扁茎黄芪转录组测序及生物信息学分析

doi:10.7668/hbnxb.20192406

摘要/Abstract

摘要：

为获得扁茎黄芪转录组信息及功能基因表达特征,以扁茎黄芪的幼苗叶片为材料,利用Illumina HiSeq平台进行转录组测序及生物信息学系统分析。结果显示,共获得扁茎黄芪Unigene 19 280条,总长23 472 470 bp,其中GC含量达42.74%,测序质量和组装效果较好。通过Blast分析后,分别有12 541,10 120,9 412,8 953,7 494,5 052条Unigene在Nr、Swiss-Prot、GO、KEGG、KOG、COG数据库获得注释。注释到的扁茎黄芪Unigene同源物种以豆科植物为主,尤其是与其同属蝶形花亚科的鹰嘴豆、蒺藜苜蓿和相思子匹配度较高,分别占31.49%,14.50%,11.65%;扁茎黄芪注释Unigene涉及3个GO类别,354条KEGG代谢通路和25个KOG功能分类,其中富集最多的类别分别为代谢过程、一般功能预测和嘌呤代谢,说明扁茎黄芪幼苗期的叶片细胞具有活跃的新陈代谢活动,基因表达丰富。另外,扁茎黄芪Unigene在KEGG数据库中的感染性疾病类别获得较多注释,表明其植株部位也可能具有药用价值。利用MISA软件挖掘到5 849个SSR位点,SSR出现频率30.34%。扁茎黄芪基因组内SSR位点丰富、类型多样化,单碱基至六碱基重复全部出现,其中单碱基丰度最高,占40.56%,以A/T、AG/CT和AAG/CTT为优势基元。

关键词: 扁茎黄芪, 转录组, 生物信息学, Unigene, 分子标记

Abstract:

In order to obtain transcriptome information and functional genes expression characteristics of Astragalus complanatus,the leaves of A.complanatus were used as material,the high-throughput transcriptome sequencing and bioinformatics analysis were performed using Illumina HiSeq platform.A total of 19 280 Unigenes were assembled with total sequence length of 23 472 470 bp,of which the GC content was 42.74%,indicating a high quality of sequencing and assembly.Blast analysis showed 12 541,10 120,9 412,8 953,7 494,5 052 Unigenes got annotation in Nr,Swiss-Prot,GO,KEGG,KOG and COG databases,respectively.The annotated Unigenes of A.complanatus were mainly homologuous to leguminous plants,in particular,highly matched to the subfamily Pteriformis plant Cicer arietinum,Medicago truncatula and Abrus precatorius with a respective percentage of 31.49%,14.50% and 11.65%.Annotated Unigenes were divided into 3 GO classifications,354 KEGG metabolic pathways and 25 KOG function categories.The most abundant categories in each database were metabolic process,general function prediction only and purine metabolism,respectively.These results showed that the leaf cells of Astragalus membranaceus had active metabolism and abundant gene expression in the seedling stage.Additionally,a lot of Unigenes were annotated to the infectious disease category in KEGG database,suggesting a potential medicinal value of the plant part of Astragalus membranaceus.A total of 5 849 simple sequence repeats(SSRs)were identified by MISA software,with an occurrence frequency of 30.34%.The SSRs in the genome of Astragalus membranaceus were abundant and diversified,containing single base to six base repeats,among which the mono-nucleotide SSRs had the largest number with a frequency of 40.56%.The main types of SSR motifs involved A/T,AG/CT and AAG/CTT.

Key words: Astragalus complanatus, Transcriptome, Bioinformatics, Unigene, Molecular marker

武悦, 陈阳, 王星哲, 单飞彪, 张勇, 孙鸿举. 扁茎黄芪转录组测序及生物信息学分析[J]. 华北农学报, 2022, 37(1): 42-49. doi: 10.7668/hbnxb.20192406.

WU Yue, CHEN Yang, WANG Xingzhe, SHAN Feibiao, ZHANG Yong, SUN Hongju. Transcriptome Sequencing and Bioinformatics Analysis of Astragalus complanatus[J]. Acta Agriculturae Boreali-Sinica, 2022, 37(1): 42-49. doi: 10.7668/hbnxb.20192406.

http://www.hbnxb.net/CN/Y2022/V37/I1/42

图/表 10

表1 测序数据质量分析

Tab.1 Quality analysis of sequencing data

高质量序列 Clean reads	高质量碱基/bp Clean bases	GC含量/% GC content	Q20碱基数占比/%	Q30碱基数占比/%
33 217 041	9 965 112 300	42.74	98.03	93.89

表2 扁茎黄芪转录本和单基因簇统计分析

Tab.2 The statistics analysis of Astragalus complanatus transcript and Unigene

类别 Categories	转录本 Transcript	单基因簇 Unigene
序列总数目	21 565	19 280
Total sequence number
序列总长度/bp	27 333 335	23 472 470
Total sequence length
最大长度/bp	15 713	15 713
Maximum length
最小长度/bp	201	201
Minimum length
平均长度/bp	1 267	1 217
Average length
GC含量/%	40.37	40.38
GC content
N50/bp	2 079	2 094

图1 扁茎黄芪Unigene的注释统计

Fig.1 The statistics analysis of annotated Unigene in Astragalus complanatus

图2 扁茎黄芪Unigene的Nr数据库匹配物种分布

Fig.2 Matched species distribution of Astragalus complanatus Unigene in Nr database

图3 扁茎黄芪Unigene的GO数据库功能分类
1.胞外区;2.细胞;3.类核;4.膜;5.病毒体;6.细胞连接;7.膜封闭腔;8.含蛋白复合物;9.细胞器;10.胞外区部分;11.细胞器部分;12.病毒部分;13.膜部分;14.细胞部分;15.超分子复合体;16.催化活性;17.结构分子活性;18.转运蛋白活性;19.绑定;20.抗氧化活性;21.蛋白标签;22.运货受体活性;23.蛋白质折叠伴侣;24.翻译调节活性;25.营养储存活性;26.分子转导活性;27.分子功能调节剂;28.分子载体活性;29.传译调节活性;30.小分子传感器活性;31.繁殖;32.细胞杀伤;33.免疫过程;34.代谢过程;35.细胞群增殖;36.细胞进程;37.碳利用;38.氮利用;39.生殖过程;40.生物黏附;41.信号传导;42.多细胞生物进程;43.发育进程;44.生长;45.转运;46.节律进程;47.应激反应;48.定位;49.多有机体进程;50.生物调控;51.细胞组分及来源;52.解毒。

Fig.3 Functional classification of Astragalus complanatus Unigene in GO database
1.Extracellular region;2.Cell;3.Nucleoid;4.Membrane;5.Virion;6.Cell junction;7.Membrane-enclosed lunen;8.Protein-containing complex;9.Organelle;10.Extracellular region part;11.Organelle part;12.Virion part;13.Membrane part;14.Cell part;15.Supramolecular complex;16.Catalytic activity;17.Structural molecule activity;18.Transporter activity;19.Binding;20.Antioxidant activity;21.Protein tag;22.Cargo receptor activity;23.Protein folding chaperone;24.Translation regulator actiivty;25.Nutrient reservoir activity;26.Molecular transducer activity;27.Molecular function regulator;28.Molecular carrier activity;29.Transciption regulator activity;30.Small molecule sensor activity;31.Reproduction;32.Cell killing;33.Immune system process;34.Metabolic process;35.Cell population proliferation;36.Cellular process;37.Carbon utilization;38.Nitrogen utilization;39.Reproductive process;40.Biological adhesion;41.Signaling;42.Multicellular organismal process;43.Developmental process;44.Growth;45.Locomotion;46.Rhythmic process;47.Response to stimulus;48.Localization;49.Multi-organism process;50.Biological regulation;51.Cellular component organization or biogenesis;52.Detoxification.

表3 扁茎黄芪Unigene的KOG数据库功能分类

Tab.3 KOG function categories of Astragalus complanatus Unigene

功能名称 Function name	注释Unigene数目 Number of annotated Unigene
一般功能预测 General function prediction only	1 551
翻译后修饰、蛋白转运 Posttranslational modification, protein turnover, chaperones	926
信号传导机制 Signal transduction mechanisms	683
翻译、核糖体结构和生物起源 Translation, ribosomal structure and biogenesis	498
未知功能 Unknown function	491
碳水化合物转运和代谢 Carbohydrate transport and metabolism	459
转录 Transcription	447
细胞内运输、分泌和囊泡运输 Intracellular trafficking, secretion, and vesicular transport	433
能量生产及转换 Energy production and conversion	363
RNA加工和修饰 RNA processing and modification	358
氨基酸转运和代谢 Amino acid transport and metabolism	342
脂类转运和代谢 Lipid transport and metabolism	304
次生代谢产物的合成、转运和代谢 Secondary metabolites biosynthesis, transport and catabolism	236
无机离子的转运和代谢 Inorganic ion transport and metabolism	210
细胞周期调控、细胞分裂、染色体分离 Cell cycle control, cell division, chromosome partitioning	176
复制、重组和修复 Replication, recombination and repair	145
细胞骨架 Cytoskeleton	145
染色体结构和动力学 Chromatin structure and dynamics	125
细胞壁/细胞膜/包膜的生物发生 Cell wall/membrane/envelope biogenesis	105
核酸转运和代谢 Nucleotide transport and metabolism	102
辅酶转运和代谢 Coenzyme transport and metabolism	98
防御机制 Defense mechanisms	67
核结构 Nuclear structure	27
细胞外结构 Extracellular structures	21
细胞活性 Cell motility	3

图4 扁茎黄芪KEGG通路类型注释统计
1.耐药性:抗菌;2.耐药性:抗肿瘤药;3.心血管病;4.免疫疾病;5.药物依赖;6.内分泌和代谢疾病;7.癌症:特定类型;8.神经退行性疾病;9.癌症:总览;10.感染性疾病:寄生;11.感染性疾病:细菌;12.感染性疾病:病毒性;13.人类疾病类;14.感觉系统;15.开发与再生;16.循环系统;17.排泄系统;18.消化系统;19.老化;20.神经系统;21.内分泌系统;22.免疫系统;23.有机系统类;24.细胞群落-原核生物;25.细胞运动;26.细胞群落-真核生物;27.细胞生长与死亡;28.运输和分解代谢;29.细胞过程类;30.信号分子与相互作用;31.膜运输;32.信号转导;33.环境信息处理类;34.复制和修复;35.转录;36.折叠、分类和降解;37.翻译;38.遗传信息处理类;39.萜类和聚酮化合物的代谢;40.异生物素的生物降解和代谢;41.其他氨基酸的代谢;42.糖的生物合成与代谢;43.辅助因子和维生素的代谢;44.核苷酸代谢;45.能量代谢;46.脂质代谢;47.氨基酸代谢;48.碳水化合物代谢;49.新陈代谢类。

Fig.4 Functional classification of Astragalus complanatus Unigene in KEGG database
1.Drug resistance:antimicrobial;2.Drug resistance:antineoplastic;3.Cardiovascular disease;4.Immune disease;5.Substance dependence;6.Endocrine and metabolic disease;7.Cancer:specific types;8.Neurodegenerative disease;9.Cancer:overview;10.Infectious disease:parasitic;11.Infectious disease:bacterial;12.Infectious disease:viral;13.Human diseases;14.Sensory system;15.Development and regeneration;16.Circulatory system;17.Excretory system;18.Digestive system;19.Aging;20.Nervous system;21.Endocrine system;22.Immune system;23.Organismal systems;24.Cellular community-prokaryotes;25.Cell motility;26.Cellular community-eukaryotes;27.Cell growth and death;28.Transport and catabolism;29.Cellular processes;30.Signaling molecules and interaction;31.Membrane transport;32.Signal transduction;33.Environmental information processing;34.Replication and repair;35.Transcription;36.Folding sorting and degradation;37.Transcription;38.Genetic information processing;39.Metabolism of terpenoids and polyketides;40.Xenobiotics biodegradation and metabolism;41.Metabolism of other amino acids;42.Glycan biosynthesis and metabolism;43.Metabolism of cofactors and vitamins;44.Nucleotide metabolism;45.Energy metabolism;46.Lipid metabolism;47.Amino acid metabolism;48.Carbohydrate metabolism;49.Metabolism.

表4 扁茎黄芪转录组Unigene数量最多的10个通路注释

Tab.4 Top ten metabolic pathways involving Astragalus complanatus Unigene

序号 Serial number	代谢通路 Pathway	Unigene数量 Number of Unigene	通路ID Pathway ID	占比/% Percentage
1	嘌呤代谢	309	ko00230	3.45
2	淀粉和蔗糖的代谢	252	ko00500	2.81
3	核糖体	227	ko03010	2.54
4	RNA转运	224	ko03013	2.50
5	剪接体	223	ko03040	2.49
6	NF-kappa B信号通路	220	ko04064	2.46
7	植物激素信号转导	213	ko04075	2.38
8	内质网中的蛋白质加工	205	ko04141	2.29
9	内吞作用	202	ko04144	2.26
10	Toll样受体信号通路	200	ko04620	2.23

图5 扁茎黄芪SSR类型及主要基元统计

Fig.5 The statistics of SSR types and main unit in Astragalus complanatus

表5 扁茎黄芪转录组 Unigene SSR 分析

Tab.5 SSR analysis of Astragalus complanatus transcriptome Unigene

单元重复数 Repeats	重复数 Repeats											合计 Total
单元重复数 Repeats	单核苷酸 Mono-nucleotide	二核苷酸 Di-nucleotide		三核苷酸 Tri-nucleotide		四核苷酸 Tetra-nucleotide		五核苷酸 Penta-nucleotide		六核苷酸 Hexa-nucleotide		合计 Total
5	0		0		762		99		49		24	934
6	0		317		420		53		8		3	801
7	0		233		224		7		1		0	465
8	0		225		150		1		0		1	377
9	0		154		31		2		0		0	187
10	1 026		109		34		1		0		0	1 170
11	464		151		19		1		0		0	635
>11	882		364		34		0		0		0	1 280
合计Total	2 372		1 553		1 674		164		58		28	5 849
占比/% Percentage	40.56		26.55		28.62		2.80		0.99		0.48	100

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