CLONING, MOLECULAR CHARACTERISTIC AND PHYLOGENETICS OF HEMOGLOBIN GENES IN CULTER ALBURNUS
-
摘要: 研究通过克隆翘嘴鲌(Culter alburnus)血红蛋白基因, 分析血红蛋白的分子特征及系统进化, 探讨鱼类耐低氧的可能成因。研究克隆了翘嘴鲌血红蛋白家族中Hba1/2和Hbb1/2 cDNA序列, 翻译后分别得到143、143、147和147个氨基酸。蛋白二级结构分析表明Hba1/2和Hbb1/2蛋白分别包含7和8个螺旋域、14和13个α1β2结合残基、12和16个亚铁血红素结合残基, 均有16个α1β1结合残基, 只有Hba1/2蛋白具有6个Bohr效应残基。与两栖类和哺乳类相比, 鱼类Hba和Hbb蛋白功能域上分别有10和5个氨基酸残基替换位点, 这很可能是为了使其适应水中的低氧环境。而耐低氧与不耐低氧鱼类相比, 其蛋白二级结构并未发现一致性的替换, 这表明鱼类的耐低氧特征很可能是受到上游信号通路的调控。通过系统发育关系表明Hba1/2和Hbb1/2基因亚型的复制事件很可能发生在脊椎动物全基因组复制之后且硬骨鱼类全基因组复制事件之前。值得注意的是在系统进化树上, 与其他鱼类相比翘嘴鲌和斑马鱼在Hba1/2和Hbb1基因上有更近的亲缘关系, 这很可能是由于其同属鲤形目且均不耐低氧所致。研究首次克隆了翘嘴鲌血红蛋白家族基因, 分析其分子特征及与其他物种的系统发育关系, 并探讨了鱼类耐低氧的可能成因, 为鱼类的耐低氧生物学研究提供了理论基础与潜在方向。Abstract: Hemoglobin is one of the most important proteins for aerobic metabolism in vertebrate. The studies of Hb are more in Mammalia, but less in fishes with low-oxygen environment. This study cloned Hba1/2 and Hbb1/2 cDNA sequences of Culter alburnus (C. alburnus) hemoglobin, which encode 143, 143, 147 and 147 of amino acids, respectively. The second structure analysis of proteins indicated that Hba1/2 and Hbb1/2 included 7 and 8 helical regions, 14 and 13 α1β2 interfaces, 12 and 16 heme bindings, 16 and 16 α1β1 interfaces, respectively, and 6 Bohr effect residues only for Hba1/2. Compared with amphibians and mammalia, there were 10 and 5 amino acid substitutions in fishes of Hba and Hbb functional domains, which may be used to adapt to hypoxia. However, compared with tolerant and intolerant hypoxia fishes, we did not find any coherent substitution in the second structure of proteins, indicating that the tolerant hypoxia trait of fish may be regulated by upstream signal pathways. The phylogenetic relationship showed the duplication events of Hba1/2 and Hbb1/2 isoforms may occurre after vertebrate and before teleost of whole-genome duplications. Interestingly, in the phylogenetic trees, the genetic relationships of C. alburnus and Danio rerio of Hba1/2 and Hbb1 were closer than those of C. alburnus and other fishes, probably because both C. alburnus and Danio rerio belong to Cypriniformes and intolerant to hypoxia. This study cloned Hba1/2 and Hbb1/2 cDNA sequences of C. alburnus hemoglobin, analyzed molecular characteristics and phylogenetic relationships with others, and discussed the possible causes of fish tolerance to hypoxia. These findings provide a theoretical basis and potential direction for fish tolerant hypoxia biology.
-
有氧代谢(Aerobic metabolism)是动物界基本的生理代谢之一, 基于此动物们才能展开一系列的能量代谢活动[1, 2]。从水中的无脊椎动物, 如软体动物等, 到脊椎动物鱼类和两栖类, 再到陆地上的脊椎动物爬行类、鸟类和哺乳类都利用各自的方式进行着有氧代谢活动[3－5]。从水环境到陆地环境的改变, 动物们的呼吸器官也从体腔和鳃转换到了皮肤和肺, 其呼吸方式的改变极大地促进了物种的进化[6－9]。而血红蛋白正是脊椎动物体内有氧代谢最重要的蛋白之一。
血红蛋白(Hemoglobin, Hb)是由多肽链组成, 又称为珠蛋白, 且每条链上都有一个血红素辅基[10, 11]。脊椎动物的血红蛋白分子一般是异源四聚体结构, 由两条α多肽链(α1和α2)和两条β多肽链(β1和β2)组成。α和β多肽链分别有7和8个螺旋片段(A-H, α多肽链无D螺旋), 这些螺旋片段和非螺旋片段形成转角并引起链的特征三级折叠形成内二聚体(α1β1和α2β2)和间二聚体(α1β2和α2β1)[3, 12, 13]。血红蛋白主要存在于红细胞中, 能溶解大量的氧气并将其运送至机体的各个组织器官, 是氧化分解代谢电子的最终受体[14]。当脊椎动物血红蛋白分子中的一个α亚基与氧分子结合后, 会立即使得其余亚基结构改变, 致使血红蛋白分子对氧分子的亲和力迅速增大[15]。与之类似, 血红蛋白分子在组织中释放氧分子的过程也随着一个氧分子的释放促使其他氧分子的释放。机体利用血红蛋白分子的协同效应来提高其转运和存储氧气的效率。血红蛋白分子各亚基氨基酸序列高度同源, 是研究全基因组复制事件的良好材料。有研究表明血红蛋白的α和β多肽链氨基酸序列相似度大约为50%, 且有颌类(Gnathostomata)的α和β球蛋白是在450—500 Ma奥陶纪时全基因组复制事件中从无颌类(Agnatha)中一个古基因复制分化而来的[16, 17]。而硬骨鱼类血红蛋白α和β球蛋白基因的多态性很可能是在320—400 Ma硬骨鱼类特有的全基因组复制事件中复制生成的[18—20]。
翘嘴鲌(Culter alburnus)隶属鲤形目、鲤科、鲌亚科、鲌属, 在我国平原各水系均有分布, 是长江流域优质经济鱼类之一, 享有“太湖三白”的美誉。翘嘴鲌以其个体大、生长快、鳞下脂肪多、营养价值高和味道鲜美等特点深受渔民和食客欢迎[21]。在众多水产养殖品种之中, 翘嘴鲌对氧的需求量较高。研究表明平均体重0.44—0.51 g的翘嘴鲌在水温21℃时窒息点为0.41 mg/L; 当水温上升到29℃时, 其窒息点达到0.57 mg/L[22]。鲤(Cyprinus carpio)在体长11.3—14.2 cm、水温30℃时, 窒息点为0.29 mg/L[23]; 镜鲤(Cyprinus carpio var. specularis
)在平均体重30—70 g、水温20℃时, 窒息点为0.15 mg/L[24]; 青鱼(Mylopharyngodon piceus)在平均全长55 cm、水温18℃时, 窒息点为0.31 mg/L[22]; 尼罗罗非鱼(Oreochromis niloticus)在平均全长13—14 cm、水温21℃时, 窒息点为0.07 mg/L[25]。随着集约化高密度养殖模式的扩大, 低氧的养殖环境容易使得鱼类生长缓慢, 抵抗力低, 甚至出现死亡。而且在鱼类运输中, 也很容易出现缺氧导致大规模死亡现象。 因此, 本研究以翘嘴鲌为实验对象, 克隆了其血红蛋白家族中Hba1/2和Hbb1/2 cDNA序列, 并运用了生物信息学方法分析其分子特征及与其他物种的系统发育关系, 为鱼类的耐低氧生物学研究提供理论依据。
1. 材料与方法
1.1 实验鱼与试剂
本实验翘嘴鲌(42.5±3.7) cm来源于中国水产科学院长江水产研究所荆州窑湾基地。翘嘴鲌从池塘捞起后先禁食24h, 然后置于冰上用MS-222 (100 mg/L)麻醉, 迅速取样, 包括脑、心脏、肝脏、脾脏、肠道组织、肌肉和血液, 取出的组织样品用灭菌的DEPC 处理水冲洗, 并用液氮快速冷冻, 然后保存于–80℃冰箱备用。
主要实验试剂有: 购自大连宝生物公司(TaKaRa)的RNA提取试剂盒、DNA聚合酶、逆转录试剂盒、3′-Full RACE Kit试剂盒、5′-Full RACE Kit试剂盒、琼脂糖凝胶电泳试剂盒(DNA Marker)和DNA凝胶纯化回收试剂盒等试剂; 购自Sigma公司的焦炭酸二乙酯(DEPC)和胰蛋白胨等试剂。
1.2 基因cDNA序列的克隆
所取翘嘴鲌各组织样品中的总RNA严格按照试剂盒说明书步骤进行操作提取。提取所得总RNA的纯度用核酸蛋白检测仪(ND-2000)检测, 其完整性用浓度为1.5%的琼脂糖电泳检测。以提取翘嘴鲌各组织样品的总RNA为模板, 使用TaKaRa公司的PrimeScriptTM II 1st Strand cDNA Synthesis Kit试剂盒合成cDNA的第一条链。
基于GenBank和Ensembl中已报道的斑马鱼(Danio rerio)、鲤、虹鳟(Oncorhynchus mykiss)和犀角金线鲃(Sinocyclocheilus rhinocerous)的Hba1、Hba2、Hbb1和Hbb2基因cDNA序列, 设计克隆实验用鱼翘嘴鲌的核心序列简并引物(表 1)。以合成的第一链cDNA为模板, 对翘嘴鲌cDNA核心片段进行扩增, PCR反应体系(20 µL): cDNA模板1 µL, 10×Ex-Taq Buffer 2 µL, dNTP (2.5 mmol/L) 2 µL, 简并引物各0.4 µL, Taq酶0.2 µL，ddH2O补足至20 µL。PCR反应条件: 94℃预变性4min; 94℃变性30s, 56℃退火30s, 72℃延伸60s, 30个循环; 然后72℃延伸5min。PCR产物经过凝胶回收后进行拼接至Ecoli DH5α感受态细胞, 并将PCR检测为阳性的克隆产物菌液交武汉擎科生物技术有限公司测序。测序所得结果在NCBI上进行比对分析检验其同源性。根据克隆得到的基因cDNA核心序列分别设计3′和5′ RACE特异性引物(表 1)并进行巢式反应, Outer-PCR反应程序为: 94℃预变性3min; 94℃变性30s, 55℃退火30s, 72℃延伸60s, 25个循环; 然后72℃再延伸5min。Inner-PCR反应程序为: 94℃预变性3min; 94℃变性30s, 58℃退火30s, 72℃延伸60s, 30个循环; 然后72℃终延伸5min。
表 1 Hba1、Hba2、Hbb1和Hbb2基因克隆所需引物序列Table 1. The primers used for the cDNAs cloning of Hba1, Hba2, Hbb1 and Hbb2 genes引物Primer 序列Sequence (5′—3′) Primers for partial fragment Hba1-F TCTGATAAGKACAMGGCTGTT Hba1-R ATGCCCAATGCAGGWTTAG Hba2-F GAACSACAGTGGWGAAGGGAG Hba2-R CTGACCTGGRCGAGGAACT Hbb1-F GGCAAGGGTGCTGATTSTA Hbb1-R CAGCCTGAAGTTGWCTGRATC Hbb2-F TGAGAAGAYCACCATCCAG Hbb2-R GTTAAGCAGTCAGCCWAAA Primers for 3′ RACE PCR 3′ GS-Hba1 01-F AGAATGCTGACCGTCTACCC 3′ GS-Hba1 11-F GGTCTGGTCCTGTGAAGAA 3′ GS-Hba2 01-F CATTGGGCAGACCTAAACC 3′ GS-Hba2 11-F ACGACAGTGGTGAAGGGAG 3′ GS-Hbb1 01-F TCCAGGGCAATCCCAAGGT 3′ GS-Hbb1 11-F ACTGTGCTAAATGCGTTGG 3′ GS-Hbb2 01-F CTCTACAACGCCGCTGCTA 3′ GS-Hbb2 11-F GGACAACATCAAAGCCACC 3′ RACE Outer TACCGTCGTTCCACTAGTGATTT 3′ RACE Inner CGCGGATCCTCCACTAGTGATTTCACTATAGG Primers for 5′ RACE PCR 5′ GS-Hba1 01-F CCCACAAGGTCGTCTATTTT 5′ GS-Hba1 11-F AAAGCATGAAGTTCGCTCA 5′ GS-Hba2 01-F CTGACCTGGGCGAGGAACT 5′ GS-Hba2 11-F CTCCCTTCACCACTGTCGTTCCA 5′ GS-Hbb1 01-F TTCCAACGCATTTAGCACA 5′ GS-Hbb1 11-F GTTGTGCCACCTTGGGATT 5′ GS-Hbb2 01-F CATAGGTGGCTTTGATGTTG 5′ GS-Hbb2 11-F GATAGCAGCGGCGTTGTAG 5′ RACE Outer CATGGCTACATGCTGACAGCCTA 5′ RACE Inner CGCGGATCCACAGCCTACTGATGATCAGTCGATG Note: Mixed bases: B-G/T/C; D-G/A/T; H-A/T/C; K-G/T; M-A/C; R-A/G; S-G/C; V-G/A/C; W-A/T; Y-C/T 1.3 序列分析及进化树的构建
用DNAStar软件将获得的全部cDNA片段进行序列拼接, 得到各构型基因的cDNA全长序列。将得到的全长序列在NCBI中进行在线比对, 以确定所获得的各序列所对应的基因类型(http://blast.ncbi.nlm.nih.gov/)。并运用NCBI中的开发阅读框(Open Reading Frame, ORF)搜索服务器查找翘嘴鲌血红蛋白基因的开发阅读框并翻译成氨基酸序列; 通过ExPAsy (https://web.expasy.org/protparam/)在线软件对翘嘴鲌血红蛋白序列进行分析; 运用在线软件SMART (http://smart.embl-heidelberg.de/)预测翘嘴鲌血红蛋白结构域; 序列比对和氨基酸同源性分析使用Clustal-W软件; 使用MEGA7.0软件进行进化分析, 以邻接法(Neighbor-joining, NJ)构建系统进化树, 每个节点的可信值重复次数为1000。
2. 结果
2.1 Hba1/2和Hbb1/2 cDNA序列的分子特征
本研究通过RT-PCR和RACE方法获取Hba1、Hba2、Hbb1和Hbb2基因的cDNA全长序列, 其长度分别为652、465、447和475 bp(表 2)。且序列分析表明cDNA序列ORF(开放阅读框)分别为429、429、441和441 bp, 翻译成蛋白后分别得到143、143、147和147个氨基酸。使用在线软件ExPAsy预测得到Hbs蛋白分子量分别为15479.01、15971.59、16138.58和16335.91, 分子式分别为C707H1119N183O198S4、C729H1155N183O208S5、C731H1148N196O208S4和C744H1151N193O207S7, 理论等电点分别为8.81、6.91、8.95和6.50。Hba1蛋白富含丙氨酸(Ala, 11.2%)、亮氨酸(Leu, 9.8%)和赖氨酸(Lys, 9.8%), Hba2蛋白富含亮氨酸(13.3%)、赖氨酸(9.1%)和缬氨酸(Val, 8.4%), 而Hbb1蛋白富含丙氨酸(12.2%)、亮氨酸(9.5%)和缬氨酸(9.5%), Hbb2蛋白富含丙氨酸(11.6%)、缬氨酸(10.9%)和亮氨酸(8.8%)。并利用在线软件SMART预测蛋白二级结构域, 结果表明Hba1/2蛋白包括7个螺旋域、16个α1β1结合残基、14个α1β2结合残基、12个亚铁血红素结合残基和6个Bohr效应残基(图 1), 而Hbb1/2蛋白包含8个螺旋域、16个α1β1结合残基、13个α1β2结合残基和16个亚铁血红素结合残基(图 2)。
表 2 翘嘴鲌Hba1、Hba2、Hbb1和Hbb2基因的cDNA全长序列信息Table 2. The information for full-length cDNA sequences of Hba1, Hba2, Hbb1 and Hbb2 from C. alburnus基因Gence 长度Length (bp) 5′UTR (bp) 3′UTR (bp) ORF (bp) 氨基酸的数目
Number of amino acidsHba1 652 108 115 429 143 Hba2 465 21 15 429 143 Hbb1 477 21 15 441 147 Hbb2 475 18 16 441 147 ![]()
图 1 翘嘴鲌Hba1和Hba2氨基酸序列与哺乳类、两栖类及其他硬骨鱼类比对分析黑色阴影部分表示是相同的氨基酸残基。根据哺乳类的α球蛋白, 方框表示螺旋区域(A—H, 除了D)。功能残基的位置分别表示为α1β1结合残基(1)、α1β2结合残基(2)、亚铁血红素结合残基(h)和Bohr效应残基(b)。三角形表示氨基酸替换位点。上述氨基酸序列从GenBank/EMBL/UniprotKB中获得的登录号详见图 3Figure 1. Alignment of C. alburnus amino Hba1 and Hba2 amino acid sequences with mammalia, amphibian and other teleostsThe black shade denotes the identical residues. According to mammalian α globins, the helical regions (A—H, except D) were outlined by boxes. The positions of functional residues are indicated by: α1β1 interfaces (1), α1β2 interfaces (2), heme binding (h), and Bohr effect residues (b). Triangles indicate changes at amino acids sites. Deduced amino acid sequences were obtained from GenBank/EMBL/UniprotKB, the accession numbers of which were dwelled on Fig. 3![]()
图 2 翘嘴鲌Hbb1和Hbb2氨基酸序列与哺乳类、爬行类、两栖类及其他硬骨鱼类比对分析黑色阴影部分表示是相同的氨基酸残基。根据哺乳类的β球蛋白, 方框表示螺旋区域(A—H)。功能残基的位置分别表示为α1β1结合残基(1)、α1β2结合残基(2)和亚铁血红素结合残基(h)。三角形表示氨基酸替换位点。上述氨基酸序列从GenBank/EMBL/UniprotKB中获得的登录号详见图 4Figure 2. Alignment of C. alburnus amino Hbb1 and Hbb2 acid sequences with mammalia, reptilia, amphibian and other teleostsThe black shade denotes the identical residues. According to mammalian β globins, the helical regions (A—H) were outlined by boxes. The positions of functional residues are indicated by: α1β1 interfaces (1), α1β2 interfaces (2), and heme binding (h). Triangles indicate changes at amino acids sites. Deduced amino acid sequences were obtained from GenBank/EMBL/UniprotKB, the accession numbers of which were dwelled on Fig. 42.2 Hba1/2和Hbb1/2蛋白多序列比对
将翘嘴鲌Hba1、Hba2、Hbb1和Hbb2氨基酸序列与其他鱼类(斑马鱼和鲤)、两栖类[非洲爪蟾(Xenopus laevis)]和哺乳类[大鼠(Rattus norvegicus)]相比, 我们发现其同源性分别为44.8%—93.0%、44.1%—88.8%、51.7%—87.1%和47.6%—89.8% (表 3)。其中翘嘴鲌Hba1、Hba2和Hbb2氨基酸序列与非洲爪蟾的同源性最低, 且其Hbb1氨基酸序列与大鼠的同源性最低。而翘嘴鲌Hba1/2氨基酸序列与斑马鱼同源性最高, 其Hbb1/2氨基酸序列与鲤同源性最高。
表 3 翘嘴鲌Hba1、Hba2、Hbb1和Hbb2氨基酸序列与其他物种同源性(%)分析Table 3. Identity analysis of C. alburnus Hba1, Hba2, Hbb1 and Hbb2 amino acid sequence with other species (%)氨基酸Amino acid 斑马鱼Zebrafish 鲤Crap 非洲爪蟾Xenopus 大鼠Rat Hba1 93.0 68.5 44.8 51.0 Hba2 88.8 86.7 44.1 51.0 Hbb1 84.4 87.1 54.4 51.7 Hbb2 84.4 89.8 47.6 53.7 Note: GenBank/EMBL/UniprotKB databases accession numbers: Hba1 (Q90487, KTG39806.1, XP_018092516.1, NP_037228.1); Hba2 (NP_898889.2, KTF87195.1, NP_001081493.1, NP_001007723.1); Hbb1 (NP_001003431.1, XP_018973479.1, NP_001079742.1, NP_150237.1); Hbb2 (NP_001096600.1, XP_018929000.1, NP_001081497.1, NP_001106694.1). The order of accession numbers of each gene corresponds to zebrafish, crap, xenopus, and rat, respectively 2.3 Hba1/2和Hbb1/2基因系统进化分析
通过系统进化分析发现, Hba1/2基因在鱼类、两栖类和哺乳类各自聚成一支(图 3)。其中鱼类和蝾螈类的Hba1和Hba2基因各自聚到一起。在鱼类中, 翘嘴鲌Hba1基因与斑马鱼亲缘关系最近而与虹鳟和鲤关系相对较远, 其Hba2基因也与斑马鱼亲缘关系最近而与黄鳝(Monopterus albus)和欧洲鲈(Dicentrarchus labrax)关系相对较远。此外, Hbb1/2基因也在鱼类、两栖类、爬行类和哺乳类各自聚成一支(图 4)。其中鱼类的Hbb1和Hbb2基因各自聚到一起。在鱼类中, 翘嘴鲌Hbb1基因与白鲢(Hypophthalmichthys molitrix)和斑马鱼亲缘关系最近而与鳜(Siniperca chuatsi)和欧洲鲈关系相对较远, 其Hbb2基因则与鲤和鲫(Carassius auratus)亲缘关系较近而与黄鳝和虹鳟关系相对较远。
![]()
图 3 翘嘴鲌和其他脊椎动物Hba1和Hba2蛋白系统进化树(▲所示为翘嘴鲌)Figure 3. The phylogenetic tree of Hba1 and Hba2 from C. alburnus (▲) and other vertebrate species![]()
图 4 翘嘴鲌和其他脊椎动物Hbb1和Hbb2蛋白系统进化树(▲所示为翘嘴鲌)Figure 4. The phylogenetic tree of Hbb1 and Hbb2 from C. alburnus (▲) and other vertebrate species3. 讨论
3.1 翘嘴鲌血红蛋白的分子特征
为研究翘嘴鲌血红蛋白分子特征, 我们克隆获得Hba1、Hba2、Hbb1和Hbb2基因的cDNA全长序列。虽然这些基因的全长序列碱基数目不一样, 但是翻译成氨基酸序列后发现翘嘴鲌α和β球蛋白氨基酸数目分别为143(Hba1和Hba2)和147(Hbb1和Hbb2)(表 2), 且蛋白二级结构中Hba1/2和Hbb1/2蛋白分别包括7和8个螺旋域(图 1), 此结果与草鱼、鲤、鲫[26]、斑马鱼[27]、鲈[28]、黄河裸裂尻鱼(Schizopygopsis pylzovi)[29]、半滑舌鳎(Cynoglossus semilaevis)[30]和大黄鱼(Larimichthys crocea)[31]保持一致。与人类的α球蛋白多肽链[32]相比, 翘嘴鲌α球蛋白的α1β1结合残基、α1β2结合残基和亚铁血红素结合残基数目是相似的, 除了6个Bohr效应残基并不连续。且翘嘴鲌β球蛋白多肽链的α1β1结合残基、α1β2结合残基和亚铁血红素结合残基数目与人类的β球蛋白[33]相比也是相似的。
3.2 血红蛋白二级结构分析
在血红蛋白二级结构比对分析中, 鱼类[翘嘴鲌、草鱼、白斑狗鱼(Esox lucius)、欧洲鲈、虹鳟、斑马鱼、鲫、鲤、斑点叉尾鮰(Ictalurus punctatus)、泥鳅(Misgurnus anguillicaudatus)和黄鳝]的Hba蛋白氨基酸位点与两栖类[非洲爪蟾、冠北螈(Triturus cristatus)和欧非肋突螈(Pleurodeles waltl)]和哺乳类[大鼠、长臂猿(Hylobates lar)、领狐猴(Varecia variegata)、牦牛(Bos mutus)和斑马(Equus burchelli)]相比有15个氨基酸残基发生替换, 其中包括1个氨基酸位点的插入(图 1)。在C-E螺旋区域间的第48位氨基酸上插入了丙氨酸(Ala, A)、赖氨酸(Lys, K)、天冬氨酸(Asn, N)、丝氨酸(Ser, S)、苏氨酸(Thr, T)或脯氨酸(Pro, P), 而据报道其在鲈中插入的是色氨酸(Trp, W) [28], 在半滑舌鳎中是赖氨酸(Lys, K) [30], 在黄尾(Seriola lalandi)[34]和大黄鱼[31]中是甘氨酸(Gly, G)。由于此氨基酸位点的插入是在螺旋间区, Miyata等[34]认为此次第48位氨基酸残基的插入对于血红蛋白的功能和三维结构的影响无关紧要。Okamot等[35]认为螺旋间区的氨基酸变化是由自然选择压力带来的, 很可能对于蛋白功能没有影响。与两栖类和哺乳类相比, 鱼类Hba蛋白15个氨基酸残基替换位点有2个是在Bohr效应残基上, 2个在α1β1结合残基上, 10个在螺旋区域和5个在非螺旋区域。而鱼类(翘嘴鲌、白鲢、白斑狗鱼、鳜、虹鳟、斑马鱼、斑点叉尾鮰、欧洲鲈、鲫、鲤、大口鲶(Silurus asotus)、黄鳝和泥鳅)Hbb蛋白6个氨基酸残基替换位点有5个在螺旋区域和1个在非螺旋区域。这些氨基酸位点的替换很可能是从低等脊椎动物鱼类在水中用鳃呼吸, 进化到两栖类再到高等的哺乳类具有利用空气中氧气的能力的重要原因[36, 37]。遗憾的是, 相对耐低氧的鱼类与不耐低氧鱼类在Hba/b蛋白二级结构上并未发现一致的氨基酸残基替换, 这表明鱼类的相对耐低氧特征很可能是受到血红蛋白上游通路的调控。
3.3 翘嘴鲌的系统发育分析
系统发育关系发现Hba1/2和Hbb1/2基因在鱼类、两栖类和哺乳类均各自聚成一支, 且鱼类各基因亚型也单独聚为一支(图 3和图 4)。一般认为有颌类的α和β球蛋白是在450—500 Ma奥陶纪时全基因组复制事件中从无颌类中一个古基因复制分化而来的[16, 17], 这表明Hba1/2和Hbb1/2基因亚型的复制事件很可能发生在脊椎动物全基因组复制之后且硬骨鱼类全基因组复制事件之前。有研究表明在硬骨鱼类全基因组复制事件之前, 脊椎动物中的一些基因会发生重复复制和删除[20, 38, 39]。在系统进化树中, 翘嘴鲌的Hba1/2和Hbb1基因与斑马鱼亲缘关系较近, 而与虹鳟、鲤及白斑狗鱼亲缘关系较远。从鱼类分类学上看, 虹鳟和白斑狗鱼隶属鲑形目(Salmoniformes), 而翘嘴鲌和斑马鱼隶属鲤形目(Cypriniformes), 因此翘嘴鲌与斑马鱼亲缘关系更近。对于鲤, 从生理学上看, 鲤在体长11.3—14.2 cm、水温30℃时, 窒息点为0.29 mg/L[23]。而翘嘴鲌在平均体重0.44—0.51 g、水温29℃时, 其窒息点达到0.57 mg/L, 当水温21℃时其窒息点为0.41 mg/L[22]; 斑马鱼平均体重约0.23 g、水温20℃时, 其窒息点为0.41 mg/L[40]。这表明翘嘴鲌和斑马鱼耐氧能力相近很可能导致其在Hba1/2和Hbb1基因上有更近的亲缘关系。
总之, 本研究首次克隆了翘嘴鲌Hba1、Hba2、Hbb1和Hbb2基因的cDNA全长序列。分析翘嘴鲌和其他物种血红蛋白二级结构发现, 与两栖类和哺乳类相比, 鱼类Hba和Hbb蛋白功能域上分别有10和5个氨基酸残基替换位点, 这些替换位点很可能是鱼类能适应水中低氧环境的原因。这也有待后续的基因功能试验来进行验证。通过血红蛋白基因系统发育关系表明Hba1/2和Hbb1/2基因亚型的复制事件很可能发生在脊椎动物全基因组复制之后且硬骨鱼类全基因组复制事件之前, 而这是由脊椎动物中的一些基因会发生重复复制和删除事件造成的。值得注意的是从系统进化树上看, 由于翘嘴鲌和斑马鱼同属鲤形目且均不耐低氧, 所以其在Hba1/2和Hbb1基因上的亲缘关系更近。这些发现为鱼类的耐低氧生物学研究提供了理论基础。
-
![]()
图 1 翘嘴鲌Hba1和Hba2氨基酸序列与哺乳类、两栖类及其他硬骨鱼类比对分析
黑色阴影部分表示是相同的氨基酸残基。根据哺乳类的α球蛋白, 方框表示螺旋区域(A—H, 除了D)。功能残基的位置分别表示为α1β1结合残基(1)、α1β2结合残基(2)、亚铁血红素结合残基(h)和Bohr效应残基(b)。三角形表示氨基酸替换位点。上述氨基酸序列从GenBank/EMBL/UniprotKB中获得的登录号详见图 3
Figure 1. Alignment of C. alburnus amino Hba1 and Hba2 amino acid sequences with mammalia, amphibian and other teleosts
The black shade denotes the identical residues. According to mammalian α globins, the helical regions (A—H, except D) were outlined by boxes. The positions of functional residues are indicated by: α1β1 interfaces (1), α1β2 interfaces (2), heme binding (h), and Bohr effect residues (b). Triangles indicate changes at amino acids sites. Deduced amino acid sequences were obtained from GenBank/EMBL/UniprotKB, the accession numbers of which were dwelled on Fig. 3
![]()
图 2 翘嘴鲌Hbb1和Hbb2氨基酸序列与哺乳类、爬行类、两栖类及其他硬骨鱼类比对分析
黑色阴影部分表示是相同的氨基酸残基。根据哺乳类的β球蛋白, 方框表示螺旋区域(A—H)。功能残基的位置分别表示为α1β1结合残基(1)、α1β2结合残基(2)和亚铁血红素结合残基(h)。三角形表示氨基酸替换位点。上述氨基酸序列从GenBank/EMBL/UniprotKB中获得的登录号详见图 4
Figure 2. Alignment of C. alburnus amino Hbb1 and Hbb2 acid sequences with mammalia, reptilia, amphibian and other teleosts
The black shade denotes the identical residues. According to mammalian β globins, the helical regions (A—H) were outlined by boxes. The positions of functional residues are indicated by: α1β1 interfaces (1), α1β2 interfaces (2), and heme binding (h). Triangles indicate changes at amino acids sites. Deduced amino acid sequences were obtained from GenBank/EMBL/UniprotKB, the accession numbers of which were dwelled on Fig. 4
![]()
图 3 翘嘴鲌和其他脊椎动物Hba1和Hba2蛋白系统进化树(▲所示为翘嘴鲌)
Figure 3. The phylogenetic tree of Hba1 and Hba2 from C. alburnus (▲) and other vertebrate species
![]()
图 4 翘嘴鲌和其他脊椎动物Hbb1和Hbb2蛋白系统进化树(▲所示为翘嘴鲌)
Figure 4. The phylogenetic tree of Hbb1 and Hbb2 from C. alburnus (▲) and other vertebrate species
表 1 Hba1、Hba2、Hbb1和Hbb2基因克隆所需引物序列
Table 1 The primers used for the cDNAs cloning of Hba1, Hba2, Hbb1 and Hbb2 genes
引物Primer 序列Sequence (5′—3′) Primers for partial fragment Hba1-F TCTGATAAGKACAMGGCTGTT Hba1-R ATGCCCAATGCAGGWTTAG Hba2-F GAACSACAGTGGWGAAGGGAG Hba2-R CTGACCTGGRCGAGGAACT Hbb1-F GGCAAGGGTGCTGATTSTA Hbb1-R CAGCCTGAAGTTGWCTGRATC Hbb2-F TGAGAAGAYCACCATCCAG Hbb2-R GTTAAGCAGTCAGCCWAAA Primers for 3′ RACE PCR 3′ GS-Hba1 01-F AGAATGCTGACCGTCTACCC 3′ GS-Hba1 11-F GGTCTGGTCCTGTGAAGAA 3′ GS-Hba2 01-F CATTGGGCAGACCTAAACC 3′ GS-Hba2 11-F ACGACAGTGGTGAAGGGAG 3′ GS-Hbb1 01-F TCCAGGGCAATCCCAAGGT 3′ GS-Hbb1 11-F ACTGTGCTAAATGCGTTGG 3′ GS-Hbb2 01-F CTCTACAACGCCGCTGCTA 3′ GS-Hbb2 11-F GGACAACATCAAAGCCACC 3′ RACE Outer TACCGTCGTTCCACTAGTGATTT 3′ RACE Inner CGCGGATCCTCCACTAGTGATTTCACTATAGG Primers for 5′ RACE PCR 5′ GS-Hba1 01-F CCCACAAGGTCGTCTATTTT 5′ GS-Hba1 11-F AAAGCATGAAGTTCGCTCA 5′ GS-Hba2 01-F CTGACCTGGGCGAGGAACT 5′ GS-Hba2 11-F CTCCCTTCACCACTGTCGTTCCA 5′ GS-Hbb1 01-F TTCCAACGCATTTAGCACA 5′ GS-Hbb1 11-F GTTGTGCCACCTTGGGATT 5′ GS-Hbb2 01-F CATAGGTGGCTTTGATGTTG 5′ GS-Hbb2 11-F GATAGCAGCGGCGTTGTAG 5′ RACE Outer CATGGCTACATGCTGACAGCCTA 5′ RACE Inner CGCGGATCCACAGCCTACTGATGATCAGTCGATG Note: Mixed bases: B-G/T/C; D-G/A/T; H-A/T/C; K-G/T; M-A/C; R-A/G; S-G/C; V-G/A/C; W-A/T; Y-C/T 
下载: 导出CSV
表 2 翘嘴鲌Hba1、Hba2、Hbb1和Hbb2基因的cDNA全长序列信息
Table 2 The information for full-length cDNA sequences of Hba1, Hba2, Hbb1 and Hbb2 from C. alburnus
基因Gence 长度Length (bp) 5′UTR (bp) 3′UTR (bp) ORF (bp) 氨基酸的数目
Number of amino acidsHba1 652 108 115 429 143 Hba2 465 21 15 429 143 Hbb1 477 21 15 441 147 Hbb2 475 18 16 441 147
下载: 导出CSV
表 3 翘嘴鲌Hba1、Hba2、Hbb1和Hbb2氨基酸序列与其他物种同源性(%)分析
Table 3 Identity analysis of C. alburnus Hba1, Hba2, Hbb1 and Hbb2 amino acid sequence with other species (%)
氨基酸Amino acid 斑马鱼Zebrafish 鲤Crap 非洲爪蟾Xenopus 大鼠Rat Hba1 93.0 68.5 44.8 51.0 Hba2 88.8 86.7 44.1 51.0 Hbb1 84.4 87.1 54.4 51.7 Hbb2 84.4 89.8 47.6 53.7 Note: GenBank/EMBL/UniprotKB databases accession numbers: Hba1 (Q90487, KTG39806.1, XP_018092516.1, NP_037228.1); Hba2 (NP_898889.2, KTF87195.1, NP_001081493.1, NP_001007723.1); Hbb1 (NP_001003431.1, XP_018973479.1, NP_001079742.1, NP_150237.1); Hbb2 (NP_001096600.1, XP_018929000.1, NP_001081497.1, NP_001106694.1). The order of accession numbers of each gene corresponds to zebrafish, crap, xenopus, and rat, respectively
下载: 导出CSV
-
[1] Rombough P J. 2 Respiratory gas exchange, aerobic metabolism, and effects of hypoxia during early life [J]. Fish Physiology, 1988(11): 59-161.
[2] Koch L G, Britton S L. Aerobic metabolism underlies complexity and capacity [J]. The Journal of Physiology, 2008, 586(1): 83-95. doi: 10.1113/jphysiol.2007.144709
[3] Jensen F B, Fago A, Weber R E, et al. 1-hemoglobin structure and function [J]. Fish Physiology, 1998(17): 1-40.
[4] Nikinmaa M. Haemoglobin function in vertebrates: evolutionary changes in cellular regulation in hypoxia [J]. Respiration Physiology, 2001, 128(3): 317-329. doi: 10.1016/S0034-5687(01)00309-7
[5] Ingermann R L. Vertebrate hemoglobins [J]. Comprehensive Physiology, 2011: 357-408.
[6] Unwin E E. On the Structure of the Respiratory Organs of the Terrestrial Isopoda [C]. Papers and Proceedings of the Royal Society of Tasmania. Hobart, Tasmania: Royal Society of Tasmania, 1931: 37-104.
[7] Szarski H. The structure of respiratory organs in relation to body size in amphibia [J]. Evolution, 1964, 18(1): 118-126. doi: 10.1111/j.1558-5646.1964.tb01576.x
[8] Graham J B. Air-Breathing Fishes: Evolution, Diversity, and Adaptation [M]. San Diego: Academic Press, 1997: 65-133.
[9] Damen W G, Saridaki T, Averof M, et al. Diverse adaptations of an ancestral gill: a common evolutionary origin for wings, breathing organs, and spinnerets [J]. Current Biology, 2002, 12(19): 1711-1716. doi: 10.1016/S0960-9822(02)01126-0
[10] De Souza P C, Bonilla-Rodriguez G O. Fish hemoglobins [J]. Brazilian Journal of Medical and Biological Research, 2007, 40(6): 769-778. doi: 10.1590/S0100-879X2007000600004
[11] Hardison R C. A brief history of hemoglobins: plant, animal, protist, and bacteria [J]. Proceedings of the National Academy of Sciences of the United States of America, 1996, 93(12): 5675-5679. doi: 10.1073/pnas.93.12.5675
[12] Riggs A. Hemoglobins: current opinion in structural biology [J]. Current Opinion in Structural Biology, 1991, 1(6): 915-921. doi: 10.1016/0959-440X(91)90086-9
[13] Verde C, Lecointre G, Prisco G D, et al. The phylogeny of polar fishes and the structure, function and molecular evolution of hemoglobin [J]. Polar Biology, 2007, 30(5): 523-539. doi: 10.1007/s00300-006-0217-3
[14] Giardina B, Mosca D, De Rosa M C, et al. The Bohr effect of haemoglobin in vertebrates: an example of molecular adaptation to different physiological requirements [J]. Acta Physiologica Scandinavica, 2004, 182(3): 229-244. doi: 10.1111/j.1365-201X.2004.01360.x
[15] Antonini E, Brunori M. Hemoglobin and Myoglobin in Their Reactions with Ligands [M]. Amsterdam: North-Holland, 1971: 27-31.
[16] Hoffmann F G, Opazo J C, Storz J F, et al. Gene cooption and convergent evolution of oxygen transport hemoglobins in jawed and jawless vertebrates [J]. Proceedings of the National Academy of Sciences of the United States of America, 2010, 107(32): 14274-14279. doi: 10.1073/pnas.1006756107
[17] Storz J F, Opazo J C, Hoffmann F G, et al. Gene duplication, genome duplication, and the functional diversification of vertebrate globins [J]. Molecular Phylogenetics and Evolution, 2013, 66(2): 469-478. doi: 10.1016/j.ympev.2012.07.013
[18] Sato Y, Nishida M. Teleost fish with specific genome duplication as unique models of vertebrate evolution [J]. Environmental Biology of Fishes, 2010, 88(2): 169-188. doi: 10.1007/s10641-010-9628-7
[19] Kasahara M, Naruse K, Sasaki S, et al. The medaka draft genome and insights into vertebrate genome evolution [J]. Nature, 2007, 447(7145): 714-719. doi: 10.1038/nature05846
[20] Opazo J C, Butts G T, Nery M F, et al. Whole-genome duplication and the functional diversification of teleost fish hemoglobins [J]. Molecular Biology and Evolution, 2013, 30(1): 140-153. doi: 10.1093/molbev/mss212
[21] 张小谷, 熊邦喜. 翘嘴鲌的生物学特性及养殖前景 [J]. 河北渔业, 2005(1): 27-36. doi: 10.3969/j.issn.1004-6755.2005.01.012 Zhang X G, Xiong B X. The biological characteristics and breeding prospects of Culter alburnus [J]. Hebei Fisheries, 2005(1): 27-36. doi: 10.3969/j.issn.1004-6755.2005.01.012
[22] 朱华平, 黄樟翰, 谢刚, 等. 翘嘴红鲌鱼苗耗氧率和窒息点的初步研究 [J]. 水利渔业, 2003, 23(4): 5. Zhu H P, Huang Z H, Xie G, et al. The preliminary research on the oxygen consumption rate and the asphyxiant point of Erythroculter ilishaeformis fry [J]. Journal of Hydroecology, 2003, 23(4): 5.
[23] 陈松波, 范兆廷, 陈伟兴. 温度对鲤鱼窒息点的影响 [J]. 东北农业大学学报, 2007, 38(6): 801-804. doi: 10.3969/j.issn.1005-9369.2007.06.020 Chen S B, Fan Z T, Chen W X. The effect of temperature on the asphyxiant point of carp [J]. Journal of Northeast Agricultural University, 2007, 38(6): 801-804. doi: 10.3969/j.issn.1005-9369.2007.06.020
[24] 黄玉瑶. 鲤鱼, 白鲢, 鳊鱼的鱼苗, 鱼种耗氧量的研究 [J]. 动物学报, 1975, 21(1): 78-88. Huang Y Y. Oxygen consumption of the early developmental stages of the red and the common carp (Cyprinus carpio), the silver carp (Hypophthalmichthys molitrix) and the Chinese bream (Parabramis pekinensis) [J]. Current Zoology, 1975, 21(1): 78-88.
[25] 张中英, 胡玫, 吴福煌. 尼罗罗非鱼耗氧率的初步测定 [J]. 水产学报, 1982, 6(4): 369-377. Zhang Z Y, Hu M, Wu F H. Preliminary study on the oxygen consumption of Tilapia nilotica [J]. Journal of Fisheries of China, 1982, 6(4): 369-377.
[26] 杜启艳, 常重杰, 陈颖. 3种鲤科鱼β珠蛋白基因的比较研究 [J]. 水生生物学报, 2005, 29(1): 86-90. doi: 10.3321/j.issn:1000-3207.2005.01.016 Du Q Y, Chang Z J, Chen Y. Comparison of β globin gene in three fishes of cyprinoid [J]. Acta Hydrobiologicica Sinica, 2005, 29(1): 86-90. doi: 10.3321/j.issn:1000-3207.2005.01.016
[27] Chan F, Robinson J, Brownlie A, et al. Characterization of adult α- and β-globin genes in the zebrafish [J]. Blood, 1997, 89(2): 688-700. doi: 10.1182/blood.V89.2.688
[28] 邵占涛, 丛潇, 袁金铎, 等. 鲈α-珠蛋白基因的克隆和序列分析 [J]. 水产科学, 2009, 28(5): 268-271. doi: 10.3969/j.issn.1003-1111.2009.05.007 Shao Z T, Cong X, Yuan J D, et al. Cloning and sequence analysis of α-type globin cDNA from Japanese sea bass (Lateolabrax japonicus) [J]. Fisheries Science, 2009, 28(5): 268-271. doi: 10.3969/j.issn.1003-1111.2009.05.007
[29] Xia M Z, Chao Y, Jia J L, et al. Changes of hemoglobin expression in response to hypoxia in a Tibetan schizothoracine fish, Schizopygopsis pylzovi [J]. Journal of Comparative Physiology B-biochemical Systemic and Environmental Physiology, 2016, 186(8): 1033-1043. doi: 10.1007/s00360-016-1013-1
[30] Wang Z S, Qi Z T, Tian J Y, et al. Cloning of hemoglobin-α1 from half-smooth tongue sole (Cynoglossus semilaevis) and its expression under short-term hypoxia [J]. Zoological Research, 2011, 32(6): 641-646.
[31] 褚武英, 于涟, 毛芝娟, 等. 大黃鱼α-珠蛋白cDNA的克隆及其特性分析 [J]. 科技通报, 2005, 21(6): 674-678. Chu W Y, Yu L, Mao Z J, et al. Molecular cloning and characterisation of α-Globin cDNA from large yellow croaker, Pseudosciaena crocea [J]. Bulletin of Science and Technology, 2005, 21(6): 674-678.
[32] Perutz M F. Stereochemistry of cooperative effects in haemoglobin: haem-haem interaction and the problem of allostery [J]. Nature, 1970, 228(5273): 726-734. doi: 10.1038/228726a0
[33] Lawn R M, Efstratiadis A, Oconnell C, et al. The nucleotide sequence of the human β-globin gene [J]. Cell, 1980, 21(3): 647-651. doi: 10.1016/0092-8674(80)90428-6
[34] Miyata M, Aoki T. Head-to-head linkage of carp α- and β-globin genes [J]. Biochimica et Biophysica Acta, 1997, 1354(2): 127-133. doi: 10.1016/S0167-4781(97)00111-5
[35] Okamoto K, Sakai M, Miyata M, et al. Molecular cloning and sequence analysis of α- and β-globin cDNAs from yellowtail, Seriola quinqueradiata [J]. Comparative Biochemistry and Physiology B, 2001, 130(2): 207-216.
[36] Giordano D, Vergara A, Lee H C, et al. Hemoglobin structure/function and globin-gene evolution in the Arctic fish Liparis tunicatus [J]. Gene, 2007, 406(1-2): 58-68. doi: 10.1016/j.gene.2007.06.002
[37] Ohno S, Wolf U, Atkin N B, et al. Evolution from fish to mammals by gene duplication [J]. Hereditas, 2009, 59(1): 169-187. doi: 10.1111/j.1601-5223.1968.tb02169.x
[38] Hoffmann F G, Opazo J C, Storz J F. Rapid rates of lineage-specific gene duplication and deletion in the a-globin gene family [J]. Molecular Biology and Evolution, 2008, 25(3): 591-602. doi: 10.1093/molbev/msn004
[39] Hoffmann F G, Opazo J C, Storz J F. Whole-genome duplications spurred the functional diversification of the globin gene superfamily in vertebrates [J]. Molecular Biology and Evolution, 2012, 29(1): 303-312. doi: 10.1093/molbev/msr207
[40] 范能全, 张璞, 李丹, 等. 两种药物对斑马鱼呼吸代谢速率的影响 [J]. 中国药师, 2018, 21(2): 208-210. doi: 10.3969/j.issn.1008-049X.2018.02.005 Fan N Q, Zhang P, Li D, et al. Effects of two drugs on the respiration of zebrafish [J]. China Pharmacist, 2018, 21(2): 208-210. doi: 10.3969/j.issn.1008-049X.2018.02.005
计量
- 文章访问数: 2258
- HTML全文浏览量: 776
- PDF下载量: 127