大菱鲆MKK基因家族全基因组鉴定及其在生物和非生物应激下的表达分析

为了阐明大菱鲆丝裂原活化蛋白激酶激酶(mitogen-activated protein kinase kinases,MAPKKs或MKKs)基因家族在生物和非生物应激响应中的作用,本实验首先通过生物信息学方法对大菱鲆MKK基因家族进行了全基因组水平的鉴定,利用多个应激相关转录组数据集分析了大菱鲆MKK家族成员在不同组织及不同生物和非生物应激下的表达模式。结果显示,本研究在大菱鲆全基因组水平上共鉴定出9个MKK基因家族成员,它们不均匀地分布在7条染色体上,并分别对其编码蛋白的理化性质、蛋白二级结构和亚细胞定位进行了预测。基于系统发育分析,将SmMKKs划分为5个亚家族。内含-外显子结构、保守基序和多重序列比对分析结果不仅为大菱鲆MKK亚家族分类提供了证据,而且表明SmMKKs在进化上高度保守。SmMKKs在不同组织及不同生物和非生物应激下的基因表达模式分析表明,SmMKKs具有明显的组织特异性表达。另外,结果显示,粘孢子虫和肿大细胞病毒感染后,SmMKK6a呈极显著差异表达;热应激处理后,SmMKK6a呈极显著差异表达;高盐或低盐胁迫后,SmMKK4a、SmMKK4b、SmMKK6a...     

Alteration of the haemocyte parameters,expression of immune‐related and neurohormone bursicon genes of giant river prawn Macrobrachium rosenbergii (de Man) in the response to salinity stress and ammonia stress

We examined the effects of salinity stress and ammonia stress on alteration of the haemocyte count, phenoloxidase activity, expressions of immune‐related genes including prophenoloxidase (proPO), crustin, heat shock protein 70 (HSP70), and expressions of stress‐responsive neurohormone (Bur‐α and Bur‐β) in the thoracic and abdominal ganglia of giant river prawn Macrobrachium. These parameters of prawn that subjected to salinity stress (transferred from 0‰ to 5‰ and 10‰), and subjected to ammonia‐nitrogen (ammonia‐N) stress (transferred from 0 to 0.262 mg/L and 0.786 mg/L) were examined after 0, 3, 6, 24, 72 and 168 hr respectively. During the initial period of 3, 6 and 24 hr, granulocyte haemocyte (granular and semi‐granular hemocyte) count and PO activity significantly decreased, while expressions of Bur‐α and Bur‐β significantly increased. After 24 hr, granulocyte haemocyte count and PO activity significantly increased, whereas expressions of Bur‐α and Bur‐β significantly decreased. The expressions of proPO, crustin and HSP70 were significantly downregulated in the prawn that subjected to salinity stress and ammonia‐N stress at all time periods of 3–168 hr. In conclusion, changes in the granulocyte haemocyte count of M. rosenbergii following salinity stress and ammonia‐N stress are closely associated with the changes of Bur‐α and Bur‐β expressions.     

浅色黄姑鱼线粒体16S rRNA基因片段序列特征分析

PCR扩增100个浅色黄姑鱼个体的线粒体16SrRNA基因片段序列,得到大约620bp的扩增产物。将其中5个个体的扩增产物进行测序和同源比对,得到468bp可供比对分析的片段。比对结果表明,5条序列包括两个单倍型,两个单倍型之间有1个碱基突变。PCR-RFLP分析结果显示,两个种群的100个样品中98%的个体为其中一种单倍型,只有2%的个体呈另一种单倍型,表明这两个种群的遗传多样性较低。两个单倍型平均碱基组成为:T22.0%,C26.3%,A29.8%,G21.9%,GC含量平均为48.2%。与GenBank中石首鱼科7属9种的11条同源序列比对,得到429个比对位点,其中包括69个简约信息位点、55个单突变子和16个插入/缺失位点。聚类分析显示,浅色黄姑鱼与黄姑鱼亲缘关系较近,与形态分类相符。     

凡纳滨对虾不同养殖密度高位池水体细菌群落动态

通过对养殖水体环境基因组DNA中细菌16S rRNA基因V4–V5区的高通量测序和生物信息学分析,研究了两种养殖密度的凡纳滨对虾(Litopenaeus vannamei)高位池水体中细菌群落在养殖过程中的动态。结果显示,养殖过程中各菌群相对丰度变化明显,细菌多样性随时间逐渐提高,优势菌群为变形菌门(Proteobacteria)、蓝藻门(Cyanobacteria)、拟杆菌门(Bacteroidetes)、放线菌门(Actinobacteria)和浮霉菌门(Planctomycetes)。随着养殖时间增长,蓝藻丰度所占比例逐渐减少,而变形菌、拟杆菌和浮霉菌丰度逐渐增大,同时养殖前期高密度池浮霉菌丰度显著高于低密度池(P0.01),而其他菌群无显著差异。结果表明,养殖期前50 d不同养殖密度水体细菌群落差异较大,而后30 d内细菌群落的时间异质性大于空间异质性,这意味着高位池水体菌相被划分为两类,到养殖后期菌相快速转变,养殖密度所带来的影响被减弱。     

Detection and molecular characterization of infectious spleen and kidney necrosis virus from major ornamental fish breeding states in Peninsular Malaysia

‘Gold standard’ OIE reference PCR assay was utilized to detect the presence of infectious spleen and kidney necrosis virus (ISKNV) in freshwater ornamental fish from Malaysia. From total of 210 ornamental fish samples representing 14 species, ISKNV was detected in 36 samples representing 5 fish species. All positive cases did not show any clinical signs of ISKNV. Three restriction enzymes analyses showed that the fish were infected by identical strains of the same virus species within Megalocytivirus genus. Major capsid protein (MCP) genes of 10 ISKNV strains were sequenced and compared with 9 other reference nucleotide sequences acquired from GenBank. Sequence analysis of MCP gene showed that all strains detected in this study were closely related to the reference ISKNV with nucleotide sequence identity that was ranging from 99.8% to 100%. In addition, phylogenetic analysis of MCP gene revealed that viruses from genus Megalocytivirus can be divided into three genotypes: genotype 1 include reference ISKNV and all other strains that were detected in this study, genotype 2 include viruses closely related to red sea bream iridovirus (RSIV), and genotype 3 include viruses closely related turbot reddish body iridovirus (TRBIV).     

大鳞副泥鳅mtDNA Cytb基因序列分析及分子系统发育研究

对15尾大鳞副泥鳅的线粒体细胞色素b基因进行PCR扩增和正反测序,获得长度为1140 bp的细胞色素b基因同源片断。所有序列中共检测到15种单倍型,序列中共出现81个变异位点,总变异率为7.1%。各单倍型的变异全部是转换或颠换,无插入和缺失,转换/颠换比为6.36。变异位点在3位密码子中的分布呈现偏倚,密码子第3位的变异占总变异的85.18%,而第1和第2位均只占7.41%。A、T、C、G碱基的平均含量分别为27.4%、30.8%、26.5%、15.3%,A+T>C+G。在所得序列中,密码子第1位上4种碱基使用较为均衡;第2位上碱基T的使用率高达41%,碱基G的使用率低至13.2%;第3位上碱基A的使用率为37.9%,而碱基G的使用率仅为7.0%。以红尾副鳅为外群用邻接法构建分子系统进化树。结果显示,在系统地位上大鳞副泥鳅与黑龙江泥鳅有较近的亲缘关系。     

斑节对虾GLUT1基因cDNA的克隆与表达分析

该研究利用RACE技术克隆获得了斑节对虾(Penaeus monodon) GLUT1 (glucose transporter type 1)的cDNA全长序列;采用实时荧光定量的方法研究了PmGLUT1在斑节对虾幼体发育过程中、各个组织中及低盐胁迫下的差异表达情况。该基因cDNA开放阅读框(ORF)全长1 476 bp,可编码491个氨基酸。检测PmGLUT1基因从受精卵至仔虾期发育过程的表达情况,结果显示,PmGLUT1在幼体发育各期的表达量有所波动,但总体呈现上升趋势。组织表达分析发现,PmGLUT1在鳃组织中的表达量最高,肝胰腺次之,在卵巢中的表达量最低。急性低盐胁迫后,PmGLUT1在肝胰腺中的表达水平显著高于对照组(P<0.05),但在鳃中的表达水平显著低于对照组(P<0.05)。研究结果表明,Pm GLUT1可能在斑节对虾幼体发育及机体应对低盐胁迫过程中具有重要作用,这为进一步研究葡萄糖转运蛋白基因在斑节对虾幼体发育调控和耐低盐胁迫应答中的分子机制提供了理论依据。     

厚壳贻贝5-羟色胺2A受体(5-HT2AR)基因克隆和时空表达

为探究5-羟色胺2A受体（5-HT 2A receptor, 5-HT2AR）基因在海水贝类生长发育中的作用,本研究通过RACE技术和实时荧光定量PCR（qRT-PCR）克隆了厚壳贻贝5-HT2AR 基因的cDNA全长,并分析该基因的时空表达。结果显示,5-HT2AR基因全长2 636 bp,开放阅读框（ORF）2 124 bp,共编码707个氨基酸。序列分析结果显示该序列与人、小鼠、斑马鱼、长牡蛎和虾夷扇贝等物种分别具有45%、45%、48%、49%和67%的同源性。厚壳贻贝雌雄成体各组织和器官中均有5-HT2AR基因表达,雄性中的鳃表达量最高,而在雌性鳃、外套膜和性腺中的表达稍高于其他组织和器官;推测该基因可能与厚壳贻贝的摄食、对外界环境的感知及促进卵母细胞成熟有关。5-HT2AR基因在厚壳贻贝各发育阶段均有表达,且稚贝的表达量为眼点幼虫的1.4倍,推测5-HT2AR基因可能参与了调控厚壳贻贝幼虫的生长发育过程。本研究为进一步了解5-HT基因家族在双壳贝类中的功能奠定了基础。