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三角帆蚌肝脏瘟病感染期抑制性消减cDNA文库的构建与分析 总被引：1，自引：0，他引：1
采用抑制性消减杂交技术构建了三角帆蚌肝脏瘟病感染期抑制性消减cDNA文库。经检验,差异表达基因均被富集了 210倍左右,证明构建的 cDNA 消减文库具有很强的消减效率。PCR 鉴定发现,在随机挑取的阳性克隆中,95% 的克隆均含有 0.2～1.0 kb 的插入片段,这些片段可能是三角帆蚌瘟病病毒感染后差异表达基因的cDNA片段。测序共获得 214 个有效 cDNA 序列,分别属于8 大类,共 98 个基因。其中细胞分裂基因 2个、细胞结构与运动基因 9 个、代谢基因 10 个、信号传导基因 7 个、细胞防疫基因 10 个、基因与蛋白表达基因 20 个、未知功能蛋白基因 26 个,GenBank中找不到任何同源序列的基因 14 个。结果说明,构建的差异表达cDNA文库,可较好地反映三角帆蚌瘟病病毒对三角帆蚌影响的基因信息。 相似文献
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为了发掘更多三角帆蚌具有EF-hand结构域的功能基因及其蛋白质,本研究运用RACE-PCR技术,克隆得到了三角帆蚌包含EF-hand结构域钙结合蛋白1基因(EF-hand calcium-binding domain-containing protein 1,EFCB1)的cDNA全长并进行了生物信息学分析;通过real-time Q-PCR技术,分析了EFCB1基因在三角帆蚌10个组织,以及内脏团、外套膜插核后不同时间点的时空表达特点。结果表明三角帆蚌EFCB1基因cDNA序列全长981 bp,ORF为531 bp,编码176个氨基酸残基,5'-UTR 239 bp和3'-UTR 211 bp。EFCB1分子式为C877H1348N238O270S10,分子量约19.9 ku,等电点为4.70,不稳定系数为62.65,属亲水蛋白。其序列无信号肽序列,存在1个跨膜区域和2个EF-hand结构域,EF-hand模块分别为DLNDDKLISPEE(98-109)和DTNGDDKLDGEE(129-140)。荧光定量结果显示三角帆蚌EFCB1基因在各组织中均有表达,其中在肠和鳃中表达量最高(P<0.05),外套膜中表达量显著高于内脏团(P<0.05)。EFCB1基因在插核后不同时期的外套膜和内脏团育珠部位组织中表达具有显著差异(P<0.05),在外套膜中的表达量均显著高于内脏团(P<0.05),在插核后第20 天时表达量显著高于各时期(P<0.05)。研究表明,EFCB1在三角帆蚌Ca2+的吸收过程中发挥调节作用,在珍珠囊形成过程中以及珍珠形成初期具有重要功能。 相似文献
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三角帆蚌是我国特有的淡水育珠蚌,在养殖业中占有重要地位,在实际养殖过程中,雄性个体有着明显的产珠优势,因此对性别决定的相关研究至关重要。研究发现Sox9基因在许多物种中起到性别决定的作用,kinase X基因是蛋白激酶(PKA)合成中至关重要的基因,而Sox9基因很可能受到PKA激酶的调控。实验通过RACE法克隆kinase X基因的全长cDNA序列,使用荧光定量PCR的方法检测该基因在2龄雌雄三角帆蚌各组织中的相对表达量,利用RNA干扰技术研究干扰链对kinase X基因及下游基因Sox9基因表达的影响。结果显示,kinase X基因全长1 652 bp,编码430个氨基酸;荧光定量PCR结果显示,kinase X基因在雄性性腺中表达量最高,雌雄间差异极显著;RNA干扰结果显示,合成的干扰链均对kinase X基因有着一定的干扰效率,其中干扰链1的干扰率最高,在雌性中的干扰率为83.1%,雄性中为81.9%,同时干扰链1干扰后Sox9基因的表达量在雌性中下降了90.3%,雄性中下降了56.6%,推测这两个基因可能参与性别决定过程。本实验为三角帆蚌性别决定和雄性单性化育种研究提供理论基... 相似文献
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三角帆蚌HcCUBDC基因cDNA的全长克隆与表达分析 总被引：1，自引：1，他引：0
利用cDNA末端快速扩增（Rapid Amplification of cDNA Ends,RACE）方法,获得了三角帆蚌HcCUBDC基因的全长cDNA序列,共5158bp,开放阅读框（ORF）1920bp,编码639个氨基酸,5′端非编码区576bp,3′端非编码区2662bp,GeneBank登陆号为KP067952。生物信息学分析表明,三角帆蚌HcCUBDC蛋白含有一个由19个氨基酸组成的信号肽和4个CUB结构域,但未能比对上已知的蛋白。经荧光定量PCR检测,HcCUBDC基因在紫色和白色蚌前端缘膜、后端缘膜、中央膜、鳃、斧足、肝胰腺、肠和肾8个组织中均有表达,并且都是肝胰腺表达量最低。在紫色蚌中,后端缘膜表达量极显著高于前端缘膜;在白色蚌中,前端和后端缘膜之间表达差异不显著。HcCUBDC基因在紫色蚌后端缘膜中的表达量极显著高于白色蚌,在紫色和白色蚌前端缘膜中的表达量差异不显著。外套膜原位杂交结果显示,HcCUBDC基因主要在缘膜外上皮细胞中表达。研究表明,HcCUBDC基因在三角帆蚌内壳色形成中发挥作用,可为进一步深入研究该基因在珍珠颜色形成过程中的功能及其调控机理提供基础资料。 相似文献
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为了解淡水贝类是否存在组织蛋白酶L的亚型及其亚型的免疫相关作用,本实验利用已构建的池蝶蚌血细胞全长c DNA文库,筛选获得与之同源的EST序列,结合RACE技术进一步克隆了池蝶蚌一个新的组织蛋白酶L基因的c DNA全长,命名为Hs Cts L1-like基因(Gen Bank登录号为KF015273)。该序列全长为1280 bp,5′-非翻译区(5′UTR)为31 bp,3′-非翻译区(3′UTR)为256 bp,开放阅读框区(ORF)为993 bp,编码330个氨基酸,预测蛋白相对分子量为36.86 ku,理论等电点为6.23。序列分析结果显示,Hs Cts L1-like与其他软体动物相对应序列具有共同结构特征,包含信号肽、前肽抑制域和成熟肽三部分,在其他物种中已鉴定的Cts L签名序列标签(ERF/WNIN、GNFD、GCXGG和QCHN等)在Hs Cts L1-like中均可找到。其氨基酸序列同缢蛏Cts L1(AGL33704.1)同源性最高,达67%;与报道的三角帆蚌Cts L(ADV03094)和池蝶蚌中另一个Cts L(AEX88474)仅均为52.91%;系统进化分析表明,Hs Cts L1-like与缢蛏、长牡蛎和合浦珠母贝的Cts L1聚为一分支,推测Hs Cts L1-like属于Cts L家族中的亚型1。实时荧光定量PCR(q RT-PCR)检测显示,Hs Cts L1-like m RNA在肝脏中表达量最高,其次是卵巢和精巢。注射鳗弧菌后,血细胞和肝脏Hs Cts L1-like m RNA转录水平显著升高,暗示其是一个免疫有关的基因,参与了池蝶蚌的先天免疫应答反应。 相似文献
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转录组测序研究三角帆蚌珍珠颜色相关基因 总被引：3，自引：2，他引：1
为筛选影响三角帆蚌珍珠颜色的候选基因,采用转录组测序平台Illumina Hi Seq TM2500对蚌壳珍珠层颜色为紫色和白色的三角帆蚌中央膜组织进行高通量测序。所得序列经质控、组装后比对到NR、Swiss-Prot、COG、KOG、KEGG、GO和Pfam数据库中注释,并进行差异基因聚类分析。结果显示,获得干净数据共72 800 581 bp,拼接获得了89 529个Unigene,差异表达基因2644个,其中上调表达基因1323个,下调表达基因1321个。根据GO功能分类可分为分子功能、细胞组分、生物过程等3大类50分支;根据KEGG代谢通路分析可以分为187类。对差异表达基因分析发现,部分基因与色素合成(眼黄素、黑色素、喋啶色素)及金属离子转运相关,还有6个细胞色素P450,3个细胞色素b561和1个细胞色素b560。研究表明,这些基因可能在珍珠颜色形成中发挥作用。 相似文献
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Reyna de Jesús Romero‐Geraldo Norma Yolanda Hernández‐Saavedra 《Aquaculture Research》2014,45(9):1512-1522
Harmful algal blooms (HABs) are natural phenomena with different effects on the aquatic environment that affect both human economy and health. Several genomic studies have been done to characterize the effects of contaminants on Crassostrea gigas. However, oysters’ molecular response to HABs exposure needs to be studied more. In this study, we challenged C. gigas with Prorocentrum lima, a diarrhoetic toxin producer, under controlled experimental conditions considering dinoflagellate density and exposure time (acute and sub‐chronic) as variables. The expression profile of six stress response genes was analysed by semiquantitative RT‐PCR: Glutamine synthetase (GS), Glutathione S‐transferase (GST), heat shock protein 70 (HSP70), and heat shock protein 90 (HSP90), CuZn superoxide dismutase (SOD) and melanogenic peroxidase (POX). The results revealed that these gene expressions depend on exposure time and cell concentration. In general, an increased expression was observed in all tested cell densities as immediate response to exposure (0–3 h); whereas lower expression levels than control were observed after 6 h. Treatment with 3,000 cells mL‐1 promoted higher expression than control on most genes after 336 h of exposure. This is the first report providing information at molecular level on C. gigas response to dinoflagellate blooms. 相似文献
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To understand the detoxification and bioactivation mechanisms for organic contaminants, it is essential to identify the cytochrome P450 (CYP) complement. Therefore, this study aimed to clone a partial cDNA sequence of the novel CYP1D1 gene from the fish Oreochromis niloticus and examine whether intraperitoneal injection of benzo-a-pyrene (BaP), a potent AHR agonist, is capable of inducing CYP1D1 mRNA expression in different tilapia fish tissues. The cloned nucleotide sequence consisted of 713 bp representing a portion of the tilapia CYP1D1 cDNA ORF, encoding 237 amino acids. Amino acid sequence comparison of O. niloticus CYP1D1 with the sequences of CYP1D1 from other species showed that this gene shared the highest identity of 81% with Fundulus heteroclitus CYP1D1. Furthermore, analysis of the percent identities shared by the deduced amino acid sequence of O. niloticus CYP1D1 with the sequences of CYP1 from other species revealed that the highest identities were shared with fish CYP1As. Real-time PCR results revealed that the highest expression level of CYP1D1 mRNA was found in muscles, followed by gills, liver, and intestine, while there was no detectable expression recorded in bile acid. These results indicate that tilapia CYP1D1 plays an important role in the metabolism of xenobiotics, expanding our knowledge regarding the diversity of CYP1 genes in this important model fish species.
相似文献13.
Wei‐Wei Li Ya‐Nan Gong Xing‐Kun Jin Lin He Hui Jiang Fei Ren Qun Wang 《Aquaculture Research》2010,41(11):e828-e837
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根据已知杂色沙蚕(Nereis diversicolor)金属结合蛋白Ⅱ(MPⅡ)部分基因序列设计引物,应用RACE技术从双齿围沙蚕(Perinereis aibuhitensis)中克隆得到MPⅡcDNA序列全长为904 bp,其中,开放阅读框为357 bp,编码119个氨基酸。Blast比对结果显示,由双齿围沙蚕MPⅡcDNA序列推导的氨基酸序列与多毛类Periserrula leucophryna蚯蚓血红蛋白(Hr)、杂色沙蚕(Nereis diversicolor)蚯蚓肌血红蛋白(MHr)及星虫类Themiste zostericola Hr氨基酸序列的同源性分别为81.51%、77.12%和61.02%,由此推断该cDNA序列可能属于血红蛋白家族。应用Real-time PCR方法,分别研究了沙蚕暴露于Cd2+质量浓度为40 mg/L的环境中12、24、48和72 h后MPⅡ基因表达水平的变化,以及3种质量浓度5、10和20 mg/L Cd2+暴露15 d后MPⅡ基因的表达水平。实验结果表明:(1)沙蚕暴露于40 mg/LCd2+72 h后,MPⅡmRNA表达量显著增加,为空白对照组的12.6倍,表... 相似文献
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Javier Pérez‐Robles Ana Denisse Re Ivone Giffard‐Mena Fernando Díaz 《Aquaculture Research》2012,43(9):1372-1383
A physiological study was performed to explore the effects in Sphoeroides annulatus (Jenyns 1842) juveniles of exposure to different salinities 10, 17, 23, 29, 35 and 41‰ on oxygen consumption rate (OCR), ammonium excretion rate (AER), the oxygen‐nitrogen atomic ratio (O:N), osmoregulation and Na+/K+‐ATPase expression. The OCR values ranged from 27.9 to 30.9 mg O2 h?1 kg?1, displaying a Type 1 response pattern Kinne (1977). AER ranged from 0.60 to 0.69 mg h?1 kg?1, and O:N values were from 53.12 to 59.26, indicating that the puffers use proteins and lipids as an energy‐substrate. Osmoregulation in S. annulatus was hyposmotic in salinities of 23, 29, 35 and 41‰ and hyperosmotic in a salinity of 10‰. The isosmotic point of the bullseye puffer was 356 mmol kg?1 (10.5‰). The enzyme expression analysis indicates that the fish acclimated to 41‰ had the highest Na+/K+‐ATPase expression level, whereas, the lowest expression level was found close to the isosmotic point. Na+/K+‐ATPase expression in the gills was found to have a U‐shaped relationship with environmental salinity. We conclude that the bullseye puffer is strongly euryhaline and can be cultivated in a wide range of environments that has important economic implications. 相似文献
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Adrian Di Natale Maureen Jarau Spencer K. Russell Ryan A. Horricks Brandon N. Lillie Brian Dixon John S. Lumsden 《Journal of fish diseases》2019,42(7):975-984
A previous proteomic study examining the plasma acute‐phase response of rainbow trout to sterile inflammation highlighted an unidentified 9.5‐kDa spot using 2D‐PAGE, which was dramatically increased. The 15 amino acid sequence obtained from this protein spot allowed rapid amplification of cDNA ends PCR to generate a 443‐bp nucleotide sequence that was 98.6% similar to type‐4 ice‐structuring protein LS‐12 from Atlantic salmon Salmo salar Linnaeus. Quantitative reverse translation PCR and an ELISA were used to measure gene expression and plasma concentrations of LS‐12 following experimental intraperitoneal injection of rainbow trout with either 106 or 108 colony‐forming units (CFU) of Flavobacterium psychrophilum. There was no significant change in the plasma concentration of LS‐12 up to 15 days post‐infection in any group. Hepatic LS‐12 gene expression was significantly reduced at 3 and 6 days (p < 0.001) post‐infection in fish injected with 108 CFU of F. psychrophilum relative to control fish, while branchial or head kidney expression was unchanged. Infected fish had significantly increased hepatic gene expression of serum amyloid A, confirming an acute‐phase response. Under the conditions used, LS‐12 is not a positive acute‐phase protein in rainbow trout. 相似文献
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C型凝集素是一种依赖于Ca~(2+)而发挥功能的糖蛋白,在一线的固有免疫防御过程中发挥着重要作用。围绕对虾C型凝集素开展深入研究,不仅可以丰富无脊椎动物固有免疫学内容,还有望将其开发为具有免疫增强效果的活性饵料,应用于对虾的健康养殖。本实验根据实验室前期转录组信息提示克隆获得了凡纳滨对虾一种新的C型凝集素基因(LvLc1,Gen Bank注册号:KY937940)。生物信息学分析显示LvLc1基因的开放阅读框全长891 bp,编码296个氨基酸,该基因编码的蛋白质含有一个保守的糖识别结构域(carbohydrate recognition domain,CRD),该结构域中具有潜在的半乳糖结合位点(QPD motif),进化发生分析显示LvLc1与来自节肢动物的甘露糖结合凝集素家族成员聚类在一起。对LvLc1基因的CRD结构域进行了原核重组表达与蛋白活性分析研究,结果显示:重组目的蛋白(rLvLc1)在Ca~(2+)存在的条件下,对多种病原菌(G~+、G~–和真菌)具有凝集作用,其凝集活性可被半乳糖、甘露糖、脂多糖等多种病原相关分子模式所抑制。研究表明,LvLc1作为C-型凝集素家族一个新成员,可能通过重要的模式识别受体作用,参与机体应答病原微生物侵染的防御过程。 相似文献
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Qinglin Yang Lijun Luo Yanhong Jiang Jiaojiao Zhang Xiaoqi Tang Mi Jiang Zhengli Wu Yanhong Li 《Aquaculture Research》2020,51(10):4315-4326
Amylase‐producing bacteria could improve water quality contaminated by waste from feed residue and fish metabolism, thereby increasing the efficiency of aquaculture systems. The objective of this research was to screen and optimize fermentation conditions of a high amylase‐producing strain. Four amylase‐producing bacterial strains (named S458‐1, G05, H38 and B09) were isolated from a grass carp pond, and the strain S458‐1 showed the highest amylase‐producing ability, with 19.58 ± 0.38 mm hydrolysis circle diameter. The strain S458‐1 was identified as Bacillus cereus based on morphological identification, biochemical identification and 16S rDNA sequence analysis. The optimal culture medium formula included (in g/L) Ca2+ 0.8, Mg2+ 0.2, Mn2+ 0.4, Fe2+ 0.6, Al3+ 0.2, 1% soluble starch and 1% peptone. The optimal fermentation conditions were determined as initial pH 9, culture temperature 37°C, fermentation time of 60 hr and 2% inoculum. Under the optimal formula and condition, its enzyme activity increased from 32 U/ml to 173.01 U/ml, a 5.41‐fold increase. Surprisingly, our research found that the strain S458‐1 also had phosphorus degradation capabilities. Its phosphorus‐dissolving ability was both time‐ and concentration‐dependent. Thus, this study will make a contribution to the bacterial amylase based on the fermentation process and provide a theoretical basis for further research of aquatic probiotics. 相似文献