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   

福建菜田氮磷积累状况及其淋失潜力研究

本文通过采集460个福建菜田代表性耕层土样,采用土壤测试和土柱渗漏水模拟试验的方法研究菜田土壤硝态氮和Olsen P含量状况和淋失临界指标及其淋失潜力。结果表明,耕层土壤硝态氮含量为47.455.5 mg/kg,Olsen-P含量则为61.743.2 mg/kg, 其中瓜果类蔬菜种植地土壤硝态氮和Olsen-P含量明显高于叶菜类和根茎类蔬菜种植地。 应用双速率转折点建模法,得到氮、 磷淋失临界指标X0分别为土壤硝态氮76.3 mg/kg和Olsen-P 42.8 mg/kg。 淋失临界值相当于或略高于满足蔬菜营养的农学指标。当土壤硝态氮或Olsen-P含量低于X0时,随着其含量增加,渗漏水硝态氮或总磷浓度以线性方式缓慢增加,反之,则以非线性形式急剧增大。土壤硝态氮和Olsen-P含量高于其X0的土样数分别占17.9%和81.3%, 表明这些样点具有较高的氮、 磷淋失潜力,是氮、 磷污染控制的关键地块。 瓜果类菜田土壤硝态氮和Olsen-P含量高于其X0的土样数分别占到32.3%和96.3%,淋失潜力明显高于叶菜类和根茎类菜田,是氮、 磷污染控制的优先区域。     

不锈钢管的焊接技术

汽轮机润滑油系统油管道焊接是汽轮机专业焊接的一个重点.为了保证汽轮杌润滑油管道系统的清洁度及汽轮机主机润滑系统、调节保安系统安全可靠地工作,其材质全部由不锈钢组成,所以油管道安装的内在质量格外重要.总结出一套焊接组装工艺措施,提高了生产效能,满足了汽轮机油管的质量要求.     

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.     

RACE法扩增梅花鹿β-防御素-1(siBD-1)全长cDNA

为了获得梅花鹿β-防御素-1（sika deer β-defensin-1,siBD-1）cDNA全序列,本试验以梅花鹿舌黏膜组织内提取的总RNA为模板,根据前期已获得的siBD-1 cDNA的已知部分序列设计引物,采用5'-RACE和3'-RACE技术分别扩增5'-和3'-末端序列,将此扩增产物克隆入pMD18-T载体,进行PCR、双酶切鉴定及序列测定与分析。结果表明,成功克隆出长度约为172和299 bp的siBD-1 cDNA 5'-和3'-末端序列,从而得到418 bp的siBD-1 cDNA全序列（GenBank登录号：HM588696.1）,其中包含89 bp 5'-非翻译区（UTR）、192 bp的开放阅读框（ORF）、终止密码子TAA、118 bp的3'-UTR和Poly（A）16。同源性比对结果显示,siBD-1 cDNA与水牛的肠防御素（BEBD）同源性最高,为90.6%,与牛（EBD、LAP、TAP、BNBD-4）、山羊（GBD-1、GBD-2）、驯鹿（reBD-1）、绵羊（sBD-1、sBD-2）和骆驼（caBD-1）的防御素cDNA的同源性较高,分别为83.2%、83.1%、87.3%、87.0%、87.5%、87.5%、84.4%、79.9%、77.1%和70.5%;与马（hoBD-1）和猪（pBD-1）的同源性较低,为60.3%和72.4%;而与人（hBD-2）的同源性最低,为16.0%。siBD-1成熟肽由38个氨基酸残基组成,其中包含9个带正电荷的氨基酸残基。     

邻二氮菲-铁(Ⅲ)测定半胱氨酸

研究了邻二氮菲-Fe3+与半胱氮酸的反应,发现半胱氨酸浓度在1.0×10-5~1.8×10-4mol.L-1范围内其产物吸光度值符合朗伯比耳定律。检测限1.0×10-6mol.L-1,表观摩尔吸光系数7.9×103L.mol-1.cm-1。采用本法测定谷氨酸、赖氨酸、半胱氨酸混合试样中半胱氨酸含量,相对标准偏差≤2%,结果令人满意。     

肝癌细胞系中Runx3基因表达及启动子区异常甲基化分析

目的：通过检测6种肝癌细胞中Runx3基因异常甲基化状态及表达情况,探讨药物5-aza-2＇-deoxycytidine（decitabine）激活Runx3基因重新表达的能力及对肝癌细胞生长的影响。方法：采用DNA甲基化特异性PCR（MSP）技术分别对6种肝细胞癌细胞系Runx3基因启动子区域甲基化状态进行检测,同时采用逆转录一聚合酶链反应（RT—PCR）检测5种肝癌细胞中Runx3的表达情况及药物5-aza-2＇-deoxycytidine处理其中3种肝癌细胞前后Runx3表达变化。结果：6种肝癌细胞系中,有3种细胞Runx3基因启动子区域存在甲基化异常,药物5-aza-2＇-deoxycytidine处理3种肝癌细胞后,Runx3基因明显表达或表达活性增强。结论：启动子区异常甲基化是导致Runx3基因失活的主要原因之一,与肝细胞癌发生早期密切相关,可作为肝细胞癌早期诊断的分子标记物及分子治疗的靶点。     

樟树、檫树、闽楠种子的休眠和萌发特性

对樟科Lauraceae 3个属的代表性树种的种子休眠与萌发特性进行研究．结果表明:新采集的樟树Cinnamoumcamphora (linn．)Presl、檫树Sassafras Tzumu(Hemsl．)Hemsl和闽楠Phoebe bournei(Hemse．)Yang的种子胚形态发育正常,除檫树种子外,樟树和闽楠种子的离体胚能正常萌发,在黑暗条件下,3个树种的完整种子于15-35℃温度范围内,无论恒温和变温均不能萌发,闽楠种子仅在光照下30、35℃恒温和30℃日／20℃夜变温中较窄的温度范围内能萌发,但发芽迟缓、过程长、萌发率低;3个树种的种子均具生理休眠特性,种皮透气性差和存在发芽抑制物质是樟树种子休眠的原因,低温(5℃)层积80 d,不能完全解除休眠,在30℃日／20℃夜最适变温条件下．并给予适当光照,萌发率仅为41．00％;采用0．5％KNO3+0．10％GA3溶液浸种2 h后低温层积80 d,在光照条件下,萌发率可提高到74．00％;胚存在发芽抑制物质和种皮透气性差是引起檫树种子休眠的最初原因,经低温层积后,种皮防碍抑制物质向外渗透是种子不能完全解除休眠的关键因素,低温层积240 d,在25℃日／15℃夜最适温度下,光暗中的萌发率仅为59．00％-54．00％．而采用4％H2O2+0．10％GA3溶液浸种后低温层积．萌发率可高达87．00％;种皮透气性差,发芽要求特殊的温度和一定的光照是闽楠种子休眠的原因,低温层积60 d,在光照条件下,20、25℃恒温和30℃日／15℃夜变温适宜温度中,萌发率能达77．33％-85．33％．