三疣梭子蟹FAMeT基因克隆及其在蜕皮周期中的表达水平

法尼酸甲基转移酶(farnesoic acid O-methyl transferase, FAMeT)是甲基法尼酯(methyl farnesoate, MF)生物合成途径中最后一步的关键酶,MF是一种类似昆虫保幼激素(juvenile hormone, JH)的倍半萜,在甲壳动物的生长发育及繁殖过程中起到了重要作用。本文克隆得到了三疣梭子蟹FAMeT的全长cDNA序列（GenBank登录号：KC192659）,它包括一个201bp 的5非编码区、一个318bp的 3非编码区,和一个825bp的开放阅读框（ORF）,编码274个氨基酸;推导的氨基酸序列与已公布的其他甲壳动物FAMeT进行比对,发现一致性达75%-97%,其中与远海梭子蟹FAMeT的一致性最高;而且该氨基酸序列由两个CF（CPAMD8/FAMeT）区域组成,这两个CF区域是FAMeT的标志,在所有甲壳动物的FAMeT里均有发现,因此推导的氨基酸序列是三疣梭子蟹FAMeT基因。我们运用实时荧光定量PCR(qRT-PCR)的方法分析了其在不同组织中、不同蜕皮周期中的表达量变化,发现FAMeT在三疣梭子蟹的各个组织里均有表达且在胸神经节(Taoracic ganglia)里表达最强;在三疣梭子蟹蜕皮过程中,大颚器FAMeT在D1期表达最强,然后逐渐下降至D4最低。该结果表明FAMeT在三疣梭子蟹蜕皮调控中起着重要的作用。     

Sequence Variation in the Farnesoic Acid O‐methyltransferase Gene and its Associations with Growth of Shrimp,Penaeus chinensis

The aim of this study is to screen single nucleotide polymorphisms (SNPs) of the farnesoic acid O‐methyltransferase gene in shrimp, Penaeus chinensis (PcFAMeT), and analyze the potential association between PcFAMeT gene polymorphisms and growth traits in a cultured population. Four SNPs (A163G, G392T, A708G, and T752C) were tested for association with six growth traits in 240 individuals using the polymerase chain reaction‐restriction fragment length polymorphism (PCR‐RFLP) method. The association analysis of SNPs of PcFAMeT gene with the six growth traits was carried out using general linear model estimation. Results indicated that SNP2 (G392T) of the PcFAMeT gene was significantly associated with body weight (P < 0.05) and carapace width (P < 0.05). The individuals of genotype GG grew faster than those of genotype GT and TT. The results would provide potential application in future shrimp breeding programs and may be used to improve the efficiency in shrimp, P. chinensis, breeding programs.     

The responsive expression of a caspase gene in Chinese shrimp Fenneropenaeus chinensis against pH stress

Caspases are a family of proteases, which play an important role in apoptosis. To evaluate the relationship between apoptosis and pH stress in crustaceans, a caspase gene (FcCasp) was cloned from the Chinese shrimp Fenneropenaeus chinensis. The full length of FcCasp was 1329 bp with a 972 bp ORF, encoding a polypeptide of 323 amino acids with a calculated molecular weight and pI of 36.0 kDa and 6.27 respectively. The deduced amino acid sequence of FcCasp contained a potential active site (QACRG pentapeptide) conserved in most caspases and two profile hits (p20 and p10 domain profile). Comparison of amino acid sequences revealed that FcCasp had an overall similarity of 76–83% with other penaeid shrimp caspases. The amino acid sequence of recombinant FcCasp protein expressed in Escherichia coli was identified by matrix‐assisted laser desorption/ionization‐time of flight‐mass spectrometer analysis. High‐level expression of FcCasp in six different tissues was detected by real‐time polymerase chain reaction after exposure to pH stress for 96 and 148 h. TUNEL analysis indicated that apoptosis began to appear in F. chinensis hepatopancreas exposed to extreme pH for 12 h. The amount of apoptosis seems positively correlated with the length of exposure to the pH stressor. The results suggested that FcCasp was involved in the response to environmental pH stress.     

脊尾白虾(Exopalaemon carinicauda)FAMeT基因在卵巢发育周期中的表达分析

为了研究法尼酸甲基转移酶(Farnesoic acid O-methyltransferase,FAMeT)在脊尾白虾卵巢发育中的作用,采用实时荧光定量PCR方法分析连续3次繁殖过程中卵巢不同发育时期(Ⅰ-Ⅴ期)大颚器、卵巢和肝胰腺中FAMeT基因的表达规律.结果显示,FAMeT基因在鳃、心脏、肌肉、肝胰腺、卵巢、大颚器等组织中均有稳定的表达,其中,在鳃和大颚器的表达量显著高于其他组织;在卵巢发育周期内,FAMeT基因在大颚器、卵巢、肝胰腺中的表达量变化趋势基本一致,从Ⅰ-Ⅳ期逐渐下降,到Ⅴ期时表达量上调;在每个卵巢发育时期,FAMeT基因在大颚器中表达量最高,约为卵巢中表达量的20-30倍.研究结果表明,大颚器是合成FAMeT的主要组织,脊尾白虾繁殖过程中FAMeT基因主要在卵巢发育前期表达,可以合成大量甲基法尼酯(Methylfarnesoate,MF),促进卵巢发育.     

Cloning of the Ecdysone Receptor Gene from the Chinese Mitten Crab,Eriocheir sinensis,and Sexually Dimorphic Expression of Two Splice Variants

The ecdysone receptor (EcR), a member of the nuclear receptor superfamily, plays an important role in molting in crustaceans and insects. Here, we report the cloning of the full‐length cDNA of the EcR gene from Eriocheir sinensis (EsEcR). A 477 bp alternatively spliced intron was identified, with two splice variants. One, EsEcR‐L, is 2522 bp long and encodes a 577 amino acid protein. The second, EsEcR‐S, is 2045 bp long and encodes a 422 amino acid protein. Among eight different tissues, the expression of EsEcR was highest in the hepatopancreas and lowest in the heart. EsEcR was expressed during different embryonal developmental stages, and its expression reached the highest level at the original zoea stage. EsEcR‐L was specifically expressed in testis among eight examined tissues and was also the predominantly expressed form in testis, while in other tissues the predominantly expressed form was EsEcR‐S, indicating different roles of these splice variants in males and females. EsEcR‐L was also expressed in embryos. The responses of EsEcR and EsEcR‐L after eyestalk ablation (ESA) were investigated in four tissues, including hepatopancreas, muscle, testis, and ovary. The expression of EsEcR was upregulated after ESA in hepatopancreas, muscle, and ovary, while the expression of EsEcR‐L was downregulated after ESA.     

中国明对虾NHE3基因克隆及其在pH胁迫下的表达

为研究钠/氢交换体(Na+/H+-exchanger,NHE)在中国明对虾(Fenneropenaeus chinensis)响应pH胁迫过程中发挥的作用,首先采用静水毒性实验方法确定了中国明对虾酸碱半致死pH,然后利用RACE技术克隆了中国明对虾Na+/H+-exchanger isoform 3(命名为FcNHE3)基因,并通过荧光定量PCR及RNA干扰技术分析了其在pH胁迫下的表达特征及功能。结果显示,72 h酸性半致死pH和碱性半致死pH分别为5.2和9.1。克隆获得FcNHE3基因(Gen Bank:MF373587)cDNA序列全长3508 bp,开放阅读框2805 bp,编码934个氨基酸,具有信号肽和12个跨膜结构域;蛋白同源分析发现,FcNHE3与青蟹(Carcinus maenas)同源性最高,达到74%;系统进化分析显示,FcNHE3与三疣梭子蟹(Portunus trituberculatus)和青蟹亲缘关系最近。荧光定量PCR分析表明,FcNHE3基因在鳃组织中表达量显著高于其他组织(P0.05);酸性半致死pH(pH 5.2)胁迫下,FcNHE3基因在整个胁迫过程中显著上调表达(P0.05);碱性半致死pH(pH 9.1)胁迫下,FcNHE3基因在前48 h显著下调表达(P0.05),12 h表达量最低,仅在72 h出现上调表达。RNA干扰后,FcNHE3基因表达受到抑制,pH 5.2胁迫下对虾存活率相比对照组显著下降。研究表明相较于高pH胁迫,FcNHE3基因在中国明对虾响应低pH胁迫过程中可能发挥更重要的调节作用。     

斑节对虾Chitinase-2基因的克隆及其在蜕皮和幼体发育过程中的表达分析

研究以斑节对虾(Penaeus monodon)转录组获得的几丁质酶基因(Pm Chi)片段,运用RACE技术克隆了PmChi基因c DNA全长,命名为PmChi-2。PmChi-2基因c DNA全长2 050 bp,其中5'-非编码区(5'-UTR)144 bp、3'-UTR 319 bp和开放阅读框(ORF)1 587 bp,编码528个氨基酸。生物信息学分析显示,PmChi-2与其他甲壳动物Chi-2的相似性为78%~97%。实时荧光定量PCR结果显示,PmChi-2在蜕皮前期(D期)表皮中表达水平最高,在胃、鳃、腹神经节和眼柄中表达水平依次降低,在其他组织(肝胰腺、肠、肌肉、心脏)中几乎不表达。不同蜕皮阶段,PmChi-2在鳃、胃和表皮3种组织中的表达变化模式基本一致,均在蜕皮期(E期)表达量最低,而最高表达量在鳃中出现在蜕皮后期(A期),在胃和表皮中为D期。幼体不同发育阶段分析揭示PmChi-2 mRNA在幼体发育阶段的无节幼体到糠虾幼体第二期表达量维持在一个低水平,在糠虾幼体第三期PmChi-2 mRNA表达水平显著升高,在仔虾期又显著下降,推测PmChi-2 mRNA可能与斑节对虾幼体发育密切相关。研究结果表明PmChi-2基因可能在斑节对虾蜕皮以及幼体的变态发育中发挥重要作用,为深入研究斑节对虾几丁质酶发育调控提供了重要信息。     

三疣梭子蟹几丁质酶基因的克隆及其在蜕皮过程中的表达分析

为探究几丁质酶基因在三疣梭子蟹蜕皮过程中的生理作用,本研究通过转录组测序和RACE技术克隆了三疣梭子蟹几丁质酶基因（PtChi）cDNA全长（登录号：KF914663）,并通过荧光定量PCR（qRT-PCR）技术研究了该基因在三疣梭子蟹不同组织及不同蜕皮阶段的表达情况。结果表明,(1)PtChi基因cDNA全长2 200 bp,包括5’非编码区（5’-UTR）16 bp、3’-UTR 714 bp和开放阅读框1 470 bp,编码489个氨基酸,预测分子量和等电点为53.97 ku和4.76。(2)BlastP 结果显示,PtChi推导氨基酸序列与已知甲壳动物Chi-3的一致性为61%~96%,系统进化树分析表明PtChi与其他甲壳动物Chi-3聚为一支。(3)qRT-PCR结果显示PtChi基因在C期三疣梭子蟹肝胰腺中表达水平最高,胃、大颚器、心脏和眼柄中PtChi-mRNA表达水平依次降低,在其他组织中PtChi-mRNA表达量最低,且表达水平无显著差异。(4)不同蜕皮阶段,PtChi在肝胰腺、肠、胃和大颚器4种组织中的表达变化模式有所不同,肝胰腺中PtChi-mRNA表达水平在AB期最高,C期最低,暗示PtChi可能参与三疣梭子蟹蜕皮后期对病原体的免疫防御;肠中的PtChi-mRNA表达水平在E期最高,C期最低,推测PtChi参与蜕皮过程中肠道围食膜的分解和免疫功能;胃中PtChi表达水平在C期最高,暗示其参与了食物消化。以上结果表明,本研究克隆的PtChi可能为甲壳动物Chi-3型,其准确生理学功能及其在蜕皮过程中的调控机制有待进一步深入研究。     

罗氏沼虾细胞色素P450家族CYP302a1基因克隆及其在蜕皮周期中的表达

蜕皮是甲壳动物重要的生理活动,与其蜕皮激素的合成密切相关,细胞色素P450(CYP)302a1是甲壳动物蜕皮激素合成通路中的关键酶之一。本研究克隆了罗氏沼虾CYP302a1基因(Mr-CYP302a1),cDNA全长1859 bp,开放阅读框(ORF)为1629 bp,编码543个氨基酸(aa),分子量大小为61.09 ku,等电点为8.42。氨基酸序列分析显示CYP302a1基因的保守结构域含有5个P450基因家族特征保守区域:heme-binding、helix-K、helixC、helix-I及PERF。系统进化分析结果显示Mr-CYP302a1首先与绿虾CYP302a1聚为一支,然后与凡纳滨对虾及三疣梭子蟹等十足目甲壳动物的CYP302a1聚为一支,与甲壳动物的亲缘关系最近。实时荧光定量PCR(qRT-PCR)检测表明Mr-CYP302a1在罗氏沼虾的多个组织中均有表达,其中在Y器官中的表达量最高,性腺中次之。同时研究发现,MrCYP302a1基因在罗氏沼虾的蜕皮后期(A期和B期)表达量很低,蜕皮间期(C期)表达量开始上升,在蜕皮前期D1亚期达到峰值。对Mr-CYP302a1进行蛋白表达及多克隆抗体制备,蛋白印迹法(Western blot,WB)检测表明Mr-CYP302a1蛋白在罗氏沼虾Y器官中的表达量最高,在蜕皮过程中的蜕皮前期D1亚期达到峰值。综上所述,该基因在罗氏沼虾的蜕皮过程中扮演着十分重要的角色。     

Response of facilitative glucose transporter 1 to salinity stress and dietary carbohydrate nutrition in white shrimp Litopenaeus vannamei

Facilitative glucose transporter 1 (GLUT1) is a transporter protein for glucose transport via the plasma membrane of the cells to provide energy through carbohydrate metabolism. GLUT1 cDNA from Litopenaeus vannamei was obtained and analysed in this study. Full‐length GLUT1 cDNA is 2062 bp long and contained a 1506‐bp ORF encoding a 502 amino acid protein, a 270‐bp 5′UTR and a 284‐bp 3′UTR. When shrimp were under acute low salinity stress, the expression in hepatopancreas, muscle, gill and eyestalk was all up‐regulated at 12 h (P < 0.05) and 96 h (P < 0.05), while the expression in the four tissues was all down‐regulated at 6 h (P < 0.05) and 48 h (P < 0.05) . The expression in the muscle of shrimp at water salinity of 3 was lower than that at water salinity of 30 independent of dietary carbohydrate levels, while expression in hepatopancreas, gill and eyestalk was up‐regulated at 200 and 300 g kg^?1 carbohydrate levels. The expression in all tissues fed glucose was up‐regulated when compared to the expression in shrimp held at a water salinity of 30. This study suggests that GLUT1 is a conserved protein in L. vannamei, and changes in expression due to environmental salinity and dietary carbohydrate level and source.     

中国明对虾calcineurin B基因的克隆及其在竞争行为中作用的初探

中国明对虾在养殖中表现出较强的竞争行为,对其生长性能产生了较大的影响,然而,到目前为止其发生的分子机制尚不清楚。钙调神经磷酸酶(calcineurin,CN)是高度保守的Ca2+/钙调蛋白(calmodulin,CaM)依赖性丝氨酸/苏氨酸磷酸酶,由催化亚基(CNA)和调节亚基(CN-B)组成,是参与许多重要生理过程的多功能蛋白质。CNB在Ca2+/CaM的介导下主要在动物的中枢神经系统发挥重要作用。另外,前期通过比较转录组分析筛选出的与中国明对虾竞争行为相关的候选基因中包括CNB基因。为了进一步明确CNB在中国明对虾竞争过程中的作用,实验通过RACE技术克隆了中国明对虾CNB基因(FcCN-B)的全长cDNA序列,并利用Real-time PCR技术分析了其在高竞争能力组(HCG)和低竞争能力组(LCG)组间不同组织(神经节、心脏、胃、肝胰腺和肠)中的表达情况。结果显示,FcCN-B的cDNA全长序列为2867 bp,包括95 bp的5′非编码区(UTR),540 bp的开放阅读框(ORF),和2232 bp的3′UTR,其中ORF中具有4个保守的EF-hand Ca2+结合结构域。蛋白质同源性分析显示,FcCN-B的氨基酸序列与其他物种具有较高的同源性(78.8%~93.8%),其中最高的是中华绒螯蟹(93.8%)和黑腹果蝇(90.5%);系统进化关系分析显示,脊椎动物和无脊椎动物分别独立为一支,且中国明对虾与中华绒螯蟹单独聚为一支,之后与黑腹果蝇聚类关系最近,提示FcCN-B在中国明对虾中可能具有与其在中华绒螯蟹和果蝇中相类似的功能。Real-time PCR定量结果显示,FcCN-B在HCG组的神经节中的表达极显著高于LCG组,而其在HCG组的心脏中的表达极显著低于LCG组。研究结果表明,calcineurin B基因在中国明对虾的竞争行为中可能发挥一定的作用,将为解析中国明对虾竞争行为的分子机制奠定重要的基础。     

Effects of Limited Dissolved Oxygen Supply on the Growth and Energy Allocation of Juvenile Chinese Shrimp,Fenneropenaeus chinensis

Information about the effect of hypoxia exposure on energy allocation is helpful for better understanding how aquatic animals tolerate and adapt to a hypoxic environment. The growth, molting, and energy allocation were investigated in juvenile Fenneropenaeus chinensis exposed to five different dissolved oxygen (DO) seawaters (2.09 ± 0.15, 3.10 ± 0.29, 4.13 ± 0.25, 4.73 ± 0.12, and 5.48 ± 0.09 mg/L DO) for 30 d. When DO was below 4.13 mg/L, the growth of shrimp was depressed. Feed ingestion and feed conversion efficiency decreased with the decrease of DO. Less feed ingestion was caused by lower daily metabolic energy. Higher proportion of ingested energy lost in metabolism, exuviations, and nitrogen excretion caused lower feed conversion efficiency. More energy lost in exuviations, lower survival rate, soft carapaces of dead shrimp, and cannibalism were found in oxygen deficient groups and it implicated that hypoxia could delay the shrimp recovering from molting and cause cannibalism and higher mortality. The results indicated that hypoxia was an important factor affecting the amount and fitness of shrimp stock because it could cause a high rate of mortality and growth depression.     

Cloning,characterization, expression analysis and RNAi of Retinoblastoma‐like gene from black tiger shrimp (Penaeus monodon)

Retinoblastoma (Rb) is a multifunctional regulator involved in several key cellular processes, such as cell cycle control, cell differentiation, tumorigenesis and senescence. In this study, an Rb‐like gene, PmRBL, was cloned from black tiger shrimp (Penaeus monodon). The full‐length cDNA sequence of PmRBL is 4,069 bp with an open reading frame of 3,243 bp, which encodes 1,080 amino acids. Quantitative real‐time PCR (qRT‐PCR) analysis indicated that PmRBL was highly expressed in the gills, hepatopancreas and ovaries of P. monodon. The highest PmRBL expression levels were observed in stage III of the ovarian development in P. monodon. RNA interference experiments were conducted to examine the expression profiles of PmRBL, PmCDK2 and PmCyclin E. The knockdown of PmRBL in the ovary and hepatopancreas by dsRNA‐RBL was successful. After dsRNA‐RBL was injected into the shrimp, the relative expression levels of PmCDK2 and PmCyclin E were upregulated at 12–72 hr in the ovaries and hepatopancreas. The localization and level of PmRBL expression in the ovary and hepatopancreas were investigated through in situ hybridization, which revealed consistent results with those of qRT‐PCR. Therefore, PmRBL, PmCDK2 and PmCyclin E may be involved in vitellogenin synthesis and ovarian maturation in P. monodon.     

脊尾白虾E75基因克隆及E75、ECR和RXR在不同蜕皮分期的表达分析

本研究在克隆脊尾白虾(Exopalaemon carinicauda)E75基因(EcE75,GenBank No.KY471317)的基础上,探讨了其在蜕皮过程中的作用,同时也探讨了克隆脊尾白虾的蜕皮激素受体基因(EcECR)和维甲酸X受体基因(Ec RXR)在不同蜕皮分期的表达特征。克隆的EcE75基因全长为3944 bp,包括2520 bp的开放阅读框(ORF)。该开放阅读框编码一个由839个氨基酸组成的蛋白质,分子量为92.307 k Da,理论等电点为7.48。EcE75蛋白包含1个C4锌指结构,1个配体结合结构域,并且有1个明显的跨膜螺旋。EcE75基因在进化上具有一定的保守性,与甲壳动物聚为一支,与三疣梭子蟹(Portunus trituberculatus)、黑背地蟹(Gecarcinus lateralis)、凡纳滨对虾(Litopenaeus vannamei)、中国明对虾(Fenneropenaeus chinensis)等亲缘关系最近。EcE75基因在脊尾白虾的各组织中均有表达,其中,眼柄中的表达量最高,卵巢次之。在不同蜕皮分期中,EcRXR与EcECR、EcE75的表达规律基本一致,生殖蜕皮前期和后期表达量都比生长蜕皮高。生长蜕皮和生殖蜕皮因为卵巢发育而存在明显不同,蜕皮激素对卵巢发育的刺激作用,通过EcECR、EcRXR和EcE75基因的表达上调可以体现出来。对EcECR、EcRXR和EcE75基因在不同蜕皮分期的表达研究,为了解虾蟹类蜕皮机制提供了参考信息。     

Development of a multiplex PCR system for the simultaneous detection of white spot syndrome virus and hepatopancreatic parvovirus infection

A multiplex PCR kit for simultaneous detection of white spot syndrome virus (WSSV) and hepatopancreatic parvovirus (HPV) was developed and field testing was conducted. A 604‐bp target sequence was selected from the vp28 gene of WSSV. A primer set was developed to amplify a 338‐bp DNA fragment at the junction of the NS2 and NS1 protein genes of HPV after alignment of eight sequences from different strains. Another internal positive control primer set produced a 139‐bp PCR fragment from the β‐actin gene by alignment of this gene from Litopenaeus vannamei, Fenneropenaeus chinensis and Penaeus monodon. The detection limits, tested using purified plasmids, for WSSV and HPV were 21.4 and 19.0 copies respectively. The optimum ratio for HPV, WSSV and β‐actin was 3:1:1, with an optimum annealing temperature of 57°C. Field test of the multiplex PCR with 170 L. vannamei individuals from 17 aquaculture farms showed 41.8% coinfection with WSSV and HPV, and 40.0% and 3.5% single infection with WSSV and HPV respectively. No virus‐free shrimp farm was found. Ten wild catch F. chinensis individuals showed 60% coinfection, and 40% were infected with HPV.     

A real‐time PCR for the detection of hepatopancreatic parvovirus (HPV) of penaeid shrimp

Hepatopancreatic parvovirus (HPV) causes a common shrimp disease that occurs in many shrimp farming regions, especially in the Indo Pacific, and infects most of the cultured penaeid species. There are seven geographic HPV isolates known, so a method to detect different HPV types is needed. We developed a sensitive and generic real‐time PCR assay for the detection of HPV. A pair of primers and TaqMan probe based on an HPV sequence obtained from samples of Fenneropenaeus chinensis from Korea were selected, and they were used to amplify a 92 bp DNA fragment. This real‐time PCR was found to be specific to HPV and did not react with other shrimp viruses. A plasmid (pHPV‐2) containing the target HPV sequence was constructed and used for determination of the sensitivity of this assay. The assay could detect a single copy of plasmid DNA, and it was used successfully in finding HPV in shrimp samples from the China‐Yellow Sea region, Taiwan, Korea, Thailand, Madagascar, New Caledonia and Tanzania.     

Dietary fulvic acid effects on survival and expression of immune‐related genes in Litopenaeus vannamei challenged with Vibrio parahaemolyticus

The effects of fulvic acid (FA) on survival and immune‐related gene expression were investigated in Litopenaeus vannamei challenged with Vibrio parahaemolyticus by immersion. Shrimp were fed with different dietary FA concentrations (1, 2, 4 and 6 g/kg feed) for 20 days (first bioassay) or 8 days (second bioassay, 2 g/kg feed of FA added every 2 days) and then challenged with V. parahaemolyticus. In a third bioassay, the expression of three immune‐related genes (translationally controlled tumour protein [TCTP], superoxide dismutase [SOD] and heat‐shock protein 70 [HSP70]) in haemocytes or hepatopancreas of experimental shrimp was measured by real‐time quantitative PCR at 0, 6, 12, 24, 48, 72 and 96 hr after FA (2 g/kg feed) administration. Fulvic acid increased survival at a concentration of 2 g/kg feed supplied every two days. Interestingly, TCTP gene expression was upregulated, whereas gene expression of SOD and HSP70 was downregulated. In conclusion, dietary fulvic acid improves survival in white shrimp challenged with V. parahaemolyticus and modulates the immune response. Therefore, FA merits further evaluation as prophylactic treatment in commercial shrimp farms.     

中国明对虾caspase2基因克隆及其在抗白斑综合征病毒中的表达分析

采用RACE技术克隆获得中国明对虾caspase2基因cDNA序列全长,并对该序列进行分析。结果显示,中国明对虾caspase2基因全长为1517 bp,开放阅读框长924 bp,5'非编码区长78 bp,3'非编码区长515 bp,命名为FcCasp2。推测该基因编码307个氨基酸,预测分子量为34.21 ku,理论等电点为7.62。同源性和系统进化分析发现,FcCasp2基因与凡纳滨对虾caspase2和斑节对虾caspase的相似性分别为88%和80%,与其他节肢动物caspase家族基因聚为一类。荧光定量RT-PCR结果显示,FcCasp2基因在肝胰腺中的相对表达量最高,在肌肉中表达量最低。WSSV感染后该基因在中国明对虾肌肉、肝胰腺和鳃丝中的表达量有不同的时空表达趋势,表明FcCasp2基因可能参与中国明对虾生物胁迫的应答反应。