青花菜锌指蛋白基因BoCCCH1的克隆和表达分析

CCCH锌指蛋白广泛存在于真核生物中,在植物的生长、发育和逆境胁迫响应中起着重要作用。为获知青花菜CCCH转录因子基因的序列特征和表达特性,从青花菜叶片中克隆到1个CCCH基因,命名为Bo CCCH1,同时,利用RT-PCR方法研究了其在不同器官及霜霉菌侵染叶片中的表达模式。序列分析结果表明,Bo CCCH1的基因组全长为1 568 bp,包含1个长度为599 bp的内含子,2个长度分别为627 bp、342 bp的外显子,编码322个氨基酸,蛋白质的分子量与等电点分别为35 296.42 Da和8.47;Bo CCCH1有2个锌指结构,类型均为C-X8-C-X5-C-X3-H。系统发育分析结果表明,Bo CCCH1与其它植物的21条同源序列在进化树上分为6组,来自十字花科的CCCH与Bo CCCH1处于同一分支。基因表达结果表明,Bo CCCH1在叶、花茎、嫩角果、花蕾和花中表达,其中花茎和嫩角果的表达量最高,但在根中未检测到表达;在霜霉菌侵染下,叶片Bo CCCH1的表达量升高,到24 h和36 h时达到最大,推测Bo CCCH1与霜霉病抗性相关。本研究为进一步探明Bo CCCH1在霜霉病抗性反应中的功能奠定了基础。     

Evaluation of two commercial PRRSV antibody ELISA kits with samples of known status and singleton reactors

Two commercial PRRSV ELISA kits (IDEXX and Bionote) were evaluated for their sensitivity and specificity using 476 PRRS-positive serum samples collected from 7 animal challenge experiments and 1,000 PRRS-negative sera. Both ELISA kits exhibited 100% sensitivity with sera collected 14 to 42 days post-infection, and the results from the kits were highly correlated (R²=0.9207). The specificity of IDEXX or Bionote kit was 99.9% or 99.7%, respectively. In addition, the Bionote ELISA kit was used to examine 100 sera that were determined to be falsely positive either by IDEXX 2XR or 3XR ELISA, and only 7 of these samples were found to be positive. These results indicate that both ELISA kits exhibited similar levels of sensitivity and specificity and would complement one another for the verification of false-positive samples.     

加州鲈弹状病毒三水株全基因组序列测定及分析

【目的】对加州鲈弹状病毒弱毒三水株(MSRV-SS-7)及其母源株(MSRV-SS)进行全基因组克隆和测序分析。【方法】采用分段扩增方法对MSRV-SS-7及MSRV-SS基因组核心序列进行PCR扩增,第1轮根据鳜鱼弹状病毒(SCRV)基因序列(NC_008514.1)设计10对引物进行扩增并测序;在第1轮扩增产物测序、拼接基础上,设计8对引物进行第2轮扩增,拼接后获得基因组核心序列。同时用cDNA末端快速扩增法(RACE)获得3′和5′末端序列。采用Vector NTI 8.0对基因组核心序列和末端序列进行拼接,获得基因组全序列,使用Clone Manager 8.0对MSRV-SS-7和MSRV-SS进行序列比对分析;同时使用DNAMAN将基因组G基因序列转换为氨基酸序列,并通过MEGA 5.0与其他鱼类弹状病毒的G蛋白氨基酸序列进行进化树分析。【结果】MSRV-SS-7及其母源株MSRV-SS基因组全长均为11 548 bp,包含N、P、M、G、L等5个结构基因,基因组结构为3′Leader-N-P-M-G-L-Trailer 5′;MSRV-SS-7与MSRV-SS全基因组中共有10处核苷酸不同。G蛋白进化分析表明,MSRV-SS-7及母源株MSRV-SS与SCRV亲缘关系最近,且与鲈鱼弹状病毒属(Perhabdovirus)聚为一支。【结论】克隆分析了MSRV-SS-7的全基因组序列,可以用于后续活疫苗的开发。     

Influence of paricalcitol on renal tubulointerstitial fibrosis in diabetic nephropathy

AIM: To investigate the effect of paricalcitol (P) on renal tubulointerstitial fibrosis and the underlying mechanisms in diabetic nephropathy (DN).METHODS: DN rat model was induced by a single intraperitoneal injection of streptozotocin after fasting. The animals were randomly divided into 2 groups:the DN rats in paricalcitol-intervened group (group P) were injected intraperitoneally with paricalcitol dissolved in propylene glycol after the day when the model was induced successfully at a dose of 0.4 μg/kg (3 times a week); the DN rats in DN group (group D) were given isopyknic propylene glycol. Normal control group (group C) was also set up. The samples of blood, urine and renal tissue were collected after intervention of paricalcitol for 12 weeks. The biochemical indexes were measured. The renal tissues were used for pathologic observation and determining the expression of transforming growth factor-β1 (TGF-β1), Wnt-4, β-catenin and Klotho by immunohistochemistry and Western blotting. In addition, the correlation among the above indexes was analyzed.RESULTS: (1) Scr, BUN and 24 h urine protein increased significantly in group D compared with group C, while decreased in group P compared with group D (P<0.05). (2) The area of renal tubulointerstitial fibrosis increased in group D compared with group C, while decreased in group P compared with group D (P<0.05). (3) The expression of Klotho decreased, while the expression of TGF-β1, Wnt-4 and β-catenin increased in group D compared with group C (P<0.05). Compared with group D, the expression of Klotho increased, while the expression of TGF-β1, Wnt-4 and β-catenin decreased in group P (P<0.05). (4) The expression of Klotho was negatively correlated with the fibrosis area, TGF-β1, Wnt-4 and β-catenin (P<0.05).CONCLUSION: Paricalcitol inhibits renal tubulointerstitial fibrosis in DN by promoting the expression of renal Klotho, and inhibiting Wnt/β-catenin signaling pathway activation and TGF-β1 synthesis.     

菜薹蛋白精氨酸甲基转移酶基因BrcuPRMT5的克隆及表达分析

以‘油青49’和‘油青甜菜薹80’菜薹茎尖为材料,采用RT-PCR和RACE技术克隆获得组蛋白甲基转移酶基因Brcu PRMT5的全长cDNA和gDNA序列。BrcuPRMT5 cDNA序列全长为2 117 bp,其中完整开放阅读框为1 929 bp,编码642个氨基酸,相对分子量为71.55 kD,理论等电点(p I)为5.87;多序列比对结果表明,Brcu PRMT5编码的氨基酸序列含有高等植物PRMT5基因1个高度保守的结构域;系统发育分析结果显示与大白菜、油菜及甘蓝的亲缘关系最近;亚细胞定位软件分析得知,Brcu PRMT5蛋白无跨膜区域,可能定位于线粒体中;对应的gDNA全长为4 151 bp,含有23个外显子和22个内含子,最长的外显子长度为140 bp,内含子的长度范围为50~150 bp。利用半定量RT-PCR和实时荧光定量PCR技术分析基因的表达,BrcuPRMT5在菜薹不同组织中均有表达,其中在花中表达量最高,叶次之,根中最低;BrcuPRMT5从苗期至完全抽薹开花期的表达量呈现上升趋势。BrcuPRMT5在菜薹花发育过程中可能起一定的调控作用。     

Bioinformatics Analysis of Bone Morphogenetic Protein 4 (BMP4) of Sheep

The assay was aimed to explore the biological characteristics of bone morphogenetic protein 4 (BMP4) of sheep,NCBI,DNAMAN DNAStar,TMHMM Server v.2.0,PsortⅡ,SignalP various bioinformatical softwares were used to speculate the physical and chemical properties,hydrophobic property,phosphorylation site,conservative structure domain,protein secondary structure of BMP4 protein.Also,the three-dimensional structure was forecasted with the SWISS-MODEL Workspace software.The results indicated that the BMP4 of sheep had high homologies with the BMP4 of various species.The encoded protein was a hydrophilic protein which was unstable.There was no transmembrane regions and it was likely to be located in the nucleus.What was more,there was signal peptide and eighteen phosphorylation sites.Through the forecast of functional domains,the protein had two functional domains,including the transforming growth factor-beta (TGF-beta) superfamily and TGF-beta propeptide superfamily.The result was consistent with the function of BMP4 gene family,it also demonstrated that BMP4 was a growth factor and it had the function of signal transduction.The amino acid homology between the predicted 3D structure of protein and template 3bmp.1.A was 88.29%.The bioinformatics analysis of BMP4 gene could provide reference for the further study in practice.     

基于Geomagic Studio 12.0的采棉部件设计

为缩短产品研制周期、增强企业竞争能力,逆向工程技术也正逐步从其他产品设计制造业领域引入到我国农业机械领域。以统收式采棉机的采摘部件梳齿为测绘对象,通过逆向工程技术,采用青岛雷顿数控设备有限公司生产的先进的NC(865)型三坐标测量仪进行扫描与测绘,获取了梳齿的点云数据,然后对其进行预处理、多边形阶段处理、参数化处理,最终运用SolidWorks软件绘制出三维模型,完成了梳齿部件的逆向设计。通过实例运用,证实了逆向工程技术在农业机械研究领域的可行性,其研究结果将对我国农业机械化的发展起到积极的推动作用。