三倍体虹鳟实时荧光定量PCR内参基因稳定性分析

采用实时荧光定量PCR(qRT-PCR)方法比较甘油醛-3-磷酸脱氢酶(gapdh)、核糖体大亚基蛋白基因(rplp2)、真核延伸因子基因(ef1-α)、β-肌动蛋白(β-actin)和18S核糖体核酸基因(18S rRNA)等5个候选内参基因在体质量约800 g三倍体虹鳟脑、眼、鳃、皮肤、心脏、肾脏(头肾、中肾、后肾...     

Stability comparison of three candidate internal reference genes in Chinese giant salamander (Andrias davidianus)

The Chinese giant salamander (Andrias davidianus) as food and medicinal product has been an important aquaculture object in China. Study of gene function in the Chinese giant salamander requires accurate normalization though the use of appropriate reference genes. In this study, the expression levels of three candidate reference genes including β‐actin, GAPDH and cytb of different tissues, different developmental stages and different challenges in Chinese giant salamander were evaluated by qPCR. The stabilities of these three reference genes were analysed by geNorm, NormFinder and BestKeeper software. The results showed that the expression of GAPDH was more stable than that of β‐actin and cytb in four tissues and at two developmental stages of Chinese giant salamander. Compared with GAPDH and cytb, β‐actin was the most stable in spleen of Chinese giant salamander treated with LPS or GSIV. Therefore, the result showed that GAPDH was the suitable reference gene in different tissues and at different developmental stages of Chinese giant salamander. The β‐actin could be used as a reference gene in spleen of Chinese giant salamander challenged with LPS and GSIV. This study provides convincing information for the GAPDH and β‐actin as suitable reference gene in Chinese giant salamander of different tissues, different developmental stages and different challenges respectively.     

大口黑鲈leptin A基因的组织表达及其对急性低氧胁迫的响应

瘦素(leptin)是一种重要的激素,参与调节动物的摄食、繁殖和能量消耗,其可以通过抑制食欲和促进能量代谢的方式维持机体能量稳态。大多数鱼类具有双重瘦素基因。在本研究中,利用PCR技术克隆了大口黑鲈leptin A基因的编码区,其开放阅读框(ORF)为486 bp,编码161个氨基酸蛋白。通过与其他物种进行同源性比对,发现鲈形目的 leptin基因的保守性较高,一致性高达91.46%,在大口黑鲈leptin A中几乎不存在基因特异性突变位点,而且大口黑鲈leptin A氨基酸序列与鳜同源性最高(92.59%),与花鲈(89.51%)、布氏鲳鲹(87.04%)、斜带石斑鱼(83.87%)的同源性次之。组织分布结果显示,leptin A基因在肝脏中的表达量最高,在心脏、头肾、脑、中肾、肠、脾脏、鳃、肌肉和性腺中的表达量均较低。此外,对大口黑鲈进行不同程度急性低氧胁迫[重度低氧(1.2±0.2)mg/L和中度低氧(3.5±0.2)mg/L],发现低氧胁迫初期时,严重低氧组和中度低氧组的leptin A的表达量都升高,显著高于对照组Leptin A的表达。这表明急性低氧能够诱导大口黑鲈leptin A在肝脏中的表达。综上所述,大口黑鲈leptin A基因与鳜leptin A基因的同源性最高,leptin A主要在大口黑鲈肝脏中显著表达,急性低氧胁迫能显著诱导leptin A的表达。     

太平洋鳕RAG1和IgM基因荧光定量PCR方法的建立及初步应用

为研究太平洋鳕发育早期特异免疫系统形成的机制,通过RAG1和IgM基因的转录水平衡量特异免疫系统的发育特点.根据GenBank中RAG1和IgM的序列信息,分别设计1对特异引物,从太平洋鳕头肾中扩增得到RAG1和IgM的基因片段.将所获基因片段分别插入到克隆载体pMD18-T中,从而构建太平洋鳕RAG1和IgM基因的质粒标准品.建立并优化太平洋鳕RAG1和IgM基因绝对荧光定量PCR方法.为进一步验证该方法的可靠性,分别利用绝对定量和相对定量检验目的基因在太平洋鳕早期发育过程不同组织内的表达差异.以优化后的绝对荧光定量PCR方法检测不同发育时期太平洋鳕RAG1和IgM的表达情况.结果显示,RAG1的回归方程为y=-3.266x+33.77,回归系数R2=0.996;IgM的回归方程为y=-3.119x +27.61,回归系数R2 =0.998.绝对定量和相对定量结果在基因转录趋势上显现出一致性,即RAG1基因在胸腺和头肾中表达,且在胸腺中的表达量显著高于头肾中的表达量,在肝脏和脾脏中无表达;IgM基因在胸腺、头肾、肝脏和脾脏中均有表达,其中脾脏中表达量最高,其次是头肾.RAG1基因在太平洋鳕发育早期的表达水平很低,到61日龄(days posthatching,dph)至95 dph表达量显著提高;IgM基因在早期表达水平同样很低,到33 dph至61 dph才有明显表达,在95 dph时表达量显著提高.研究表明,本实验方法可靠,特异性较强,可成功对目标基因转录水平进行检测.     

患腹水病牙鲆病原菌分离、鉴定及病原菌的特性

为确定引起河北北戴河地区养殖牙鲆患腹水病死亡的病原,本实验从患腹水病牙鲆体内分离到3株优势菌,对分离株进行生理生化鉴定和16S rRNA序列比对来确定分离株的生物学地位,进一步通过毒力基因(toxR、vhhA、vhhB)鉴定及组织病理学分析其病理特征,并通过人工回感实验分析分离株毒性。结果显示,通过生理生化实验及16S rRNA序列比对,确定此次从患病牙鲆中分离到的3株菌株均为哈维氏弧菌;经鉴定,3株分离株毒力基因(toxR、vhhA、vhhB)结果均为阳性,病理组织切片结果显示,该分离株对牙鲆多器官(肠、肾脏、脾脏和肝脏)均可造成不同程度的病理损伤,呈全身性感染;人工回感实验显示,菌株BDHYPFS-Y1G对牙鲆的半致死浓度为LD50=5.88×106 CFU/mL,低于自然状态毒性;药敏实验表明,3株分离株均对呋喃妥因高度敏感。本实验确认了此次牙鲆腹水病的病原菌,并初步研究了病原菌致病性以及药物敏感性,以期为牙鲆工厂化养殖疫病防控提供科学依据。     

Isolation and selection of suitable reference genes for real-time PCR analyses in the skeletal muscle of the fine flounder in response to nutritional status: assessment and normalization of gene expression of growth-related genes

In the present study, different reference genes were isolated, and their stability in the skeletal muscle of fine flounder subjected to different nutritional states was assessed using geNorm and NormFinder. The combinations between 18S and ActB; Fau and 18S; and Fau and Tubb were chosen as the most stable gene combinations in feeding, long-term fasting and refeeding, and short-term refeeding conditions, respectively. In all periods, ActB was identified as the single least stable gene. Subsequently, the expression of the myosin heavy chain (MYH) and the insulin-like growth factor-I receptor (IGF-IR) was assessed. A large variation in MYH and IGF-IR expression was found depending on the reference gene that was chosen for normalizing the expression of both genes. Using the most stable reference genes, mRNA levels of MYH decreased and IGF-IR increased during fasting, with both returning to basal levels during refeeding. However, the drop in mRNA levels for IGF-IR occurred during short-term refeeding, in contrast with the observed events in the expression of MYH, which occurred during long-term refeeding. The present study highlights the vast differences incurred when using unsuitable versus suitable reference genes for normalizing gene expression, pointing out that normalization without proper validation could result in a bias of gene expression.     

大口黑鲈鲁氏耶尔森氏菌的分离鉴定、主要毒力基因及致病性研究

为探明2021年3月福建某水库养殖大口黑鲈疾病暴发的原因,实验从患病大口黑鲈肝脏、肾脏、脾脏中分离优势菌,通过人工感染实验确定病原菌,并综合16S rDNA基因序列分析、生理生化和质谱特征等技术对该病原菌进行种属鉴定,同时,进行毒力基因检测、药物敏感性实验以及组织病理学观察。结果显示,从病鱼脾脏中分离获得1株优势菌并鉴定为鲁氏耶尔森氏菌;该菌株对大口黑鲈的半致死剂量为3.8×10⁵ CFU/尾;该菌株携带yrP1、yhl A、yhl B等毒力基因,对恩诺沙星、盐酸多西环素、氟甲喹、硫酸新霉素、氟苯尼考等5种药物相对敏感。组织病理学观察发现,鲁氏耶尔森氏菌的感染造成大口黑鲈肝脏、肾脏、脾脏不同程度的损伤,表现为明显的变性、坏死及炎症细胞的浸润等。本研究首次报道了鲁氏耶尔森氏菌对养殖大口黑鲈的致病性,可为养殖大口黑鲈鲁氏耶尔森氏菌病的诊断和药物防治提供参考依据。     

Gene structure and expression analyses of multiple vitellogenesis-inhibiting hormones in the whiteleg shrimp <Emphasis Type="Italic">Litopenaeus vannamei</Emphasis>

In the whiteleg shrimp Litopenaeus vannamei, the existence of six sinus gland peptides (SGPs) possessing vitellogenesis-inhibiting hormone (VIH) function has been previously demonstrated. Genomic and cDNA sequence information for two of these is already known. Here, we cloned cDNAs for the remaining three, i.e., those encoding Liv-SGP-A, -B, and -F. The deduced amino acid sequences were comprised of a signal peptide, a CHH precursor-related peptide, a mature peptide, and a C-terminal amidation signal. The genomic DNA for Liv-SGP-A, -B, -C, -F, and -G, was found to be organized as three exons and two introns. The insertion of the two introns were conserved in all VIH genes, with intron I being inserted six residues following the signal peptide, and intron II after the 40th residue of the mature peptide. We also quantified the relative simultaneous expression of Liv-SGP-A, -B, -C, -F, and -G in relation to molting and unilateral eyestalk ablation. The expression levels of each gene in the eyestalks of adults did not change significantly during molting or following unilateral eyestalk ablation. However, unilateral eyestalk ablation significantly decreased expression levels at 10 and 20 days for Liv-SGP-A and -C, and at 20 days for Liv-SGP-G in the eyestalks of subadults. This study has therefore clarified the genomic structure of the five major subtype I VIHs in L. vannamei, and elucidated their expression levels in the eyestalks in relation to molting and unilateral eyestalk ablation.     

Multilocus sequence analysis revealed a high genotypic diversity of Aeromonas hydrophila infecting fish in Uganda

A multilocus sequence analysis (MLSA) was carried out to delineate Aeromonas hydrophila from fish in Uganda. Five housekeeping genes including recA, gyrB, metG, gltA and pps; and the 16S rRNA gene were amplified and sequenced from a total of nine A. hydrophila isolates. The obtained sequences were edited, and consensus sequences generated for each gene locus. The housekeeping gene sequences were concatenated and phylogenetic analysis performed in MEGA version 7.0.2. Pairwise distances ranged from 0.000 to 0.118, highest within the gltA gene locus and lowest within the 16S rRNA gene. The average evolutionary diversity within isolates from the same source ranged between 0.002 and 0.037, and it was 0.033 between the different sources. Similar tree topologies were obtained from the different gene loci with recA, metG and gyrB being more consistent in discriminating isolates according to sources while the 16S rRNA gene had the lowest resolution. The concatenated tree had the highest discriminatory power. This study revealed that A. hydrophila strains infecting fish in Uganda are of diverse genotypes suggesting different sources of infection in a given outbreak. Efforts to minimize spread of the bacteria across sources should be emphasized to control infections of mixed genotypes.     

青鲫sIgZ重链基因的克隆及对嗜水气单胞菌的免疫响应

免疫球蛋白是鱼类适应性免疫中主要的效应因子之一, 研究鱼类免疫球蛋白对鱼类疾病的免疫防控尤为重要。本研究利用 PCR 与 RACE 技术克隆获得青鲫(Carassius auratus indigentiaus)分泌型免疫球蛋白 Z (secretory immunoglobulin Z, sIgZ)重链基因 cDNA 全长序列 (基因序列登录号: MZ274344.1), 青鲫 sIgZ 重链基因 cDNA 序列全长 2083 bp, 其开放阅读框长 1668 bp, 编码一条由 556 个氨基酸组成的肽链, 相对分子质量为 61.59 kD, 理论等电点为 7.03。青鲫 sIgZ 重链基因结构由 1 个可变区(VH), 4 个恒定区(CH1-CH4)以及 1 个分泌型的尾部(secretory tail, Sec tail)构成。在 GenBank 中进行同源序列检索, 青鲫 sIgZ 氨基酸序列与其他鱼类 IgZ 的相似性依次为团头鲂 (Megalobrama amblycephala) (63.17%)、草鱼(Ctenopharyngodon idella) (60.82%)、鲤(Cyprinus carpio carpio) (52.98%)、虹鳟(Oncorhynchus mykiss)(37.06%)和金头鲷(Sparus aurata)(35.01%)。利用 MEGA 5.1 软件进行多重序列比对分析, 采用邻接法构建遗传系统进化树, 发现青鲫 sIgZ 基因与同属鲤科(Cyprinidae)鱼类的 IgZ 聚为一支, 青鲫 sIgZ 与团头鲂的 sIgZ 亲源关系最近。通过 qPCR 检测青鲫不同组织中 sIgZ 基因的表达, 发现青鲫头肾中 sIgZ 基因的表达量最高, 中肾、脾和肝脏次之。利用嗜水气单胞菌(Aeromonas hydrophila)对青鲫进行浸泡感染, sIgZ 在头肾、脾脏、中肾、肝、肠、鳃和皮肤中的表达水平在感染的 28 d 内先升高后降低, sIgZ 的相对表达量在黏膜免疫组织(肠和皮肤)中达到峰值的时间要早于系统免疫组织(头肾和脾脏), 肠和皮肤中 sIgZ 在峰值时的相对表达量显著高于头肾中 sIgZ 在峰值时的相对表达量, 实验结果显示, 青鲫 sIgZ 在肠和皮肤黏膜免疫组织中可能发挥重要的免疫功能。     

黑棘鲷内脏结节病病原的分离、鉴定及致病性研究

2017年4月,浙江台州某海水养殖公司跑道式养殖池黑棘鲷大量发病,病鱼活力下降、食欲减退、体表溃疡,剖检可见肝脏、脾脏、肾脏肿大且有不同程度的白色结节,同时伴随大量腹水。用胰蛋白胨大豆琼脂(TSA)从典型结节病濒死黑棘鲷器官分离到革兰阴性短杆菌。分离病原菌AS15对健康黑棘鲷的致病力结果显示,AS15腹腔注射可使健康黑棘鲷发病死亡,死亡黑棘鲷可出现自然发病症状,在(24±1)°C的条件下,(25±2) g黑棘鲷的半数致死浓度(LD_(50))为6.5×10~4 CFU/尾。经API 20E鉴定,分离菌株AS15生理生化特性与类杀鱼爱德华氏菌LADL05-105和迟缓爱德华氏菌典型菌ATCC15947相似度为86.2%,与杀鱼爱德华氏菌ET~T883的相似度为82.8%;AS15的16S rDNA与杀鱼爱德华氏菌ET~T883同源性达99%,gyrB与类杀鱼爱德华氏菌LADL05-105和杀鱼爱德华氏菌ETT883同源性分别为100%和98%;16S rDNA进化树显示,AS15与ETT883、LADL05-105聚为一簇,gyrB进化树与LADL05-105、NCIM2056聚为一簇;采用4种爱德华氏菌属种特异性引物对AS15进行PCR分析,结果显示,AS15可扩增出类杀鱼爱德华氏菌的种特异性片段,不能扩增出迟缓爱德华氏菌、鲇鱼爱德华氏菌、杀鱼爱德华氏菌的种特异性片段,表明AS15属类杀鱼爱德华氏菌成员。分析了AS15的菌毛基因、sodB等毒力基因,发现AS15具有fimA、fimB、fimC、fimD等4种菌毛基因和sodB、citC、esrB、mukF、katB等毒力基因。本实验首次从黑棘鲷上检出致病性类杀鱼爱德华氏菌,该菌对黑棘鲷的发病机制和毒力机理还需进一步研究。     

Disease of rainbow trout caused by Pseudomonas luteola

Bacteria isolated from rainbow trout, Oncorhynchus mykiss, kept in a farm, in Turkey. During the outbreak, 40% of the rainbow trout (10-40 g) died. Typical clinical signs were exophthalmia, dark pigmentation, hemorrhage at the base of the pectoral, pelvic, anal fins and around the vent. Internal signs were enlarged spleen, pale liver and intestine filled with yellowish fluid. Liver, kidney and spleen of diseased fish, were aseptically streaked on Tryptic Soy Agar. After incubation, pure cultured colonies were observed and biochemically characterized with API 20 NE and other biochemical tests. Cultured bacterial 16 S rDNA gene was sequenced. Based on biochemical characteristics and sequence of 16 S rRNA, the causative bacteria were identified as Pseudomonas luteola. This study reports the first P. luteola infection in fish.     

Aeromonas hydrophila and Aeromonas veronii cause motile Aeromonas septicaemia in the cultured Chinese sucker,Myxocyprinus asiaticus

In August 2017, a serious disease causing high mortality occurred in a Myxocyprinus asiaticus aquaculture farm. According to necropsy findings, bacteriology experiments and phylogenetic analysis based on 16S rRNA, cpn60, gyrB and rpoB genes and concatenated alignment sequences (cpn60, gyrB and rpoB genes), two isolates, that is, BBAh1 and BBAv1, were identified as Aeromonas hydrophila and Aeromonas veronii respectively. Artificial infection experiments were carried out, showing that the BBAh1 and BBAv1 strains can cause similar symptoms and have LD50 values of 1.93×10⁵ and 8.77×10⁵ cfu/g respectively. In addition, some virulent genes coding for AerA, Alt, Ast, AscV, AexT, AspA, HlyA, OmpA, Lip and FlaA were detected in the two strains. Furthermore, BBAh1 and BBAv1 showed the same sensitivities to 28 antibiotics, some of which, such as cefotaxime, aztreonam, ceftriaxone and tetracycline, may be used to control the disease. However, the strains were also resistant to many antibiotics. These results provide a scientific reference for the diagnosis, prevention and treatment of motile Aeromonas septicaemia caused by A. hydrophila or A. veronii in cultured Chinese sucker.     

拟态弧菌的绿色荧光蛋白标记及其在感染草鱼体内的动态分布

为了示踪研究拟态弧菌感染草鱼的动态过程,将增强型绿色荧光蛋白编码基因EGFP克隆至质粒pBAD24,并转化到拟态弧菌04-14菌株构建荧光标记重组菌.重组菌经阿拉伯糖诱导后,能高效表达EGFP蛋白;荧光显微镜观察和流式细胞仪检测均发现重组菌能够发出明显的绿色荧光信号,且传至30代后质粒稳定率仍为100％;生物学特性检测结果显示,与野生株相比,重组菌的形态、生长特性和细胞黏附性均未发生明显改变.用标记重组菌浸泡感染草鱼,定点采集鳃、肠道、肌肉、头肾、脾脏和肝脏,借助荧光信号检测4d内细菌在不同组织脏器中的动态分布.结果发现感染4h后即可在肠道和鳃中检测到绿色荧光信号,标记菌检出量分别为3.60×108和2.36×106 CFU/g,直至10 h,其含量无明显变化,12 h后含菌量逐渐下降,但持续存在直至鱼死亡.标记菌在肌肉、头肾、脾脏和肝脏中呈现相似的动力学,感染24 h后才检测到荧光信号,24～ 85 h时间段含菌量呈现先增加后下降的变化,48 h达到峰值,检出量分别为9.58×104(肌肉)、8.75×104(头肾)、1.50×104(脾脏)和4.50×104 CFU/g(肝脏),但均低于肠道中的检出量,结果表明肠道是拟态弧菌黏附定植与繁殖的主要靶器官.     

Oral bovine serum albumin immune‐stimulating complexes improve the immune responses and resistance of large yellow croaker (Pseudosciaena crocea,Richardson, 1846) against Vibrio alginolyticus

This study aimed to investigate effects of bovine serum albumin immune‐stimulating complexes (BSA ISCOMs) on immune‐related genes expression, serum nonspecific immunity and disease resistance of large yellow croaker (Pseudosciaena crocea). Fish were fed diets containing 3.5 ml of BSA ISCOMs per kg feed (experimental group) or 3.5 ml of phosphate‐buffered saline per kg feed (control group) for 1 week. The liver, spleen, head‐kidney tissues were sampled for determining gene expression of myxovirus‐resistant protein (Mx), major histocompatibility complex class II alpha chain (MHC II α), tumour necrosis factor‐alpha (TNF‐α) and interleukin‐10 (IL‐10) 30 and 90 days after feeding. Also, blood samples were collected for determining activities of serum superoxide dismutase (SOD), interferon alpha (IFN‐α), TNF‐α and alkaline phosphatase (ALP). TNF‐α and MHCⅡα gene expression in the liver, spleen, head‐kidney, as well as IFN‐α, TNF‐α and ALP activities in the serum, of experimental fish were significantly higher 30 days after feeding; while only TNF‐α and MHC II gene expression in the head‐kidney remained upregulated 90 days after feeding. The cumulative mortality of the experimental fish was significantly lower than control. This study indicated that BSA ISCOMs improved the immune response and induced protective immunity in large yellow croaker.     

尼罗罗非鱼雌雄鱼肌肉组织差异表达基因的筛选

应用引物退火控制技术(ACP)筛选尼罗罗非鱼雌雄鱼肌肉组织差异表达基因,寻找与雌雄鱼肌肉生长发育相关的候选基因。本实验从同等条件下养殖的尼罗罗非鱼群体中随机选取雌、雄鱼各5尾组成RNA池,采用引物退火控制技术,分析了两组个体肌肉组织差异表达基因。利用20对随机引物差异显示扩增,共获得8条ESTs,其中5个已知的ESTs分别为转录变体3(LOC100691543)、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、肌型肌酸激酶M2-CK和转录因子Sox4,其余3个为未知的ESTs。实时定量PCR分析各差异表达基因在尼罗罗非鱼雌雄肌肉组织中的表达发现,8个差异表达基因中转录变体3与ACP6-Y在尼罗罗非鱼雄鱼肌肉组织中的表达均极显著高于雌鱼(P0.01),ACP3-X、60S核糖体蛋白(RL3)、小白蛋白β样蛋白、ACP15-X、肌型肌酸激酶M2-CK与转录因子Sox4在尼罗罗非鱼雌鱼肌肉组织中的表达均极显著高于雄鱼(P0.01)。结果表明,应用引物退火控制技术筛选获得了8个可能参与了雌雄鱼肌肉生长发育调控的ESTs,为进一步筛选雌雄鱼肌肉生长发育相关候选基因奠定了基础。     

CD3ε is involved in host immune response against challenges in Nile tilapia (Orechromis niloticus)

CD3 is an important membrane molecule of mature T cells. TCR‐CD3 complex plays important roles in the activation and signal transduction of T cells. In this study, the CD3ε (OnCD3ε) gene was cloned from Nile tilapia (Oreochromis niloticus), and its tissue distribution was detected by qPCR. The expression changes in CD3ε were analysed after three challenges (S. agalactiae, A. hydrophila and poly (I:C)) in vivo and in vitro. The open reading frame (ORF) of OnCD3ε contains 531 bp, encoding 176 amino acids, which found that OnCD3ε consists of conserved amino acid residues and motifs (cysteine residues, CXXC motif, immunoreceptor tyrosine‐based activation motifs and PxPDY). In addition, tissue distribution displayed that OnCD3ε has the highest expression in thymus and significant expression level in mucosal immune‐related tissues (intestine and gills). In vivo, all three challenges could significantly up‐regulate the expression of CD3ε in the head kidney and spleen. For further in vitro assays, after poly (I:C) stimulation, OnCD3ε appeared significant up‐regulation at 3 hr post stimulation, and two pathogenic bacteria also induce significant up‐regulation of OnCD3ε in spleen leucocytes (12 hr). These results suggested that OnCD3ε was likely to get involved in host immune response against pathogenic challenges.