杜仲ARF基因家族全基因组鉴定和表达分析

doi:10.11707/j.1001-7488.20210318

摘要/Abstract

摘要：

目的: 杜仲是我国特有的传统药用和胶用木本植物，其生长发育相关基因研究对杜仲育种有重要意义。生长素响应因子（ARF）是生长素信号转导途径中重要的转录因子，了解ARF在杜仲器官发育中的作用具有重要的理论和实际应用价值。方法: 利用杜仲全基因组测序数据鉴定杜仲ARF基因，并对这些基因进行生物信息学分析；根据杜仲EuARFs与拟南芥、水稻和欧美杨ARF蛋白的序列比对，构建系统进化树；根据转录本全长测序数据分析杜仲叶片不同生长时期的ARF基因表达谱，并利用qRT-PCR分析杜仲ARF基因与调控ARF基因的miRNA在不同器官不同发育时期的表达模式。结果: 共鉴定出18个杜仲ARF基因，根据系统进化树可以将EuARFs分为5类；miRNA靶位点分析表明eu-miR160s调控Ⅱ亚族的EuARFs，eu-miR167s调控除了EuARF8.2的V亚族的EuARFs。表达谱结果显示EuARFs在叶片不同生长时期的表达不同，其中V亚族EuARFs与Ⅵ亚族EuARF19.2在3 cm长的生长叶和完全展开的幼叶中表达量较高。qRT-PCR试验结果显示，EuARFs在正常生长条件下不同发育阶段（幼嫩和成熟）的器官（根、茎和叶）中均有表达，在根中表现为在成熟阶段上调表达，叶和茎段中表现为在成熟的器官中下调表达；eu-miR160s主要在幼嫩的根中表达，eu-miR167s则主要在成熟的叶片中表达。结论: 共鉴定出18个杜仲ARF基因，这些EuARFs在正常生长条件下不同发育阶段（幼嫩和成熟）的器官（根、茎和叶）中均有表达，根据表达量可以预测V亚族的EuARFs对幼苗的生长发育具有调控作用，EuARF19.2对杜仲叶片生长具有重要调控作用。

关键词: 杜仲, 生长素响应因子, 基因表达模式, miRNA调控

Abstract:

Objective: Eucommia ulmoides is a traditional medicinal and rubber woody plant that is endemic to China. It is important to study the genes related to growth and development for breeding. Auxin response factor(ARF) is an important transcription factor in the auxin signaling pathways. Study of the role of ARF in E. ulmoides organ development has great values both in theory and practice. Method: Identification of putative EuARF genes from genome data and bio-informatics analysis were performed. The phylogenetic tree was generated using a multiple sequence alignment of the EuARF, AtARF (Arabidopsis thaliana ARF), OsARF (Oryza sativa ARF) and PeARF (Populus×euramericana ARF) proteins. RNA-seq data were used to analyze EuARFs expression profiles. qRT-PCR were used to analyze the expression patterns of these genes and miRNAs in organs at different developmental stages. Result: A total of 18 EuARFs were identified in the E. ulmoides genome. These EuARFs were divided into five classes by phylogenetic tree and predicted that eu-miR160s target EuARFs of class Ⅱ and eu-miR167s target EuARFs of class Ⅴ except EuARF8.2. The expression patterns showed that the expression of EuARFs in different growth stages of leaves was different, and the expression of EuARFs in class Ⅴ and EuARF19.2 in class Ⅵ were higher in 3 cm long growing leaves and unfolded young leaves. The qRT-PCR indicated that EuARFs expressed in organs (leaf, root and stem) at different developmental stages (young and mature). In addition, EuARFs were up-regulated in mature roots and down-regulated in mature leaves and stems. Meanwhile eu-miR160s was mainly expressed in young roots, and eu-miR167s was mainly expressed in mature leaves. Conclusion: A total of 18 EuARFs were identified in the genome of E. ulmoides. These EuARFs were expressed in organs (roots, stems and leaves) at different developmental stages (young and mature) under normal growth conditions. The expression level indicates that EuARFs in class V has a regulating role for seedlings growth and development and EuARF19.2 plays an important role in regulating the leaf growth.

Key words: Eucommia ulmoides, auxin response factor, gene expression patterns, miRNA regulation

中图分类号:

S718.46

刘闵豪,李龙,叶靖,周轩辕,李周岐,范睿深,徐郡儡. 杜仲ARF基因家族全基因组鉴定和表达分析[J]. 林业科学, 2021, 57(3): 170-180.

Minhao Liu,Long Li,Jing Ye,Xuanyuan Zhou,Zhouqi Li,Ruishen Fan,Junlei Xu. Genome-Wide Identification and Expression Analysis of the ARF Gene Family in Eucommia ulmoides[J]. Scientia Silvae Sinicae, 2021, 57(3): 170-180.

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miRNA	序列Sequences (5′—3′)	茎环引物Stem ring primer (5′—3′)	正向引物Forward primer (5′—3′)
eu-miR160d	TGCCTGGCTCCCTGCATGCCA	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCTGGCATGC	TCGGCAGGTGCCTGGCTCCCT
eu-miR160f	TGCCTGGCTCCCTGTATGCC	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCGGCATACA	TCGGCAGGTGCCTGGCTCCC
eu-miR167a	TGAAGCTGCCAGCATGATCTC	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCGAGATCAT	TCGGCAGGTGAAGCTGCCAGC
eu-miR167d	TGAAGCTGCCAGCATGATCT	CTCAACTGGTGTCGTGGAGTCGGCAATTCAGTTGAGCAGATCATG	TCGGCAGGTGAAGCTGCCAG

基因 Gene	正向引物Forward primer (5′—3′)	反向引物Reverse primer (5′—3′)
EuARF1	CTTCAGGTGCCAAGCCTAGTTCTC	GCCGGTCTTCTTATGCCAGTGTC
EuARF2.1	CCATAGTGAACAGGTGGCAGCATC	GGTTGGAGCGTCATCTGAGCATAC
EuARF2.2	GCATGGCATACTTGCGAGTG	CCGAACGAGTAATCATATTCAACAG
EuARF3.1	ACGAAGGTGACATGATGCTGATGG	TCGTTGCCGCTGCCAATTCC
EuARF3.2	AGCCAGCACCATCACTTCC	AGCCAGCACCATCACTTCC
EuARF4	GAAGGTTCAGCGGTCTTGTG	TCCCATTTCACTATCAAGCATCTC
EuARF6.1	TCGCCACAAGAGGTGCAGGAG	GCATGGTCATCGCAGCTACTATCG
EuARF6.2	GATACTATCGCCGCTGGAAGTGC	TGTCGCCGTTGCCGTGATTAAG
EuARF8.1	CATTGGAGATCCGTCAAGGTTGGTT	ATGGGAAAGGTTGTCAAAGGCTCAA
EuARF8.2	GGGCTTGTCCATCTGAGTTTG	GCACCAGTTTGACGACCTTC
EuARF9	TCCACAGCAACAGATGACACAACC	GCTGCGGAAGCTGAGAGTATAACG
EuARF10.1	GCGTCCTGGGATGAGGTTTA	CTGAAGACACTGCCATGAAGAAT
EuARF10.2	TTGGAACAGACGATACAGCAGACAA	GAGCCAGGCCATCTAGCAGGAT
EuARF16	TCGTCAACCGCAAGAAGCTCATC	GCGTTGGCTGAAGGTAGAGGAAG
EuARF17	ATGTGAAGCGTGTCAGCCCTTG	AGGTGGTGAGAACTGTGGAATCTGA
EuARF18	ACCGACAGCGAGAATGACATGATG	GCCGACAGATGAAGACTTGGACAG
EuARF19.1	GGTGCGGTGGGATGAAACTTCTAC	CAGGAGTCAGGGCAGGCTCTATT
EuARF19.2	TTGCCTCAGGTTGGAAGTCTTGTC	GTCTGCGTGTAATGTGGCGTTATG

基因 Gene	基因编号 Gene ID	蛋白长度 Protein length (aa)	等电点 pI	分子量 Molecular weight/ kDa	结构域 Domain
EuARF1	GWHGAAAL013439	668	6.15	74.75	DBD, MR, CTD
EuARF2.1	GWHGAAAL012635	822	7.62	91.97	DBD, MR, CTD
EuARF2.2	GWHGAAAL023550	577	6.18	65.12	DBD, MR, CTD
EuARF3.1	GWHGAAAL026509	638	6.27	70.13	DBD, ARF
EuARF3.2	GWHGAAAL025681	823	6.69	90.37	DBD, ARF
EuARF4	GWHGAAAL008814	846	5.91	94.44	DBD, MR, CTD
EuARF6.1	GWHGAAAL000541	892	5.75	99.40	DBD, MR, CTD
EuARF6.2	GWHGAAAL008938	858	5.76	95.04	DBD, MR, CTD
EuARF8.1	GWHGAAAL000541	892	5.75	99.40	DBD, MR, CTD
EuARF8.2	GWHGAAAL000012	273	9.68	30.64	DBD, ARF
EuARF9	GWHGAAAL016769	653	5.68	73.19	DBD, MR, CTD
EuARF10.1	GWHGAAAL008845	631	6.19	69.97	DBD, ARF
EuARF10.2	GWHGAAAL008846	633	5.36	70.70	DBD, ARF
EuARF16	GWHGAAAL011358	682	6.96	74.95	DBD, ARF
EuARF17	GWHGAAAL001628	526	5.63	57.54	DBD, ARF
EuARF18	GWHGAAAL025932	623	6.04	70.23	DBD, MR, CTD
EuARF19.1	GWHGAAAL003011	1 062	6.05	117.84	DBD, MR, CTD
EuARF19.2	GWHGAAAL003601	1 013	6.12	111.39	DBD, MR, CTD

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