RNA干扰对LCDV-cn在牙鲆体内复制的影响

海水养殖生物病毒病是世界性难题,淋巴囊肿病毒中国分离株(LCDV-cn)是牙鲆(Paralichthys olivaceus)淋巴囊肿病的病原。以mcp基因为靶基因,设计siRNA-mcp1、siRNA-mcp2、siRNA-mcp3等3条siRNA序列,研究RNA干扰(RNAi)对淋巴囊肿病毒在牙鲆体内复制的影响。结果表明,牙鲆体内注入siRNA-mcp1和siRNA-mcp3后,mcp基因的表达有所下调;统计分析表明,注入siRNA-mcp1后,mcp基因的表达下调明显,具有显著性差异(p<0.05),注入siRNA-mcp2、siRNA-mcp3后,mcp基因的表达下调不明显,差异不显著(p>0.05)。本实验中初步实现了RNAi对LCDV-cn复制的抑制作用。     

一株H_5N_1亚型禽流感病毒株NA基因的克隆与序列分析

用RT-PCR方法从1个H5N1亚型禽流感病毒分离株A/Chicken/Guangdong/DH/1997扩增NA基因cDNA片段,将其克隆至pMD18-T载体,获得重组质粒pMD-NA,并对其核苷酸序列进行测定和分析。结果表明,该毒株的NA基因长度为1350bp,编码449个氨基酸,与其它H5N1亚型AIV分离株的核苷酸序列同源性为97.0%～99.4%,氨基酸序列同源性为97.7%～99.1%,提示禽流感病毒NA基因保守性较高。NA基因氨基酸序列的聚类分析表明该毒株与来自香港的A/Pheasant/HK/FY155/01和A/Ch/HK/FY150/01两个分离株处于同一进化枝,亲缘关系较近。     

一株H_5亚型禽流感病毒血凝素基因的克隆与序列分析

根据GenBank中发表的H5亚型禽流感病毒HA基因序列设计2对引物,用RT-PCR方法从禽流感病毒广东分离病毒株(A/Chicken/Guangdong/1997)中扩增HA基因cDNA片段,并将其克隆至pMD-18T载体进行核苷酸序列测定。结果表明:用2对引物所扩增的片段大小分别约为1300 bp和800 bp,经序列拼接获得的HA基因cDNA长度约为1601 bp,编码533个氨基酸,与国内己发表的11个代表株的核苷酸和氨基酸序列同源性为分别为96.9%~99.9%和86.5%~93.0%;HA基因编码的氨基酸序列的系统进化树也表明A/Chicken/Guang-dong/1997、A/Goose/Huadong/01/2000、A/Ck/Hk/37.4/2002、A/Chicken/Zhoukou/2/02、A/Duck/Guangxi/53/2002、A/Duck/Fujian/01/2002等毒株处于同一进化枝,亲缘关系较近;而与A/Silly/Chicken/Hongkong/SF189/01株处于不同进化枝,亲缘关系较远。     

草鱼呼肠孤病毒096vp7基因生物信息学分析及其酵母表达载体的构建

草鱼呼肠孤病毒（Grass carp reovirus, GCRV）是草鱼出血病的病原.从患病草鱼体内分离到一株草鱼呼肠孤病毒GCRV 096,经反转录PCR,克隆得GCRV 096 长度为855 bp 的vp7 基因,并分析该基因编码蛋白的特性.结果表明,同一基因型分离株VP7 蛋白间的差异不大,但不同基因型分离株VP7 蛋白间存在很大的差异.对GCRV 096 VP7 蛋白生物信息学分析表明,信号肽位点位于20 氨基酸处,且VP7 含有4个潜在的抗原决定簇.构建GCRV 096 vp7 基因的酵母表达载体pGBKT7-S10,并成功转化至酵母中.     

对虾病毒病暴发前期病毒和弧菌相互作用关系

模拟天然继发感染研究对虾病毒病暴发前期病毒和弧菌的关系。结果显示 ,弧菌先感染再病毒感染组比病毒先感染再弧菌感染组死亡率高、死亡快、生长慢、免疫功能低下。表明弧菌的潜伏感染对病毒的增殖有利 ,而病毒的潜伏感染对弧菌的继发感染没有明显的促进作用。说明病毒性流行病的暴发前期可能是由弧菌先感染 ,使对虾体内环境发生变化而助长病毒感染 ,其中对虾的免疫力下降是关键     

大口黑鲈抗菌肽hepcidin cDNA序列和结构分析

以大口黑鲈为材料,提取肝脏总RNA,经RT-PCR扩增出hepcidin cDNA的开放阅读框(ORF)及3′端非编码区序列,应用5′RACE方法得到大口黑鲈hepcidin cDNA5′末端。将所获得的两个片段分别克隆到T载体后进行测序,并拼接成大口黑鲈hepcidin全长cDNA。序列分析表明:大口黑鲈hepcidin全长cDNA为564bp,含有一个258bp的ORF,编码86个氨基酸残基,由信号肽(24个残基)、前肽(42个残基)和成熟肽(20个残基)3部分组成hepcidin前体。在前肽部分具有前肽转化酶典型的RX(K/R)R基元,成熟肽部分含有8个保守的半胱氨酸残基,可形成四个链内二硫桥,使β-折叠结构保持稳定。大口黑鲈Hepcidin与其他鱼类的同源性在29.7%~90.5%间,尤其是信号肽区域,与鳜、尼罗罗非鱼、真鲷、花鲈、黑鯛、金眼狼鲈仅有2~3个氨基酸的差别。     

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China (LCDV-cn) genome

An open reading frame (lcn61) of iymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector.Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene.     

Expression of putative zinc-finger protein lcn61 gene in lymphocystis disease virus China （LCDV-cn） genome

An open reading frame (lcn61) of lymphocystis disease virus China (LCDV-cn), probably responsible for encoding putative zinc-finger proteins was amplified and inserted into pET24a (+) vector. Then it expressed in E. coli BL21 (DE3), and His-tag fusion protein of high yield was obtained. It was found that the fusion protein existed in E. coli mainly as inclusion bodies. The bioinformatics analysis indicates that LCN61 is C2H2 type zinc-finger protein containing four C2H2 zinc-finger motifs. This work provides a theory for functional research of lcn61 gene. Supported by High Technology Research and Development Program of China (863 Program, No. 2006AA100309)     

Effects of High Hydrostatic Pressure on the Solubilities and Structures of Alaska Pollock Protein

Alaska pollock is an important protein source which is extensively used in the food industry. Pollock protein isolates(PPI) with significantly enriched protein contents could be prepared using isoelectric solubilization/precipitation(ISP) processing; however, the functional properties of this process is limited by the large amount of water-insoluble proteins. In this study, we investigated the influence of high hydrostatic pressure(HHP) treatment on the solubility and structural changes of PPI. PPI obtained using ISP is treated with hydrostatic pressures of 200, 300, 400, and 500 MPa for up to 15 min, and the HHP-treated samples were observed to exhibit significantly improved solubilities. Further biochemical assays reveal that the continuous HHP treatments reduce the contents of free sulfhydryl groups and promote the formation of macromolecules with better water solubilities, which may induce the solubility improvements of the HHP-treated PPI. Our results indicate that HHP can be utilized to effectively prepare highly water-soluble Alaska pollock protein in food processing.     

荷那龙罗非鱼两种GHSR基因的克隆与序列分析

用RT-PCR和RACE方法,从荷那龙罗非鱼(Oreochromis hornorum)垂体中克隆到生长激素促分泌素受体(GHSR)cDNA全序列。荷那龙罗非鱼GHSR基因具有GHSR-1a与GHSR-1b两个高度保守cDNA序列。GHSR-1a序列全长1 646 bp,包括225 bp的5′非编码区,266 bp的3′非编码区和1 155 bp的开放阅读框,编码384个氨基酸残基,具有7个跨膜结构域结构(transmembrane domains,TM);GHSR-1b序列全长1 877 bp,包括225 bp的5′非编码区,755 bp的3′非编码区和897 bp的开放阅读框,编码298个氨基酸残基,只具有前5个TM,在第6个TM的第4个氨基酸处开始缺失。将GHSR cDNA序列与基因组序列比较发现,这两种cDNA转录本来自同一个基因的不同变体。     

Partial Sequence Analysis of Mitochondrial COI Gene of the Chinese Shrimp, Fenneropenaeus Chinensis

Eight hundred and thirty eight base pair fragment of mitochondrial COI gene of wild and cultured populations (CP1, CP4, CP5 and CP6) of Fenneropenaeus chinensis was amplified and sequenced. The A, T, G and C contents of the sequence were 235bp (28.0% ),.307bp (36.6%), 138bp (16.5%) and 158bp (18.9%), respectively. Furthermore, 556bp fragment of the sequence was used to discuss the phylogenetic relationship among 14 Penaeidae species using Alpheus armillatus as the outgroup. From the molecular phylogenetic tree constructed by neighbor-joining method, we obtained three large shrimp groups: Farfantepenaeus, Litopenaeus and Fenneropenaeus group. The results also indicated that there were a closer genetic relationships between F. aztecus and F. paulensis, L. schmitti and L. setiferus, F. indicus and F. merguiensis, and the genus Farfantepenaeus was closer to Litopenaeus.     

军曹鱼群体遗传结构的AFLP分析

筛选出10对扩增片段长度多态分析技术(Amplified fragment length polymorphism,AFLP)的选择性扩增引物(E-aga/M-cgt、E-aga/M-cga、E-agc/M-cga、E-agg/M-cga、E-act/M-cgc、E-aag/M-cgc、E-aca/M-cga、E-aac/M-cgc、E-aac/M-cac和E-aac/M-cat),分析了4个军曹鱼(Rachycentron canadum)全人工繁育群体海南1(HN1)、海南2(HN2)、湛江(ZJ)和流沙湾(LS)的遗传结构。结果表明:总体多态位点比例(83.9%)、分化指数(0.427 8)和Shannon信息指数(0.614 9)均处于较高水平,遗传多样性丰富;主坐标分析和邻位连接树中LS-HN2支与ZJ-HN1支明显分离,各群体内多样性水平存在差异(遗传多样性大小依次为HN1、HN2、LS、ZJ),但总体基因流仍然较大(Nm=10.327),分子方差分析(AMOVA)获得的群内方差(53%)也略大于群间(47%)。     

Sequence analysis and quantitative detection of Norwalk-like viruses in cultured oysters of China

We isolated 4 Norwalk-like viruses (NLVs) contaminated oysters from 33 Chinese oysters collected from local commer-cial sources of Shandong Province. After amplification of the RNA-dependent RNA polymerase (RdRp) region of NLVs genomes with RT-PCR, the open reading frame 1 (ORF1) of the RdRp was sequenced and subjected to multiple-sequence alignment. The re-suits showed that NLVs in the four isolates belong to genogroup Ⅱ. The sequence comparison showed that the similarity between four Chinese oyster isolates were higher than 99.0%, which indicated that NLVs prevalent in close areas have high homogeneity in genome sequences. In addition, the most conserved sequences between diverse NLVs were used to design primers and TaqMan probes, then the real-time quantitative PCR assay was performed. According to the standard curve of GII NLVs, the original amounts (copies) of NLVs in positive patient's fecal isolate, positive Japanese oyster isolate, and the Chinese oyster isolate were 8.9×108, 1.25×108 and 4.7×101 respectively. The detecting limit of NLVs was 1×101 copies. This study will be helpful for routine diagnosis of NLVs pathogens in foods and thus for avoiding food poisoning in the future.     

主成分分析技术在遥感蚀变信息提取中的应用研究综述

主成分分析是目前遥感蚀变异常信息提取常用方法之一,该方法具有对影像大气校正质量要求不高、实现简单、提取效果好、效果稳健等优点,广受地质工作者的青睐。根据输入影像的数量及类型、主成分分析的次数等,本文将主成分分析分为标准主成分分析、特征向量主成分分析、定向主成分分析、二次主成分分析、不同影像间的主成分分析等5种类型。其中,特征向量主成分分析又可细分为4个波段特征向量主成分分析和3个波段特征向量主成分分析。在上述分类的基础上,系统介绍了各种主成分分析及蚀变信息主分量的选择,尤其是对特征向量主成分分析的Crosta技术和定向主成分分析的软落叶技术进行了详细阐述。并以TM/ETM+、ASTER影像为例,对部分应用主成分分析提取蚀变异常信息的实例进行了分析,认为：在基岩裸露区,不同主成分分析都可以很好地提取铁化、泥化蚀变信息;在中、低植被覆盖区,采用标准主成分分析、Crosta技术、改进的Crosta技术、软落叶技术、“掩膜/抑制干扰信息+主成分分析”等方法可以有效地提取蚀变异常信息;高植被覆盖区多采用主成分分析生成的蚀变信息主分量进行彩色合成,再通过对彩色影像进行目视解译的方式判断蚀变的类型和范围。其中,“掩膜+Crosta技术”、“掩膜+软落叶技术”、二次主成分分析等方法在高植被覆盖区也可以取得较好的应用效果;对于干扰信息种类众多、岩性复杂的地区,可根据干扰信息、岩性种类划分成若干个小区,再根据每个小区实际情况采用不同的蚀变提取方法,最后将每个小区内提取的蚀变信息进行合并。