上清中表达重组融合蛋白的大肠杆菌,提取蛋白并用Ni-NTA对其进行纯化,之后再用纯化后的重组特异抗原免疫小鼠4次,获得anti-LmKnk3-5′多克隆抗体,ELISA法对其进行效价检测,并用Western blot法检测其特异性。取飞蝗5龄若虫第8天表皮,制备石蜡切片,通过免疫组化法对LmKnk3-5′蛋白进行组织定位。【结果】通过氨基酸序列比对,选取LmKnk3-5′的特异抗原区域R1、R2和R3,分别包含208、147和131个氨基酸残基,蛋白理论分子量分别为24.0、17.0和14.8 kD。酶切连接后,成功获得 pET-32a-R1、pET-32a-R2 和pET-32a-R3重组质粒。诱导表达检测,发现只有含pET-32a-R2的表达菌在菌体裂解液的上清液中大量表达重组蛋白。将其扩大培养纯化后获得 R2重组融合蛋白,免疫小鼠取抗血清进行效价检测,抗体效价高达1﹕512 000,可满足抗体使用需求。Western blot结果表明,注射dsLmKnk3-5′后飞蝗体壁LmKnk3-5′的蛋白表达显著减少,表明anti-LmKnk3-5′抗体特异性良好。免疫组化试验结果表明,表皮蛋白LmKnk3-5′在飞蝗腹部体壁皮细胞及新表皮中均有分布,特别是新合成的外表皮顶端。【结论】成功获得飞蝗LmKnk3-5′多克隆抗体,证明其特异性良好。LmKnk3-5′在飞蝗腹部体壁新合成的外表皮顶端分布较多。研究结果为飞蝗表皮蛋白LmKnk3-5′功能研究提供了蛋白水平的证据。     

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.