西藏山溪鲵转录组组装与分析

doi:10.14012/j.cnki.fjsc.2023.03.001

摘要/Abstract

摘要：

目前有关小鲵属物种的基因组信息和转录组序列报道尚少。本研究利用Illumina HiSeq高通量测序技术对西藏山溪鲵(Batrachuperus tibetanus)进行了转录组测序与数据分析,再经拼接和组装,最终获得了36 252条unigenes,序列的平均长度为937 bp,N50为1 549 bp。此外,对unigenes进行GC含量、长度分布和基因表达量等分析,结果显示测序数据质量较好。使用Swiss-prot、Nr、Pfam、KEGG、KOG和GO数据库注释西藏山溪鲵转录组unigenes,分别有16 465、18 749、13 983、10 607、15 704和14 242条unigenes 获得注释。根据注释结果,共检测出17 472条CDS,鉴定出2 779个SSR标记和3 100个SNP 位点。KEGG通路分析结果表明:获得注释的10 607条unigenes又被划分到257个代谢通路中,其中涉及信号转导通路的unigenes比例最大。本研究通过对西藏山溪鲵进行转录组测序,得到的大量转录本信息,为进一步了解西藏山溪鲵遗传多样性分析、分子标记开发、种群资源评价与保护和功能基因的克隆等研究提供了丰富的数据资源。

关键词: 西藏山溪鲵, 转录组, unigenes, 基因注释

Abstract:

The genomic information and transcriptome sequences of Batrachuperus tibetanus are rarely reported at present.In this study,the transcripts of this species were sequenced and assembled using Illumina HiSeq platform.A total of 36 252 unigenes were obtained through sequencing and assembling,with an average length of 937 bp and a N50 of 1 549 bp.Additionally,GC content,length distribution and expression level of unigenes were assessed.The result showed that the sequencing data was of high quality.Blasted against the Swiss-prot,Nr,Pfam,KEGG,KOG and GO,a total of 16 465,18 749,13 983,10 607,15 704 and 14 242 unigenes were annotated,respectively.According to the functional annotation results,17 472 coding sequence (CDS) were detected.A total of 2 779 microsatellite markers (SSR) and 3 100 single nucleotide polymorphisms (SNP) were identified.KEGG pathway analysis showed that 10 607 unigenes were annotated,and it was divided into 257 categories according to the different pathways.Among all the pathways,the number of unigenes involved in signal transduction pathway exhibited the largest proportion.In this study,the obtained transcriptome sequences provided rich data resources for further understanding of genetic diversity analysis,molecular marker development,population resource evaluation and conservation,as well as functional gene cloning of the B.tibetanus.

Key words: Batrachuperus tibetanus, transcriptome, unigenes, gene annotation

中图分类号:

S937

朱文文, 郁川, 熊建利, 黄勇. 西藏山溪鲵转录组组装与分析[J]. 渔业研究, 2023, 45(3): 213-221.

ZHU Wenwen, YU Chuan, XIONG Jianli, HUANG Yong. Transcriptome data assembly and analysis of Batrachuperus tibetanus[J]. Journal of Fisheries Research, 2023, 45(3): 213-221.

图/表 9

表1 测序组装的序列结果统计

Tab.1 Assembly result of sequences

索引 Index	总数 All number	中等GC 含量/% Median GC	平均GC 含量/% Mean GC	最小长度/bp Minor length	中等长度/bp Median length	平均长度/bp Mean length	最大长度/bp Max length	总碱基数/bp Total bases	N50 长度/bp N50 length
Transcripts	43 626	45.60	46.23	201	605	1 061	20 766	46 293 115	1 822
Unigenes	36 252	45.40	46.09	201	545	937	20 766	33 976 485	1 549

表2 SNP位点的分类

Tab.2 Type of SNP identified sequences

SNP类型 SNP type	数量/个 Number of SNPs	百分比/% Percentage
A-G	965	31.13
C-T	862	27.81
A-C	414	13.35
A-T	279	9.00
C-G	213	6.87
G-T	367	11.84
总计Total	3 100	100

表3 SSR位点统计

Tab.3 SSR locus statistics

类别Category	数量/个Number
单核苷酸重复SSR位点数 Mono-nucleotide	1 473
二核苷酸重复SSR位点数 Di-nucleotide	715
三核苷酸重复SSR位点数 Tri-nucleotide	490
四核苷酸重复SSR位点数 Tetra-nucleotide	56
五核苷酸重复SSR位点数 Penta-nucleotide	38
六核苷酸重复SSR位点数 Hexa-nucleotide	7

表4 FPKM值密度分布

Tab.4 FPKM value density distribution

FPKM值 FPKM value	基因数目 Gene number	百分比/% Percentage
0~0.1	0	0
0.1~0.3	16	0.04
0.3~3.57	13 868	38.28
3.57~15	17 099	47.20
15~60	4 000	11.04
>60	1 243	3.43

表5 序列功能注释

Tab.5 Functional annotation of sequences

数据库 Database	注释unigenes的数目 Annotated number of unigenes	百分比/% Percentage
Swiss-prot	16 465	45.42
Nr	18 749	51.72
Pfam	13 983	38.57
KEGG	10 607	29.26
KOG	15 704	43.32
GO	14 242	39.29
总计Total	36 252	100

图1 物种分布

Fig.1 Species distribution

图2 Unigenes的 KOG注释注:A.RNA 加工和修饰;B.染色质结构与动力学;C.能量产生和转换;D.细胞周期调控、细胞分裂、染色体;E.氨基酸运输和代谢;F.核苷酸运输和代谢;G.碳水化合物的运输和代谢;H.辅酶运输和代谢;I.脂质运输和代谢;J.翻译、核糖体结构和生物合成;K.转录;L.复制、重建和修复;M.细胞壁/细胞膜/膜结构的生物合成;N.细胞运动;O.翻译后修饰、蛋白质周转、伴侣;P.无机离子转运与代谢;Q.次生代谢产物的合成、转运和代谢;R.普通功能预测;S.未知功能;T.信号转导机制;U.胞内运输、分泌和囊泡运输;V.防御机制;W.胞外结构;Y.核结构;Z.细胞骨架。

Fig.2 KOG functional annotation of unigenes Notes:A.RNA processing and modification;B.Chromatin structure and impetus;C.Energy production and conversion;D.Cell cycle control,cell division,chromosome partitioning;E.Amino acid transfer and metabolism;F.Nucleotide transfer and metabolism;G.Carbohydrate transfer and metabolism;H.Coenzyme transfer and metabolism;I.Lipid transfer and metabolism;J.Translation,ribosomal structure and biosynthesis;K.Transcription;L.Replication,recombination and repair;M.Cell wall/membrane/envelope biosynthesis;N.Cell motility;O.Post-translational modification,protein turnover,chaperones;P.Inorganic ion transfer and metabolism;Q.Secondary metabolites biosynthesis,transfer and catabolism;R.General function prediction;S.Function unknown;T.Signal transduction mechanisms;U.Intracellular transport,secretion,and vesicular transport;V.Defense mechanisms;W.Extracellular structures;Y.Nuclear structure;Z.Cytoskeleton.

图3 Unigene GO功能注释注:1.转录、DNA依赖性;2.转录调控、DNA依赖性;3.蛋白质转运;4.多细胞器官发育;5.细胞分化;6.凋亡;7.细胞黏附;8.蛋白质水解;9.信号转导;10.细胞分化;11.细胞周期;12.有丝分裂;13.小G蛋白介导信号转导;14.DNA 修复;15.转运; 16.RNA加工;17.翻译;18.转录正调控;19.胞内信号转导;20.染色质修饰;21.转录负调控;22.细胞内蛋白质转运;23.跨膜输送;24.精子形成;25.RNA剪切;26.细胞核;27.必需膜;28.细胞浆;29.胞液;30.细胞质膜;31.内质网膜;32.线粒体;33 胞外区;34.胞核;35.核质;36.细胞骨架;37.高尔基体膜;38.微管;39.高尔基氏复合体;40.细胞膜;41.ATP结合;42.锌离子结合;43.蛋白质结合;44.DNA 结合;45.金属离子结合;46.RNA结合;47.钙离子结合;48.结合;49.特异序列DNA结合;50.蛋白丝氨酸/苏氨酸激酶活性。

Fig.3 GO annotation of unigenes Notes:1.Transcription,DNA-dependent;2.Regulation of transcription,DNA-dependent;3.Protein transport;4.Multicellular organ development;5.Cell division;6.Apoptosis;7.Cell adhesion;8.Proteolysis;9.Signal transduction;10.Cell division;11.Cell cycle;12.Mitosis;13.Small GTPase mediated signal transduction;14.DNA repair;15.Transport;16.RNA procession;17.Translation;18.Positive regulation of transcription;19.Intracellular signal transduction;20.Chromatin modification;21.Negative regulation of transcription;22.Intracellular protein transport;23.Across the membrane transport;24.Spermatogenesis;25.RNA splicing;26.Nucleus;27.Integral to membrane;28.Cytoplasm;29.Cytosol;30.Plasma membrane;31.Endoplasmic reticulum membrane;32.Mitochondrion;33.Extracellular region;34.Nucleus;35.Nucleoplasm;36.Cytoskeleton;37.Golgi membrane;38.Microtubules;39.Golgi complex;40.Cell membrane;41.ATP binding;42.Zinc ion binding;43.Protein binding;44.DNA binding;45.Metal ion binding;46.RNA binding;47.Calcium ion binding;48.Binding;49.Specific DNA sequence binding;50.Protein serine/threonine-specific kinase activity.

图4 Unigenes的 KEGG注释注:1.排泄系统;2.感觉系统;3.神经系统;4.环境适应;5.发育;6.循环系统;7.免疫系统;8.内分泌系统;9.糖生物合成与代谢;10.其他氨基酸代谢;11.外源物质的生物降解与代谢;12.萜类化合物与多酮类化合物的代谢;13.氨基酸代谢;14.其他次生代谢产物的生物合成;15.核苷酸代谢;16.能量代谢;17.辅因子和维生素的代谢;18.脂类代谢;19.碳水化合物代谢;20.复制与修复;21.转录;22.折叠、分选和降解;23.翻译;24.信号分子与相互作用;25.信号转导;26.膜转运;27.细胞迁移;28.细胞通讯;29.运输与代谢;30.细胞生长与死亡。

Fig.4 KEGG annotation of unigenes Notes:1.Excretory system;2.Sensory system;3.Nervous system;4.Environmental adaptation;5.Development;6.Circulatory system;7.Immune system;8.Endocrine system;9.Glycan biosynthesis and metabolism;10.Metabolism of other amino acids;11.Xenobiotics biodegradation and metabolism;12.Metabolism of terpenoids and polyketides;13.Amino acid metabolism;14.Biosynthesis of other secondary metabolites;15.Nucleotide metabolism;16.Energy metabolism;17.Metabolism of cofactors and vitamins;18.Lipid metabolism;19.Carbohydrate metabolism;20.Replication and repair;21.Transcription;22.Folding,sorting and degradation;23.Translation;24.Signaling molecules and interaction;25.Signal transduction;26.Membrane transport;27.Cell motility;28.Cell communication;29.Transport and catabolism;30.Cell growth and death.

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