对虾白斑综合征病毒中国地方株变异区基因的比较

对虾白斑综合征病毒(White spot syndromevirus,WSSV)是对虾养殖的主要病原之一,它是目前发现的基因组最大的动物病毒,为环状双链DNA病毒[1,2],全基因组序列分析结果显示,对虾白斑综合征病毒和其他杆状病毒相差甚远,最新病毒分类报告已将该病毒划归新建立的线头病毒科(Nima-viridae)白斑病毒属(Whispovirus)[3,4]。目前Gen-Bank公布有3个版本的WSSV全序列[1,2],其基因组大小的测定结果相差较大。不同的WSSV毒株可能在形态结构、理化性质上无法区分,但病毒基因组限制酶切片段长度多态性(RFLP)可以将之区分开来,Marks等[6,7]通过计…     

Comparison of Gene Expression Profiles of Fenneropenaeus chinensis Challenged with WSSV and Vibrio

Microarray technique was used to analyze the gene expression profiles of shrimp when they were challenged by WSSV and heat-inactivated Vibrio anguillarum, respectively. At 6 h post challenge (HPC), a total of 806 clones showed differential expression profile in WSSV-challenged samples, but not in Vibrio-challenged samples. The genes coding energy metabolism enzyme and structure protein were the most downregulated elements in 6 h post WSSV-challenged (HPC-WSSV) tissues. However, a total of 155 clones showed differential expression in the Vibrio-challenged samples, but not in WSSV-challenged samples. Serine-type endopeptidase and lysosome-related genes were the most upregulated elements in tissues 6 h post Vibrio challenge (HPC-Vibrio). Totally, 188 clones showed differential expression in both 6 and 12 HPC-WSSV and HPC-Vibrio samples. Most of the differentially expressed genes (185/188) were downregulated in the samples of 12 HPC-WSSV, whereas upregulated in the samples at 6 and 12 HPC-Vibrio and 6 HPC-WSSV. The expression profiles of three differentially expressed genes identified in microarray hybridization were analyzed in hemocytes, lymphoid organ, and hepatopancreas of shrimp challenged by WSSV or Vibrio through real-time PCR. The results further confirmed the microarray hybridization results. The data will provide great help for us in understanding the immune mechanism of shrimp responding to WSSV or Vibrio. Wang and Li contributed equally to this work.     

cDNA微阵列结合抑制性差减杂交研究对虾白斑综合征病毒部分表达基因

通过抑制性差减杂交技术建立了包含对虾白斑综合征病毒表达性基因的差减文库,并用cDNA微阵列技术进行了鉴定,得到255个正向克隆.对其中的184个正向阳性克隆进行了测序,测序结果通过BLAST与GenBank中序列进行比对,共得到WSSV(white spot syndrome virus,WSSV)基因30个.此次首次鉴定了5个,其中WSV184具有调控蛋白的结构特征(Cys2/Cys2型锌指),WSV321和WSV322含跨膜结构,且存在可能的糖基化位点.有3个被其它研究推断无polyA结构的阅读框所处的mRNA应有polyA结构.进一步用Dot Northern blot对克隆号PCI118(含WSSV阅读框WSV321和WSV322)进行鉴定,表明确实存在该基因的转录.进而根据已报道的WSSV基因序列设计两条引物,用快速扩增cDNA末端技术,扩增WSV321和WSV322两个阅读框所处的cD-NA的5′端片段和3′端片段,分析得到其全长共1 109bp,与已报道的WSSV全基因序列(AF332093)的相关序列完全相同.该mRNA存在polyA,并有加尾信号AATAAA;两个阅读框都没有自己的TATA盒,但都有病毒RNA聚合酶Ⅱ的结合位点-CCAAT盒;它们编码的蛋白质分别有117和227个氨基酸,都存在可能的糖基化位点,其中WSV321一个,WSV322两个.     

应用抑制消减杂交和cDNA芯片技术筛选流行性感冒病毒感染相关基因

病毒基因组有限的编码能力和以病毒蛋白为靶的抗病毒药物易出现耐药性,使从病毒感染宿主筛选病毒感染相关生物大分子作为抗病毒药靶和诊断标志物成为新的研究方向。为了筛选流行性感冒（流感）病毒感染相关基因,采用抑制消减杂交（suppression subtractive hybridization,SSH)技术,以流感病毒A／鲁防／93－9（H3N2）感染的MDCK细胞及正常MDCK细胞为材料,构建病毒感染特异性差减cDNA文库。从文库中随机挑取约800个克隆,PCR扩增其中插入片段,经纯化、紫外定量后,用基因芯片自动点样仪点在氨基片上,制备cDNA芯片。将流感病毒感染的MDCK细胞和正常MDCK细胞的总RNA分别用Cy3、Cy5反转录荧光标记后,与cDNA芯片杂交,用芯片扫描仪扫描获得芯片杂交信号,经阳性对照校正和归一化处理后,以如下条件作为判定基因差异表达的标准;（a)Cy3与Cy5的信号比值大于1．5（正常细胞用Cy5标记）或小于0．67（正常细胞用Cy3标记）;（b)Cy3和Cy5信号值之一必须大于1000。经cDNA芯片筛选获得了18个流感病毒感染特异性克隆,经测序和生物信息学分析发现均为流感病毒感染相关新基因EST。流感病毒感染相关基因cDNA片段的获得,为新型病毒药靶诊断标志物发现和功能研究提供了基础。     

对虾白斑综合征病毒新株型WSSV-CN-Pc的毒力研究

对虾白斑综合征病毒(white spot syndrome virus,WSSV)是一种能够感染虾类并且造成其大面积死亡的环状双链DNA病毒。WSSV有多种分离株,其毒力有所差异。从克氏原螯虾(Procambarus clarkii)中分离得到1株WSSV新分离株WSSV-CN-Pc,其毒力尚不清楚。本研究采用肌肉注射和经口注射的方法,以WSSVTW型作为阳性对照,分别对克氏原螯虾(P.clarkii)和罗氏沼虾(Macrobrachium rosenbergii)进行活体实验。实验结果显示:肌肉注射WSSV-CN-Pc和WSSV-TW的克氏原螯虾均在第6天出现100%的死亡;罗氏沼虾在肌肉注射WSSV-TW后未出现死亡,但在注射WSSV-CN-Pc后的第9天死亡率达100%。经口注射WSSV-CN-Pc和WSSV-TW的克氏原螯虾均在第16天出现100%的死亡;罗氏沼虾经口注射WSSV-CN-Pc后的第19天死亡率为100%,但注射WSSV-TW的实验组并未出现死亡。结果表明,对于克氏原螯虾,WSSV-CN-Pc具有和WSSV-TW相似的毒力,而对罗氏沼虾存在明显的毒力差异。提示克氏原螯虾是WSSV传播途径中的重要因素。     

白斑综合征病毒浙江分离株在动物模型克氏原螯虾组织中的分布

应用对虾白斑综合征病毒浙江分离株(WSSV-ZJ)人工口服感染实验动物模型克氏原螯虾,研究其在消化道组织和血淋巴细胞内分布及病理变化的特点。结果显示,在受感染濒死螯虾的胃、中肠和循环血淋巴中观察到大量病毒粒子,是病毒侵染的主要靶组织;此外,在肝胰腺组织的细胞中观察到少量病毒粒子。该病毒主要侵染结缔组织细胞、上皮细胞和循环血淋巴细胞等敏感细胞的细胞核。电镜和光镜观察及应用原位杂交检测表明,浙江株病毒粒子在螯虾体内的形态大小、分布特点和靶细胞组织的病理与其他地理株相似或相同。     

原位杂交研究对虾白斑杆状病毒在虾体内感染过程

应用地高辛标记的对虾白斑杆状病毒（ｗｈｉｔｅ ｓｐｏｔ ｓｙｎｄｒｏｍｅ ｂａｃｕｌｏｖｉｒｕｓ,ＷＳＳＶ）核酸探针,与人工感染后不同时间采集的对虾组织样品进行原位杂交,以动态研究病毒从侵染至对虾以病死亡的过程。将典型感染ＷＳＳＶ的病虾组织投喂健康对虾,结果显示：ＷＳＳＶ道德通过侵染消化道上皮进入虾体内增殖,此后随着细胞裂解、病毒粒子释放,游离的粒子伴随血淋巴循环进而杂其它靶组织,直至对虾发病死亡     

Microsatellite-centromere distances and microsatellite diversity in different ploidy classes of Chinese shrimp (Fenneropenaeus Chinensis)

This is the first report of microsatellite-centromere mapping in this commercial species Fenneropenaeus Chinensis, and will be important for providing fixed points in the linkage groups of genetic maps. Triploid Chinese shrimp was induced by heat shock. The fertilized eggs were treated either by retention of the first polar body or the second polar body to produce Meiosis I (MI) or Meiosis II (MII) triploid. The triploidy status in each Chinese shrimp could be confirmed by nine polymorphic microsatellite loci, in which the parents with different alleles and the female parents were each heterozygous. The nine loci were mapped in relation to their centromeres in three MII triploid families, which were induced by retention of the second polar bodies after fertilization with sperm. Microsatellite-centromere (M-C) distances ranged from 9.6 cM to 37 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in F. chinensis. Twelve polymorphic microsatellites were used to assess the heterozygosity and allelic diversity in different ploidy classes. As expected, triploids were significantly more polymorphic than diploids. The diploids had an average heterozygosity and allelic diversity value of 0.86, whereas the triploids heterozygosity averaged 0.93 and had allelic diversity value of 1.29. However, MI triploids were not significantly more polymorphic than MII in the microsatellite loci.     

斑点杂交检测对虾白斑综合征病毒青岛株在螯虾体内的动态分布

近年来 ,我国学者对人工养殖对虾暴发性病毒病的病原进行了较为系统的研究[1～ 5] ,本试验应用螯虾这一动物模型[6] ,利用斑点杂交方法 ,研究了白斑综合征病毒 (ＷＳＳＶ ,前称无包埋体对虾病毒Ｎｏｎ -Ｏｃｃｌｕｄｅｄ -ＳｈｒｉｍｐＶｉｒｕｓＮＯＳＶ )青岛株在螯虾体内的动态分布 ,为研究该病毒的传播途径、增殖致病机理提供了参考。1　材料与方法1.1　实验动物克氏原螯虾 (Ｃａｍｂａｒｕｓｐｒｏｃｌａｒｋｉｉ ,以下简称螯虾 ) 40尾 ,购自南京某农贸市场 ,实验室饲养一周以上 ,健康存活。1.2　种毒处理及接种白斑综合征病毒青岛株 (…     

抗对虾白斑综合症病毒单链抗体P1D3基因在毕赤酵母中的分泌表达

为了将可中和对虾白斑综合症病毒(WSSV)的单链抗体P1D3在酵母中实现表达,以原核表达载体M13噬菌粒为模板,设计带有SnaBⅠ和EcoRⅠ酶切位点的特异性引物,通过PCR方法扩增P1D3基因。经过酶切、连接反应将该基因连入大肠杆菌-酵母穿梭质粒pPIC9K上。重组质粒pPIC9K-scFvP1D3经BglⅡ线性化后,用电转化的方法转入毕赤酵母(Pichiapastoris)GS115中。通过PCR和DNA测序,挑选和鉴定阳性克隆。经甲醇诱导,P1D3在酵母中获得分泌表达。ELISA实验结果表明,酵母表达上清液中的单链抗体具有较高的WSSV结合活性,而且其活性要高于大肠杆菌所表达抗体的活性。表达条件优化后,单链抗体在酵母中最高表达量可达302mg/L,为开展对虾被动免疫研究提供了新的抗体来源。     

Application of suppression subtractive hybridization (SSH) to cloning differentially expressed cDNA inDunaliella salina (chlorophyta) under hyperosmotic shock

Two subtracted cDNA libraries ofDunaliella salina (Volvocales, Chlorophyceae) under different hyperosmotic shock were constructed using the suppression subtractive hybridization (SSH) method. The mRNA isolated from algae grown without stress was used as a “driver”, and the mRNAs isolated from algae 16 h (short-term treatment) or 7 d (long-term treatment) after salt stress were used as “testers”. The differentially expressed cDNA fragments inD. salina under salt stress were identified by screening these 2 libraries. Two cDNA fragments,D27 andD114, were identified from clones pL27 and pL114 after the long-term treatment. Three cDNA fragments,D21, D39, andD88, were identified from clones pSh21, pSh39, and pSh88 after the short-term treatment. The homology analysis revealed that D27 was highly similar (91%) to the subunit V of PS I reaction center inChlamydomonas reinhardtii. D21 was similar to fructose-1,6-diphosphate aldolase (78.4%). After searching GenBank with the sequences ofD39, D88, andD114, no similar sequences were found. Northern analysis revealed that the expression levels of all 5 cDNAs were increased significantly after salt stress. This means that SSH can be used in cloning differentially expressed cDNAs inD. salina under salt stress. The expression ofD27, D21, andD88 wasde novo induced by salt stress, and the expression ofD114 andD39 was increased from a relatively lower level; this indicates that all 5 cDNAs might exert an influence on the alga under hyperosmotic shock.     

Extraction of High Quality of RNA and Construction of a Suppression Subtractive Hybridization (SSH) Library from Chestnut Rose (Rosa roxburghii Tratt)

Chestnut rose (Rosa roxburghii Tratt) is a rare fruit crop of promising economical importance in fruit and ornamental exploitation in China. Isolation of high quality RNA from chestnut rose is difficult due to its high levels of polyphenols, polysaccharides and other compounds, but a modified CTAB extraction procedure without phenol gave satisfactory results. High concentrations of PVP (2%, w/v), CTAB (2%, w/v) and β-mercaptoethanol (4%, v/v) were used in the extraction buffer to improve RNA quality. The average yield was about 200 μg RNA g⁻¹ fresh leaves. The isolated RNA was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several pathogen-induced defense genes. Qiang Xu and Xiaopeng Wen - Contribute to this work equally Revisions requested 3 November 2005; Revisions received 18 January 2006