IDENTIFICATION AND EVOLUTION ANALYSIS OF TUBULIN SUPERFAMILY GENES IN EUPLOTES OCTOCARINATUS
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摘要: 为了系统分析八肋游仆虫(Euplotes octocarinatus)微管蛋白基因家族, 从八肋游仆虫大核基因组中共鉴定得到20个微管蛋白基因, 基于同源比对及系统进化分析, 将其归入α、β、γ、δ、ε及η六个微管蛋白亚家族; 多序列比对及Western blot结果显示八肋游仆虫η微管蛋白基因在翻译过程中需发生一次+1位编程性核糖体移码, 其移码位点为AAA-TAA; 所有自由生纤毛虫都含有多个α和β微管蛋白基因亚型, 可能用于组成不同的微管结构。研究为后续深入探讨八肋游仆虫微管蛋白的生物学功能及微管多样性奠定了基础。Abstract: Microtubules represent one of the major cytoskeletal filament systems of all eukaryotic cells. They play a key role in spatial arrangement of the organelles, intracellular transport, nuclear and cell division, and ciliar motility. Ciliates are ideal model organisms for studying the functional diversity of tubulins. Here, a total of 20 tubulin genes were identified in the macronuclear genome of the ciliate Euplotes octocarinatus. Based on ortholog comparisons and phylogenetic analysis, these genes were clustered into six groups: α-, β-, γ-, δ-, ε- and η-tubulins. Sequence analysis and western blots suggested that the η-tubulin gene of E. octocarinatus required a +1 programmed ribosomal frameshifting to produce complete protein product. The slippery sequence is AAATAAT. We further systematically identified tubulin genes from 9 other ciliates and compared them with E. octocarinatus. The α- and β-tubulins of all investigated free-living ciliates are encoded by multigene families, and each tubulin isotype may be used to form distinct tubulin structures. Phylogenetic analysis showed that α- and β-tubulin genes underwent multiple independent duplications and losses in ciliate. Our study lays a foundation for studying the biological function of tubulins and exploring the mechanisms of microtubule diversity.
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Keywords:
- Euplotes octocarinatus /
- Tubulin /
- Sequence analysis /
- Phylogenetic analysis
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图 1 八肋游仆虫微管蛋白基因家族序列分析
用八肋游仆虫(Eo, 粗体)、嗜热四膜虫(Tt)及第四双小核草履虫(Pt)的微管蛋白构建的最大似然树, 大肠杆菌的FtsZ蛋白作为外类群。自展值以百分比的形式列于每一个节点
Figure 1. Sequence analysis of the tubulin gene families from Euplotes octocarinatus
The maximum likelihood phylogenetic tree based on the deduced amino acid sequences of tubulin genes in Euplotes octocarinatus (Eo, in bold), Tetrahymena thermophila (Tt) and Paramecium tetraurelia (Pt). The FtsZ protein of Escherichia coli (Ec) was used as outgroup. Bootstrap values are displayed as percentages at each tree node
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图 2 八肋游仆虫微管蛋白基因微染色体的结构示意图
椭圆代表微染色体的端粒, 黑色方框表示外显子, 内含子的长度标在上方
Figure 2. Schematic representation of the Eo-tubulin nanochromosomes structure
The ellipses indicated the telomeres of the nanochromosomes. The black rectangles represented the exon. The length of the intron was listed above the line
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图 3 八肋游仆虫η微管蛋白基因的表达需要编程性核糖体移码
A. 移码区域的详细分析。标出了Eoη-tub基因的起始密码子和终止密码子的位置, 不同起始密码子和终止密码子组合相对应的三个开放阅读框。假定的滑动序列AAAUAAU用浅色字体表示, 理论上0读框和+1读框翻译的氨基酸序列分别列于mRNA序列的上方和下方, ORF表示开放阅读框(Open reading frame); B. 八肋游仆虫η微管蛋白移码位点附近的氨基酸序列与Paramecium tetraurelia (Pt)、Euplotes focardii (Ef) 及Euplotes crassus (Ec)同源蛋白的比对, 推测的移码位点用黑色圆点表示; C. 利用制备的Eoη-tub多克隆抗体进行western blot分析, 泳道1和2分别表示用和不用抗原多肽孵育得到的结果。箭头所指为Eoη-tub的位置, Eoα-tub作为内参, Mr代表蛋白Marker
Figure 3. Programmed ribosomal frameshifting is likely required for expression of gene encoding η-tubulin in Euplotes octocarinatus
A. Close-up of the frameshift region. Relative positions of the multiple start and stop codons in the Eoη-tub gene sequence were shown. The putative slippery sequence AAAUAAU motif is shown in light type. Conceptual translations in the 0 reading frame and +1 reading frame are aligned above and below the mRNA sequence, respectively. ORF, open reading frame. B. The parts of the putative frameshift site of the η-tubulin of E. octocarinatus (Eo) are aligned with the respective homologous proteins from P. tetraurelia (Pt), E. focardii (Ef) and E. crassus (Ec). The putative location of the frameshift is marked by black dot. C. Western blot of total cell lysates probed with Eoη-tub antibody in absence, or presence of the relative antigenic peptide (lanes 1 and 2, respectively). The Eoη-tub recognition is indicated by arrowhead. Eoα-tub is used as the internal control, Mr represents protein Marker
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图 4 八肋游仆虫与其他纤毛虫中微管蛋白基因的比较
系统发育树是利用不同物种的18S rRNA序列采用最大似然法构建的, 以人作为外群。自展值以百分比的形式列于每一个节点, 每个微管蛋白亚家族的基因个数列在右边
Figure 4. Comparison of tubulin genes in Euplotes octocarinatus and other ciliates
Phylogenetic tree was constructed using the maximum likelihood method based on the 18S rRNA sequences. Homo sapiens is an outgroup. Bootstrap values are displayed as percentages at each tree node. The gene number of each tubulin subfamily was indicated on the right
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图 5 纤毛虫α及β微管蛋白亚家族系统发育分析
基于不同纤毛虫α(A)及β(B)微管蛋白亚家族序列构建的最大似然树, 大肠杆菌的FtsZ蛋白作为外类群, 自展值以百分比的形式列于每一个节点。Pt. 第四双小核草履虫; Tt. 嗜热四膜虫; Pp. 水滴伪康纤虫; Im. 多子小瓜虫; Sc. 天蓝喇叭虫; Ot. 三棱尖毛虫; Sl. 浮萍棘尾虫; Ec. 厚游仆虫; Ef. 嗜冷游仆虫; Eo. 八肋游仆虫
Figure 5. Phylogenetic analysis of α and β tubulin subfamilies in ciliates
Phylogenetic tree was constructed using the maximum likelihood method based on the deduced amino acid sequences of α tubulin subfamilies sequences (A) and β tubulin subfamilies sequences (B) from different ciliates. The FtsZ protein of Escherichia coli was used as outgroup. Bootstrap values are displayed as percentages at each tree node. Pt. Paramecium tetraurelia; Tt. Tetrahymena thermophile; Pp. Pseudocohnilembus persalinus; Im. Ichthyophthirius multifiliis; Sc. Stentor coeruleus; Ot. Oxytricha trifallax; Sl. Stylonychia lemnae; Ec. Euplotes crassus; Eh. Euplotes harpa; Eo. Euplotes octocarinatus
表 1 八肋游仆虫微管蛋白基因的分子特征
Table 1 Molecular characteristics of the tubulin genes in Euplotes octocarinatus
基因/亚家族
Gene/Subfamily基因组ID
Genome ID内含子个数Introns number 5′前导序列长度
5′ leader (bp)3′尾端序列长度
3′ tailer (bp)RPKM值RPKM value 氨基酸长度Length (aa) 分子量Molecular weight (kD) α-tubulin Eoα-tub Contig18065 0 78 99 2601.66 450 49.62 Eoα-tub-like-1 Contig28852 0 51 71 0.22 445 50.73 Eoα-tub-like-2 Contig27230 0 40 31 1.91 483 55.97 Eoα-tub-like-3 Contig3451 0 57 33 1.54 459 51.62 Eoα-tub-like-4 Contig27158 0 38 46 / 456 51.56 Eoα-tub-like-5 Contig26869 0 38 57 / 457 52.34 β-tubulin Eoβ-tub1 Contig5259 0 50 127 231.98 444 49.63 Eoβ-tub2 Contig5984 0 54 51 1.15 444 49.60 Eoβ-tub3 Contig8213 0 54 143 168.77 443 49.65 Eoβ-tub4 Contig4144198 0 55 143 131.57 443 49.60 Eoβ-tub-like-1 Contig18362 2 33 44 2.19 435 49.87 Eoβ-tub-like-2 Contig25579 5 31 38 1.69 454 51.88 Eoβ-tub-like-3 Contig29123 4 35 101 1.71 444 50.90 Eoβ-tub-like-4 Contig6445 0 79 79 169.66 467 52.44 Eoβ-tub-like-5 Contig4144404 1 41 38 3.42 433 48.93 Eoβ-tub-like-6 Contig9575 0 39 41 1.08 505 58.35 γ-tubulin Eoγ-tub Contig11092 2 49 82 18.528 461 52.04 δ-tubulin Eoδ-tub Contig24288 1 44 13 1.03 423 48.54 ε-tubulin Eoε-tub Contig17307 2 80 36 3.96 499 56.51 η-tubulin Eoη-tub Contig8731 2 34 51 5.89 410 46.66 
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