上海海洋大学学报  2019, Vol. 28 Issue (5): 772-781    PDF    
两种弧菌感染大黄鱼免疫相关基因的SNP位点分析
孙明洁1, 张娜1, 徐善良2, 鲍宝龙1, 龚小玲1     
1. 上海海洋大学 省部共建水产种质资源发掘与利用教育部重点实验室, 上海 201306;
2. 宁波大学 应用海洋生物技术教育部重点实验室, 浙江 宁波 315211
摘要:为了探究大黄鱼(Larimichthys crocea)免疫相关基因的SNP与弧菌抗性关系,分别利用鳗弧菌(Vibrio anguillarum)和副溶血弧菌(Vibrio parahaemolyticus)人工感染大黄鱼。对感染前后抗感群体的转录组进行高通量测序、筛选并分析其抗病差异:(1)筛选氨基酸的非同义突变SNP位点在抗鳗弧菌组有17个,而抗副溶血弧菌组的有28个;(2)一代测序验证结果发现,染色体NW_011323507.1上白细胞介素6受体基因(IL-6R)第91 196位碱基G突变为C,导致缬氨酸突变为亮氨酸,该位点G/C在抗鳗弧菌组、对照组样本之间突变基因型CC频率分别为12.5%和0,呈显著性差异(P < 0.05);(3)补体C1q/肿瘤坏死因子相关蛋白9(CTRP9)基因在染色体NW_011323975.1上的35 665位碱基突变(A-G),在副溶血弧菌抗感易感群体中突变位点基因型GG频率分别为37.5%和0,呈极显著性差异(P < 0.01)。结果表明,IL-6R-91196-G/C位点突变与大黄鱼抗鳗弧菌有关联,CTRP9-35665-A/G位点突变与大黄鱼抗副溶血弧菌有关联,这为大黄鱼抗弧菌群体的选育提供了理论依据。
关键词鳗弧菌    副溶血弧菌    大黄鱼    转录组    免疫基因    SNP    

大黄鱼(Larimichthys crocea)属硬骨鱼纲鲈形目(Perciformes)石首鱼科(Sciaenidae)黄鱼属(Larimichthys),是我国近海主要养殖经济鱼类[1-2]。近些年,养殖环境恶化、养殖规模扩大、养殖密度过高等原因导致其病害频发,尤其是鳗弧菌(Vibrio anguillarum)和副溶血弧菌(Vibrio parahaemolyticus)感染引起的弧菌病,对大黄鱼生长危害极大。弧菌病发病时间短、死亡快、传染性极强,严重威胁着渔业经济发展和人们的健康[3]。因此,培育大黄鱼抗病品系是预防弧菌病的重要途径之一。

单核苷酸多态性(single nucleotide polymorphism,SNP)是一种理想的遗传标记,在遗传的多样性分析、关联分析、品种鉴定、高密度遗传连锁图谱的构建以及辅助育种等方面有广泛的应用[4-6]

水产品免疫基因SNP与抗病关联的研究在鱼类和贝类中已有报道[7-17]。与大黄鱼生长、繁殖性状、耐高温、溶氧等相关基因SNP标记也有一定的筛选[18-22],这将提升大黄鱼遗传育种的效率。有关大黄鱼免疫相关基因SNP的报道不多,李婵等[23]从白细胞介素8(IL-8)基因cDNA中筛选出5个同义突变SNPs,这些SNP可能被用作抗病大黄鱼的分子标记。

为了确定免疫基因中SNP是否与抗弧菌相关联,运用鳗弧菌和副溶血弧菌感染大黄鱼,观察大黄鱼感染后的病理特征,从转录组中筛选免疫相关基因的SNP位点并加以验证,寻找抗弧菌病的突变位点,为大黄鱼抗弧菌的分子选育奠定基础。

1 材料与方法 1.1 大黄鱼暂养、驯化

出膜40余天、1~2 cm左右的大黄鱼苗取自象山港水产引种育种有限公司,在水温26 ℃、水深1 m、1.0 m(内径)×1.2 m(高)的养殖桶里暂养3~4 d,每天投饵2次(早上7:00、下午6:30),换水2次,每次换水90%,清理底部食物残渣及粪便。为了防止大黄鱼患白点病,换水时加入少量甲醛并维持水体中甲醛浓度为0.005‰。待鱼苗适应环境、进食稳定后,将暂养的大黄鱼苗移入实验缸中进行实验。

1.2 弧菌对大黄鱼的感染

鳗弧菌为本实验室从双斑河鲀中分离保存的菌种[24-25],副溶血弧菌(ATCC17802)由中国工业微生物菌种保藏管理中心提供。鳗弧菌和副溶血弧菌分别在含盐1.5‰和高盐30‰的LB培养基, 28 ℃培养。

实验水体为海水70 L,加入到长80 cm、宽45 cm的玻璃缸,每缸放苗500尾,水温控制在26 ℃,待鱼苗稳定后进行实验;设置鳗弧菌1.5×108 CFU/mL和副溶血弧菌1.5×107 CFU/mL浓度感染实验组和对照组,每组设置3个平行。各缸每隔24 h投喂1次饵料,清理掉食物残渣和粪便,换水1/3,并补充感染菌以维持其恒定的浓度。每天清理、记录各缸中死苗数,RNA保护液保存死亡样本用于后续PCR验证。

1.3 转录组分析

实验第7天取对照组、鳗弧菌感染无症状(抗鳗弧菌组)、副溶血弧菌感染无症状(抗副溶血弧菌组)活鱼样本各3尾,Trizol法提取整鱼总RNA,各组样本总RNA混合后质检、建库,用TruSeq PE Cluster Kit在cBot中进行cluster generation,然后在Illumina HiseqTM 2500中进行双向测序。测序原始数据去除低质量和错误碱基后得到clean reads,以模式生物斑马鱼转录组和大黄鱼基因组数据为参考序列进行比对,对测序数据的KEGG通路、差异表达基因和SNP进行分析。从转录组中筛选免疫相关的KEGG通路,根据通路中的免疫基因从差异表达基因中筛选差异倍数较大的免疫基因,并筛选出免疫基因对应的非同义突变位点。

1.4 PCR扩增及测序

选取鳗弧菌和副溶血弧菌感染第4天死亡(易感)、第7天存活(抗感)和对照第7天存活的实验鱼各20、共100尾,分别提取全鱼的总RNA。以RNA为模版用TaKaRa公司的PrimeScriptTM Ⅱ1st和cDNA Synthesis Kit反转录成cDNA,-20℃保存备用。根据大黄鱼转录组和基因组序列设计免疫基因相关引物进行PCR扩增(表 1),PCR反应体系为25 μL:12.5 μL 2 × Taq Plus Master Mix (Dye Plus)(诺唯赞公司),Primer F(10 μmol/L)和Primer R(10 μmol/L)各1 μL,cDNA模板1 μL,无菌水9.5 μL。扩增反应器为Eppendorf PCR仪(德国)。PCR反应程序:95 ℃预变性5 min,94 ℃变性30 s、64 ℃退火30 s、72 ℃延伸30 s(45个循环),72 ℃延伸10 min。经琼脂糖凝胶检测、目的条带清晰无杂带的PCR产物送上海生工生物工程股份有限公司进行测序。

表 1 免疫基因非同义突变SNP引物设计 Tab.1 Primer design of immune gene non-synonymous mutation SNP
1.5 基因型的判断及数据分析

根据测序结果用BioEdit软件观察测序峰型图,进而确定SNP位点的基因型,单峰为纯合子基因型,双峰为杂合子基因型。用SPSS 18.0软件统计数据进行单因素方差分析(P < 0.05:显著性差异;P < 0.01:极显著差异)。

2 结果 2.1 感染实验结果

记录实验鱼每天各缸的死亡数,计算其死亡率,并绘制鳗弧菌和副溶血弧菌感染死亡趋势图(图 1):对照组和鳗弧菌实验组死亡率在实验第1、2天差别不大,从第3天开始感染组死亡率急剧增加,到第7天对照组死亡率达到最高。副溶血弧菌感染组死亡率趋势明显高于对照组(图 1)。感染组、对照组样本死亡率在实验第7天后均急剧上升。

V. AV. P分别表示V. anguillarumV. parahaemolyticus的简称 V. A is short for V. anguillarum, V. P is short for V. parahaemolyticus 图 1 两种弧菌感染大黄鱼死亡趋势 Fig. 1 The death trends of two Vibrio infected large yellow croaker

两感染组中,第4天死亡和第7天存活部分样本出现了感染后症状。如图版-1对照组,可以看出鱼体整体剔透,外部无明显症状;图版-2鳗弧菌感染组中患病样本,部分鱼苗出现鳃部、腹部内淤血、脊椎下充血和烂尾症状,大多数鱼苗表现为头部内出血症状;图版-3副溶血弧菌感染组患病鱼苗部分出现鳃部、腹部内出血的症状。

1.表示对照组样本;2.表示鳗弧菌感染患病样本;3.表示副溶血弧菌感染患病样本 1. represents the control sample; 2. represents the diseased sample of V. anguillarum infection; 3. represents the disease sample of V. parahaemolyticus infection 图版 弧菌感染大黄鱼后的症状 Plate Symptoms of large yellow croaker infected by Vibrio
2.2 转录组中免疫基因的SNP统计

根据转录组测序结果,与免疫相关的信号通路主要有Toll-like receptor signaling pathway、Arginine and proline metabolism、TGF-beta signaling pathway、P13K-Akt signaling pathway、MyD88-dependent pathway、NF-κB signaling pathway、MAPK signaling pathway、JAK-STAT signaling pathway等。根据信号通路筛选免疫差异基因(P < 0.01),从抗鳗弧菌组与对照组中共筛选出101个,其中上调基因39个、下调基因62个;39个上调基因中筛选出的14个基因编码区存在64个SNP位点,其中氨基酸非同义突变位点有17个(表 2)。从抗副溶血弧菌组与对照组中筛选出127个,其中有53个免疫上调基因、73个下调基因;53个免疫上调基因中共发现29个基因的编码区存在146个SNP位点,其中非同义突变位点有28个(表 3)。

表 2 对照组与抗鳗弧菌组差异表达的免疫基因非同义突变SNP Tab.2 Non-synonymous mutation SNP of differentially expressed immune genes in control group and V. anguillarum resistance group
表 3 对照组和抗副溶血弧菌组差异表达的免疫基因非同义突变SNP Tab.3 Non-synonymous mutation SNP of differentially expressed immune genes in control group and V. parahaemolyticus resistance group
2.3 免疫基因SNP验证

选择对照组、鳗弧菌易感组(第4天死亡)、抗鳗弧菌组(第7天存活)、副溶血弧菌易感组、抗副溶血弧菌组样本各10尾,共50尾样本的cDNA对表 2表 3中部分SNP进行一代测序验证。

数据统计分析结果(表 4)显示:tyrosine-protein kinase STYK1-like、glutathione S-transferase omega 1、NF-kappa-B inhibitor zeta-like、complement C1q and tumor necrosis factor-related protein 9A-like、APOBEC1 complementation factor基因分别在其染色体上的第261 746、1 060 606、286 278、35 665和35 767、6 206位上的SNP位点无显著性差异。补体C1q/肿瘤坏死因子相关蛋白9(complement C1q and tumor necrosis factor-related protein 9A-like,CTRP9)所在染色体上的35 767位在抗副溶血弧菌组与对照组间存在显著性差异(P < 0.05),但抗副溶血弧菌组与对照组样本却不存在显著性差异(P < 0.05);interleukin-6 receptor (IL-6R)基因所在染色体的91 196位的突变在鳗弧菌实验组样本中存在极显著性差异(P < 0.01)。

表 4 免疫基因SNP位点卡方值统计 Tab.4 Chi-square value statistics of immune gene SNP locus
表 5 IL-6R基因在鳗弧菌实验组SNP位点统计 Tab.5 Statistics of IL-6R gene in SNP sites of V. anguillarum experiment group
2.3.1 IL-6R基因SNP位点扩大样本检测

扩大样本对照组(19尾)、鳗弧菌易感组(19尾)、抗鳗弧菌组(16尾)进行IL-6R基因SNP的显著性差异验证。

数据分析显示SNP位点IL-6R -91196-G/C在抗鳗弧菌群体中CC基因型频率为12.5%,而在对照组和易感群体中CC基因型频率均为0,存在显著性差异(P < 0.05)。

大黄鱼IL-6R基因cDNA全长共1 026 bp,编码401个氨基酸。生物信息学的预测分析:第341位氨基酸缬氨酸Val(GUG)突变为亮氨酸Leu(CUG),会导致蛋白质结构发生明显变化,由缬氨酸Val变为亮氨酸Leu时,蛋白质折叠由近似球状变为椭球状;为缬氨酸时N端距离IL-6R基因的蛋白分子较近,而为亮氨酸Leu时,N端远离分子,蛋白质三级结构的变化,可能影响该基因的功能(图 2)。

图(a、b)分别表示IL-6R基因341位氨基酸是缬氨酸(Val)和亮氨酸(Leu)时的蛋白质三级结构 Figures (a, b) show the tertiary structure of the protein when the amino acid at position 341 of the IL-6R gene is valine (Val) and leucine (Leu) respectively 图 2 Swiss model预测IL-6R蛋白质三级结构 Fig. 2 Predicting IL-6R protein tertiary structure using Swiss model
2.3.2 CTRP9基因SNP位点扩大样本检测

为了更准确验证CTRP9基因SNP位点在副溶血弧菌感染组样本中的基因型,扩大样本进行分析:对照组、副溶血弧菌易感组和抗副溶血弧菌组样本分别为20、18、16尾。数据分析发现SNP位点CTRP9 - 35665-A/GAA 22.2%、AG 77.8%、GG 0,在抗副溶血弧菌组中AA 37.5%、AG 25%、GG 37.5%。CTRP9基因的Ile突变为Val位点,副溶血弧菌易感组与抗副溶血弧菌组间存在极显著性差异(P < 0.01),但抗副溶血弧菌组与对照组之间却不存在显著性差异(P>0.01)(表 6)。

表 6 CTRP9基因两个SNP位点在副溶血弧菌实验组样本中卡方值统计 Tab.6 Chi-square value statistics of two SNP loci of CTRP9 gene in V. parahaemolyticus experimental group samples

CTRP9基因cDNA全长1 368 bp,编码455个氨基酸,异亮氨酸(Ile)突变为缬氨酸(Val)发生在氨基酸序列的第383位,用Swiss model预测CTRP9蛋白质三级结构(图 3)。CTRP9氨基酸序列第383位为Ile和Val时蛋白质三级结构整体折叠层次都近似球体状,但稍有差异,为Ile时C端尾巴折叠靠近蛋白质大分子,为Val时C端尾巴则远离蛋白质大分子。位点突变导致其蛋白质三级结构改变,该SNP位点可能与大黄鱼抗副溶血弧菌有关。

图(a、b)分别表示CTRP9基因383位氨基酸是异亮氨酸(Ile)和缬氨酸(Val)时的蛋白质三级结构 Figures (a, b) show the tertiary structure of the protein when the amino acid at position 383 of the CTRP9 gene is isoleucine (Ile) and proline (Val) respectively 图 3 Swiss model预测CTRP9蛋白质三级结构 Fig. 3 Predicting CTRP9 protein tertiary structure using Swiss model
3 讨论

鳗弧菌和副溶血弧菌感染大黄鱼,激活相应组织上的TLR受体[26],与受体结合后激活细胞内相关免疫通路。从相关免疫通路中,筛选出有差异表达的免疫基因及相关的非同义突变的SNP,推测45个SNPs可能与抗弧菌有关。首次对免疫相关基因的SNP进行一代测序验证,证明白细胞介素受体IL-6R -383位SNP与抗鳗弧菌有关;补体C1q/肿瘤坏死因子相关蛋白CTRP9 -341位SNP与抗副溶血弧菌有关。

白细胞介素受体是参与免疫反应的重要基因,在启动和调节免疫反应中起中心调节作用。IL-6R主要有α链和和β链,当病原菌感染机体时,IL-6首先与B淋巴细胞、T淋巴和吞噬细胞等细胞表面的IL-6R的α链结合,后与β链上的信号转导蛋白gp130结合,形成高亲和复合体,激活JAK-STAT信号通路,发挥免疫功能[27]IL-6R基因的SNP在人、牛等生物中有相关报道。溶藻弧菌感染大黄鱼后,IL-6R基因在大黄鱼所有组织中表达量都上调,在头肾中最高[28]。本实验中两种弧菌感染大黄鱼部分出现腹腔内出血症状,推测大黄鱼的血管内皮质细胞和炎性细胞中IL-6R被激活、上调,从而发挥免疫学功能和抗菌活性。因此,研究大黄鱼IL-6R基因多态性与抗弧菌关联,不仅为大黄鱼抗菌机制研究提供新思路,也是分子育种的迫切需要。

CTRP9基因是脂肪因子超家族中与脂联素结构最相近的分子,可与脂联素形成异源多聚复合物参与炎症等诸多生理和病理过程[29]CTRP9基因还通过脂联素受体1/AMPK/Akt/内皮型一氧化氮合酶依赖的通路,增加人静脉内皮细胞一氧化氮,发挥舒张血管的作用[30]CTRP9基因在鱼类中的研究主要有石斑鱼和罗非鱼。点带石斑鱼(Epinephelus coioides)中,CTRP9的结构和蛋白功能在硬骨鱼代谢和食物摄入起重要的调节作用[31]。杨国坤等[32]在尼罗罗非鱼中证明了CTRP9对硬骨鱼类繁殖的调节作用。梁丽丽等[33]首次对CTRP9基因多态性与冠心病的相关性进行研究,本文对CTRP9基因的SNP与抗副溶血弧菌的关系进行研究,这为CTRP9基因多态性与疾病关联研究奠定基础。

陈小明等[22]在研究大黄鱼耐高温性状全基因组关联分析时,在38个SNPs附近共找到26个已知的功能基因,部分基因与免疫功能相关。本研究首次对筛选到的大黄鱼免疫基因SNPs进行验证,确定具体抗弧菌的基因型频率。SNP位点IL-6R -91196-CC和CTRP9 -35665-GG基因型CTRP9 -35665-GG基因型可以作为大黄鱼抗弧菌病的候选基因,用于指导大黄鱼的分子标记辅助选择育种。另外,HAO等[34]发现三疣梭子蟹(Portunus trituberculatus)C型凝集素中的SNP E4-205C/T与抗溶藻弧菌有关联,基因型TT可增加梭子蟹对溶藻弧菌抗性;GUO等[35]克隆出了血蓝蛋白Ig样结构域基因存在11个SNP,通过测序验证了5个SNP位于基因组DNA和cDNA中,可能与环境压力或致病性相关。免疫相关基因的SNPs在水产育种中的应用,将大大提升水产品的质量和育种速率。

综上所述,本研究首次对大黄鱼免疫相关基因非同义突变SNP进行筛选与验证,SNP的标记不仅可以评估大黄鱼遗传多样性,而且为大黄鱼的抗病选育等工作提供研发基础。同时, 本研究检测到了大黄鱼IL 6RCTRP9基因与弧菌抗性相关的SNP,为大黄鱼的选育工作提供新思路,有利于推进大黄鱼的遗传育种进程。

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Analysis of SNP loci in immune-related genes of two species of Vibrio infecting large yellow croaker (Larimichthys crocea)
SUN Mingjie1, ZHANG Na1, XU Shanliang2, BAO Baolong1, GONG Xiaoling1     
1. The Key Laboratory of Exploration and Utilization of Aquatic Genetic Resources, Ministry of Education, Shanghai Ocean University, Shanghai 201306, China;
2. Key Laboratory of Applied Marine Biotechnology, Ministry of Education, Ningbo University, Ningbo 315211, Zhejiang, China
Abstract: In order to investigate the relationship between SNP and Vibrio resistance in immune-related genes of large yellow croaker (Larimichthys crocea), Vibrio anguillarum and Vibrio parahaemolyticus were used to artificially infect large yellow croaker. High-throughput sequencing, screening and analysis of disease-resistant groups before and after infection:(1)17 non-synonymous mutation sites of amino acid were screened from V. anguillarum resistance group.A total of 28 SNPs were screened from the V. parahaemolyticus resistance group.(2)The first-generation sequencing confirmed that the SNP loci was located on the 91196th of chromosome NW_011323507.1 that interleukin-6 receptor gene (IL-6R) was from G changed to C, the proline was mutated to leucine, and the genotype CC frequencies of this site in the V. anguillarum resistance and the control groups were 12.5% and 0, which showed significant difference (P < 0.05).(3)And the 35665 base mutation (A-G) of the complement C1q/TNF-related protein 9 (CTRP9) gene on chromosome NW_011323975.1 showed a significant difference in the V. parahaemolyticus susceptibility/resistance groups (P < 0.01), the genotype GG frequencies were 37.5% and 0. The results showed that IL-6R-91196-G/C site mutation was associated with V. anguillarum resistance group, and CTRP9-35665-A/G site mutation was associated with large yellow croaker against V. parahaemolyticus.It provides theoretical support for the selection and breeding of large yellow croaker against Vibrio population.
Key words: Vibrio anguillarum     Vibrio parahaemolyticus     Larimichthys crocea     transcriptome     immune gene