基于Iso-Seq技术的野生马齿苋叶片转录组分析

doi:10.3969/j.issn.1004-1524.2023.01.08

浙江农业学报 ›› 2023, Vol. 35 ›› Issue (1): 67-78.DOI: 10.3969/j.issn.1004-1524.2023.01.08

基于Iso-Seq技术的野生马齿苋叶片转录组分析

叶梅荣(), 黄守程, 王晓鹏, 刘爱荣, 崔峰, 康健

安徽科技学院生命与健康科学学院, 安徽凤阳 233100

收稿日期:2022-09-24 出版日期:2023-01-25 发布日期:2023-02-21
作者简介:叶梅荣(1969—),女,安徽滁州人,博士,副教授,主要从事植物生理学教学和研究。E-mail: lindayyyy815@126.com
基金资助:
安徽科技学院稳定人才项目(SKWD201601);安徽省自然科学基金(1808085MC78);安徽省创新训练项目(S202110879058);安徽省创新训练项目(S202110879059);安徽科技学院创业创新项目(X202110879017)

Transcriptome analysis of leaves of wild Portulaca oleracea L. based on Iso-Seq technology

YE Meirong(), HUANG Shoucheng, WANG Xiaopeng, LIU Airong, CUI Feng, KANG Jian

College of Life and Health Sciences, Anhui Science and Technology University, Fengyan 233100, Anhui,China

Received:2022-09-24 Online:2023-01-25 Published:2023-02-21

摘要/Abstract

摘要：

基于全长转录组测序(isoform sequencing,Iso-Seq)技术对野生马齿苋叶片(对照、干旱和低温胁迫3种处理的叶片)转录组进行测序及其转录组信息分析的结果显示,总共得到641 732条原始片段,质控后得到合格的插入片段26 758 071条,总碱基量为44 Gb,利用CCS软件获得442 760条一致性序列,根据smrtlink软件得到全长的插入片段和全长的去除嵌合体的插入片段分别为391 258条和301 412条,去除冗余得到103 298条转录本,最终得到39 717条unigene,预测出40 952条LncRNA和38 419条CDS序列;并将unigene 与Uniprot、Pfam、GO、KEGG、eggNOG、Pathway和Nr等数据库进行同源比对,其中36 381条unigene被注释,从这些unigene里查找到25 898个简单重复序列标记(simple sequence repeats, SSR)位点。利用差异表达分析软件DESeq2筛选不同处理组间的差异表达基因,对照和干旱处理、对照和低温胁迫处理及干旱和低温胁迫处理间分别鉴定出16 194(上调和下调差异基因分别为2 874和13 320)、16 093(上调和下调差异基因分别为6 365和9 728)和25 498(上调和下调差异基因分别为15 163和10 335)个差异表达基因。研究结果可为马齿苋属植物基因研究和SSR开发研究提供参考。

关键词: 马齿苋, 转录组, Iso-Seq技术

Abstract:

The transcriptome of wild Portulaca oleracea’s leaves (leaves of three treatments: control, drought stress and low temperature stress) was sequenced and analyzed by Iso-Seq technology. The results showed that 641 732 polymerase reads were generated from the transcriptome of P. oleracea’s leaves. 26 758 071 subreads and 44 Gb total bases were obtained by quality control methods. 442 760 consensus sequences were generated by using CCS software. 391 258 full-length reads and 301 412 full-length non-chimeric reads were generated based on Smrtlink software. 103 298 transcripts were obtained from these consensus sequences by removing redundancy, and 39 717 unigenes were generated from these transcripts by removing redundancy. 40 952 LncRNAs and 38 419 CDS sequences were predicted from the unigenes. Unigenes were aligned to the sequences of public databases, including UniProt, Pfam, GO, KEGG, eggNOG, pathway and NR databases, in which 36 381 unigenes were annotated, and 25 898 (simple sequence repeats) SSR loci were detected in the unigenes. The differentially expressed genes (DEGs) among different treatment groups were screened by using DESeq 2.16 194(Up: 2 874, Down: 13 320), 16 093 (Up: 6 365, Down: 9 728) and 25 498 (Up: 15 163, Down: 10 335) DEGs were obtained in C vs DS, C vs LTS, and DS vs LTS, respectively. This study could provide reference for gene research and SSR development of P. oleracea.

Key words: Portulaca oleracea L., transcriptome, Iso-Seq technology

中图分类号:

叶梅荣, 黄守程, 王晓鹏, 刘爱荣, 崔峰, 康健. 基于Iso-Seq技术的野生马齿苋叶片转录组分析[J]. 浙江农业学报, 2023, 35(1): 67-78.

YE Meirong, HUANG Shoucheng, WANG Xiaopeng, LIU Airong, CUI Feng, KANG Jian. Transcriptome analysis of leaves of wild Portulaca oleracea L. based on Iso-Seq technology[J]. Acta Agriculturae Zhejiangensis, 2023, 35(1): 67-78.

图/表 15

表1 初级转录本统计

Table 1 The statistics of primary transcripts

类型 Type	片段数量 Number of reads/bp
一致序列数目 Number of consensus reads	442 760
含有5'引物的插入片段的条数 Number of 5' primer reads	430 646
含有3'引物的插入片段的条数 Number of 3' primer reads	430 907
含有poly-A的插入片段的条数 Number of poly-A reads	403 679
过滤掉的短序列数目 Number of filtered short reads	27
非全长的插入片段数目 Number of non-full-length reads	51 475
全长的插入片段数目 Number of full-length reads	391 258
全长并去除嵌合体的插入片段数目 Number of full-length non-chimeric reads	301 412
全长非嵌合序列平均长度 Mean full-length non-chimeric reads	1 577

图1 Unigenes长度分布图

Fig.1 Length distribution of unigene

图2 LncRNA长度分布图

Fig.2 All LncRNA length distribution

图3 CDS长度分布

Fig.3 Length distribution of CDS

图4 Unigene 条目的注释统计

Fig.4 The statistical of the Unigene annotation

图5 GO功能注释

Fig.5 GO function annotation

图6 KEGG功能注释

Fig.6 KEGG function annotation

图7 COG功能注释分类

Fig.7 COG function annotation

图8 Nr注释

Fig.8 Nr annotation

图9 Nr注释的值

Fig.9 The E-value of Nr annotation

图10 Nr注释的一致性分布

Fig.10 Identity distribution of Nr annotation

图11 Pfam结构域分类注释图

Fig.11 Pfam domain annotation

图12 SSR重复单元数

Fig.12 Number of SSR types

图13 差异表达基因分布 A,C vs DS 火山图;B,C vs LTS 火山图;C,DSvsLTS 火山图;D,不同处理组比较的维恩图。

Fig.13 Distribution of differentially expressed genes A, C vs DS Volcano; B, C vs LTS Volcano; C, 15 DSvsLTS Volcano; D, Venn diagram of DEGs in different comparisons.

图14 C vs DS (A), C vs TS (B), DS vs LTS (C)差异表达基因GO分类图

Fig.14 GO classification of DEGs in C vs DS (A), C vs TS (B), DS vs LTS (C)

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基于Iso-Seq技术的野生马齿苋叶片转录组分析

Transcriptome analysis of leaves of wild Portulaca oleracea L. based on Iso-Seq technology

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