蒺藜苜蓿（Medicago truncatula）全长转录组测序及分析

doi:10.13560/j.cnki.biotech.bull.1985.2021-0191

摘要/Abstract

摘要：

为了深入分析和探索豆科模式植物蒺藜苜蓿的mRNA完整结构,使用单分子长读数测序技术（single-molecule long-read sequencing technology,SMRT）对蒺藜苜蓿进行全长转录组测序及分析。共获得7 728 183个subread和509 014条全长非嵌合序列（full-length non-chimeric read,FLNC）,通过比对分析发现,94.36%的序列与93.01%的序列分别与蒺藜苜蓿R108与A17参考基因组匹配。总计存在8 406种可变性剪接,其中主要的剪接方式为内含子保留（intron retention,RI）。共发现23 926个基因,其中12 049个基因存在295 545条转录本,在这些转录本中至少存在一个poly（A）位点。此外,共鉴定出3 223条转录因子,6 595条长非编码RNA（long non-coding RNA,lncRNA）和479条融合转录本。使用SMRT技术能够深入发掘蒺藜苜蓿转录数据,也为更好地利用蒺藜苜蓿基因组资源提供数据补充。

关键词: 蒺藜苜蓿, 单分子全长读数测序, 可变性剪接, 融合转录本

Abstract:

For deep analysis and exploring of the complete structure of mRNA from Medicago truncatula,a model plant in Leguminosae,the single-molecule long-read sequencing technology（SMRT）was used to sequence and analyze the full-length transcriptome of M. truncatula. A total of 7 728 183 subreads and 509 014 full-length non-chimeric reads（FLNC）were obtained. By comparing the reference genomes of R108 and A17,94.36% and 93.01% isoforms were identified as mapped reads,respectively. A total of 8 406 alternative splicing events was identified and the majority was intron retention（RI）. The 23 926 genes were detected,of 12 049 genes had 295 545 transcripts,in which at least there was one poly（A）site. In addition,3 223 transcription factors,6 595 long non-coding RNAs（lncRNA）and 479 fusion transcripts were identified. The results showed the feasibility of deep sequencing M. truncatula transcriptome by SMRT,which provides data supplement for better utilizing the genome resource of M. truncatula.

Key words: Medicago truncatula, single-molecule long-read sequencing, alternative splice, fusion transcript

尚骁尧, 周玲芳, 尹芊芊, 晁跃辉. 蒺藜苜蓿（Medicago truncatula）全长转录组测序及分析[J]. 生物技术通报, 2021, 37(8): 131-140.

SHANG Xiao-yao, ZHOU Ling-fang, YIN Qian-qian, CHAO Yue-hui. Sequencing and Analysis of Full-length Transcriptome from Medicago truncatula[J]. Biotechnology Bulletin, 2021, 37(8): 131-140.

图/表 13

表1 SMRT测序数据统计

Table 1 Statistics of sequencing data by SMRT

类别 Category	数目 Amount	最小长度 Min length/bp	最大长度 Max length/bp	平均长度 Mean length/bp	N50长度 N50 length/bp
Subread	7 728 183	51	73 164	1 524	2 051
CCS	659 220	200	17 791	2 257	2 757
FLNC	509 014	200	17 128	2 047	2 440
ICE consensus	243 832	200	17 128	2 305	3 367
Polished consensus	243 676	166	17 128	2 321	3 380
Corrected consensus	236 243	260	17 954	2 425	3 496

图1 校正后序列与参考基因组比对分析

Fig. 1 GMAP analysis of corrected reads to reference genome

图2 RC61125序列的分类

Fig. 2 Classification of RC61125 sequence

表2 新基因功能注释

Table 2 Functional annotations of novel genes

数据库 Databases	新基因注释数量 Number of functional annotated novel genes
NR	5 183
SwissProt	2 370
KEGG	4 532
KOG	1 884
GO	1 717
NT	6 916
PFAM	1 717
总计 Total	7 209
未注释 Unannotated ones	227

图3 新基因Nr同源物种分布图

Fig. 3 Nr homologous species distribution diagram of novel genes

图4 新基因RT-PCR验证 1-5：随机挑选新基因;M：DNA marker

Fig. 4 RT-PCR validation of novel genes 1-5：Randomly selected novel genes. M：DNA marker

表3 编码区及非编码区鉴定

Table 3 CDS and UTR identification

序列来源 Sequence origin	编码区数量 Number of CDS	编码区平均长度 Mean CDS length/bp	完整编码区数量 Number of complete CDS	3'非编码区数量 Number of 3'-UTR	5'非编码区数量 Number of 5'-UTR
SMRT	36 235	1 027.03	13 451	1 990	11 071
R108	61 020	874.54	16 313	3 895	4 928

图5 转录本外显子分布 Pac-Bio：SMRT测序数据;R108：蒺藜苜蓿R108参考基因组数据;A17：蒺藜苜蓿A17参考基因组数据

Fig.5 Distribution of exons in transcripts Pac-Bio：SMRT transcripts. R108：Transcripts from M. truncatula R108 reference genome.A17：Transcripts from M. truncatula A17 reference genome

图6 AS数量及类型 SE：外显子跳跃;MX：外显子互斥;A5：可变性5'剪切位点;A3：可变性3'剪切位点;RI：内含子保留;AF：第一外显子可变剪接;AL：最后外显子可变剪接

Fig.6 Numbers and types of AS events SE：Skipped exon. MX：mutually exclusive exon. A5：Alternative 5' splice site. A3：Alternative 3' splice site. RI：Retained intron. AF：First exon is alternative splice. AL：Last exon is alternative splice

表4 SMRT测序及参考基因组转录本数目

Table 4 Number of transcripts in SMRT sequence and reference genomes

转录本数目 Number of transcript	SMRT测序 SMRT sequence	参考基因组 Reference genome
1	11 730	52 332
2	4 713	2 308
3	2 660	617
4	1 614	246
5	965	96
6	618	58
7	415	21
8	334	10
9	216	8
10	147	5
>10	514	5

图7 lncRNA鉴定 A：使用CPC、CNCI、PLEK及Pfam 4种方法预测lncRNA韦恩图;B：SMRT鉴定的：4种lncRNA类型比例

Fig.7 Identification of lncRNA A：Venn diagram of lncRNAs predicted by CPC,CNCI,PLEK and Pfam methods.B：Proportions of 4 types of lncRNA identified by SMRT

图8 融合转录本RT-PCR验证 1-5：随机挑选融合转录本;M：DNA marker

Fig. 8 RT-PCR validation of fusion transcripts 1-5：Randomly selected fusion transcripts. M：DNA marker

图9 poly（A）位点分析 A：基因poly（A）位点数目分布;B：poly（A）剪切位点附近核苷酸组成;C：poly（A）位点上游重复序列MEME分析;D：poly（A）位点下游游重复序列MEME分析

Fig. 9 Analysis of poly（A）sites A：Distribution of the number of poly（A）sites per gene. B：Nucleotide composition around poly（A）cleavage sites. C：MEME analysis of repeated sequence in the upstream of the poly（A）site. D：MEME analysis of repeated sequence in the downstream of the poly（A）site

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