铁皮石斛CLE基因家族鉴定与功能分析

doi:10.3969/j.issn.1004-1524.20231020

浙江农业学报 ›› 2024, Vol. 36 ›› Issue (7): 1583-1590.DOI: 10.3969/j.issn.1004-1524.20231020

铁皮石斛CLE基因家族鉴定与功能分析

诸燕¹(), 丁兰², 陈忆乾¹, 黄秀静¹, 姜伟伟¹, 陈东红¹^,^*()

1.浙江农林大学省部共建亚热带森林培育国家重点实验室,浙江杭州 311300
2.临安区农林技术推广中心,浙江杭州 311300

收稿日期:2023-08-28 出版日期:2024-07-25 发布日期:2024-08-05
作者简介:诸燕(1983—),女,浙江绍兴人,硕士,农艺师,研究方向为药用植物遗传育种。E-mail: zhuyan198305@163.com
通讯作者: *陈东红,E-mail: chendh212@163.com
基金资助:
浙江省农业新品种选育重大科技专项(2021C02074)

Identification and functional analysis of CLE gene family in Dendrobium officinale Kimura et Migo

ZHU Yan¹(), DING Lan², CHEN Yiqian¹, HUANG Xiujing¹, JIANG Weiwei¹, CHEN Donghong¹^,^*()

1. The State Key Laboratory of Subtropical Silviculture, Zhejiang A&F University, Hangzhou 311300 China
2. Agricultural and Forestry Technology Extension Center of Lin’an District, Hangzhou 311300, China

Received:2023-08-28 Online:2024-07-25 Published:2024-08-05

摘要/Abstract

摘要：

CLE多肽是一类小分子分泌蛋白,参与植物的生长发育和细胞间通讯,在维持干细胞的增殖与分化中发挥关键调控作用。为研究兰科药用植物铁皮石斛(Dendrobium officinale Kimura et Migo)中CLE基因家族成员的功能,从铁皮石斛叶、根、茎、花苞、果实的组织中提取RNA,反转录成cDNA,通过半定量PCR检测CLE家族基因成员在各组织中的表达情况。结果表明:在铁皮石斛基因组中鉴定出17个CLE成员,它们均拥有保守的12氨基酸CLE基序。半定量RT-PCR结果表明,CLE基因家族成员在根、茎、叶、花、果实中展示不同的组织表达谱,尤其是CLE19635在花苞和果实中特异性表达,CLE22175在果实中特异性表达。用体外合成的CLE多肽处理拟南芥,结果显示,CLE合成肽参与拟南芥根的发育,其中,CLE05351对根生长有促进作用,CLE18468对根影响不明显,其他CLE多肽均对根的生长有抑制作用。mPS-PI染色并未发现根尖分生组织结构模式受到CLE合成肽的影响。此外,CLE02038过表达的拟南芥T₂代纯合系具有短根表型,与CLE合成肽处理结果一致。研究结果为进一步挖掘CLE家族在铁皮石斛附生根中的作用及其信号调控网络奠定了基础,并为铁皮石斛理想根系的育种提供了潜在靶基因。

关键词: 铁皮石斛, CLE基因家族, 肽激素, 根发育, 拟南芥

Abstract:

CLE peptide are a class of small secreted proteins that widely participate in plant development and cell-to-cell communication, playing a key regulatory role in maintaining the proliferation and differentiation of stem cells. In order to study the function of CLE family member in Dendrobium officinale Kimura et Migo, RNA was extracted from the leaves, roots, stems, buds and fruits of D. officinale and reverse transcribed into cDNA, and the expression of CLE family genes in each tissue was detected by semi-quantitative RT-PCR. The results showed that 17 CLE members were identified in D. officinale, all of which conserved 12-amino acid CLE motifs. Semi-quantitative RT-PCR performed on roots, stems, leaves, flowers and fruits showed that these CLE members had different tissue expression profiles, particularly CLE19635 with specifical expression in flower buds and fruits, and CLE22175 with specifical expression in fruits. In vitro synthesis of CLE peptides for treatment of Arabidopsis thaliana revealed that CLE peptides impacted the growth of A. thaliana roots, CLE05351 promoted root elongation, CLE18468 had no obvious effect, but the other CLE peptides all inhibited root elongation. mPS-PI staining did not find that the cell pattern of root-tip meristem was affected by CLE peptides. In addition, T₂ homozygotes of A. thaliana overexpressing CLE02038 showed a short root phenotype, which was consistent with the result of CLE peptides treatment. The results laid a foundation for the further exploring the role of CLE family in the epiphytic root of D. officinale and its signal regulation network, and provided potential target genes for breeding of ideal root systems in D. officinale.

Key words: Dendrobium officinale, CLE gene family, peptide hormone, root development, Arabidopsis

中图分类号:

S567.239

诸燕, 丁兰, 陈忆乾, 黄秀静, 姜伟伟, 陈东红. 铁皮石斛CLE基因家族鉴定与功能分析[J]. 浙江农业学报, 2024, 36(7): 1583-1590.

ZHU Yan, DING Lan, CHEN Yiqian, HUANG Xiujing, JIANG Weiwei, CHEN Donghong. Identification and functional analysis of CLE gene family in Dendrobium officinale Kimura et Migo[J]. Acta Agriculturae Zhejiangensis, 2024, 36(7): 1583-1590.

图/表 6

参考文献 27

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基因名称 Gene name	正向引物 Forward primer (5'→3')	反向引物 Reverse primer (5'→3')	基因ID Gene ID	产物长度 Product length/bp
CLE109525	CTCTTACAGTACCTGATCATACAC	TTATGCCTGTCCAGGCAA	LOC110109525	525
CLE01792	ATGTCTCTCCAATTGCTCTTG	TTAATGGTGGAGTGGGTTGG	PKU85961	372
CLE02038	GACTTCATGAGGAAGAATAGAA	TCAGTTATGAAGAGGATTAGGTC	PKU86207	228
CLE04631	ATGGCTAAACTAAAAGAGATAGG	TCATCTGTTATGCAGAGGATTAG	PKU65016	267
CLE05351	ATGGCTTCTGCTGAAGTCTC	CTACCTATTGGAGTCTGGGTT	PKU82346	267
CLE11641	ATGGCATTCTCTTCAAGAGC	TCAATGGTGTTGAGGGTCTG	PKU78021	231
CLE5097	ATGGCAAGAGAAGTAGTGAGG	TCAATTGTGTAGCGGGTTTG	PKU64567	171
CLE15729	ATGGTTGGGCAAAGAGAGATG	TCAGTTGTGCAAAGGATTAGG	PKU75207	264
CLE18468	ATGGACACTGAGCTGATCAG	TCACCTATTTGAATCAGGATTC	PKU83835	336
CLE19635	ATGCTGCCCACTCTGCCG	CTAGTGATGGAGGGGATTCG	PKU59933	177
CLE20689	ATGCAAGGAATTAGAAGGACT	TCAGTTGTGAAGTGGATTGG	PKU74917	249
CLE22175	ATGGGGAGAATCAATATTCT	TTAATGGTGTTCAGGATCTG	PKU68119	210
CLE22943	ATGGTTGGGCTAAGAGAGA	CTATCTGTTGTGCAAAGGATT	PKU86730	267
CLE29071	ATGCTTCCCACTCTGCCG	CTAGTGATGGAGGGGATTCGC	PKU62687	177
CLE79016	ATGGCGTTCACATACTCAAGAG	TCAATGGTGTTGGGGGTCTG	PKU79016	231
CLE82107	ATGCCAACTTCAACGAGGC	TTACCGGTTGTGAAGGCGG	PKU82107	234
CLE66413	ATGGGGCTGCGTAAAAGAG	TCACAGCTCTTTTACGCAGC	PKU66413	237
DcACTIN1	GATTTGCTGGTGACGATGC	AGGATACCTCTTTTGGACTGG	LOC110111141	136

基因名称 Gene name	正向引物 Forward primer (5'→3')	反向引物 Reverse primer (5'→3')	基因ID Gene ID	产物长度 Product length/bp
CLE109525	CTCTTACAGTACCTGATCATACAC	TTATGCCTGTCCAGGCAA	LOC110109525	525
CLE01792	ATGTCTCTCCAATTGCTCTTG	TTAATGGTGGAGTGGGTTGG	PKU85961	372
CLE02038	GACTTCATGAGGAAGAATAGAA	TCAGTTATGAAGAGGATTAGGTC	PKU86207	228
CLE04631	ATGGCTAAACTAAAAGAGATAGG	TCATCTGTTATGCAGAGGATTAG	PKU65016	267
CLE05351	ATGGCTTCTGCTGAAGTCTC	CTACCTATTGGAGTCTGGGTT	PKU82346	267
CLE11641	ATGGCATTCTCTTCAAGAGC	TCAATGGTGTTGAGGGTCTG	PKU78021	231
CLE5097	ATGGCAAGAGAAGTAGTGAGG	TCAATTGTGTAGCGGGTTTG	PKU64567	171
CLE15729	ATGGTTGGGCAAAGAGAGATG	TCAGTTGTGCAAAGGATTAGG	PKU75207	264
CLE18468	ATGGACACTGAGCTGATCAG	TCACCTATTTGAATCAGGATTC	PKU83835	336
CLE19635	ATGCTGCCCACTCTGCCG	CTAGTGATGGAGGGGATTCG	PKU59933	177
CLE20689	ATGCAAGGAATTAGAAGGACT	TCAGTTGTGAAGTGGATTGG	PKU74917	249
CLE22175	ATGGGGAGAATCAATATTCT	TTAATGGTGTTCAGGATCTG	PKU68119	210
CLE22943	ATGGTTGGGCTAAGAGAGA	CTATCTGTTGTGCAAAGGATT	PKU86730	267
CLE29071	ATGCTTCCCACTCTGCCG	CTAGTGATGGAGGGGATTCGC	PKU62687	177
CLE79016	ATGGCGTTCACATACTCAAGAG	TCAATGGTGTTGGGGGTCTG	PKU79016	231
CLE82107	ATGCCAACTTCAACGAGGC	TTACCGGTTGTGAAGGCGG	PKU82107	234
CLE66413	ATGGGGCTGCGTAAAAGAG	TCACAGCTCTTTTACGCAGC	PKU66413	237
DcACTIN1	GATTTGCTGGTGACGATGC	AGGATACCTCTTTTGGACTGG	LOC110111141	136

基因名(CLE多肽名称) Gene name (Name of CLE peptide)	保守区段基序 Motif sequence	基因名(CLE多肽名称) Gene name (Name of CLE peptide)	保守区段基序 Motif sequence	基因名(CLE多肽名称) Gene name (Name of CLE peptide)	保守区段基序 Motif sequence
CLE66413 (PKU66413)	RKVPNASDPLHN	CLE05351 (PKU82346)	HEVPNGPNPDSN	CLE82107 (PKU82107)	RPVPSCPDRLHN
CLE109525 (LOC110109525)	RKVPKGPDPIHN	CLE18468 (PKU83835)	HEVPSGPNPDSN	CLE19635	RLVPSGANPLHH
				(PKU59933/PKU62687)
CLE79016 (PKU79016)	RLSPGGSDPQHH	CLE04631 (PKU65016)	RNVYTGPNPLHN	CLE20689 (PKU74917)	RLVPSGPNPLHN
CLE22175 (PKU68119)	RLSPGGPDPEHH	CLE15729	RTVYTGPNPLHN	CLE01792 (PKU85961)	RFVPCGPNPLHH
		(PKU75207/PKU86730)
CLE11641 (PKU78021)	RLSPGGPDPQHH	CLE02038 (PKU86207)	RIIYAGPNPLHN	CLE5097 (PKU64567)	RFVPQGPNPLHN

基因名(CLE多肽名称) Gene name (Name of CLE peptide)	保守区段基序 Motif sequence	基因名(CLE多肽名称) Gene name (Name of CLE peptide)	保守区段基序 Motif sequence	基因名(CLE多肽名称) Gene name (Name of CLE peptide)	保守区段基序 Motif sequence
CLE66413 (PKU66413)	RKVPNASDPLHN	CLE05351 (PKU82346)	HEVPNGPNPDSN	CLE82107 (PKU82107)	RPVPSCPDRLHN
CLE109525 (LOC110109525)	RKVPKGPDPIHN	CLE18468 (PKU83835)	HEVPSGPNPDSN	CLE19635	RLVPSGANPLHH
				(PKU59933/PKU62687)
CLE79016 (PKU79016)	RLSPGGSDPQHH	CLE04631 (PKU65016)	RNVYTGPNPLHN	CLE20689 (PKU74917)	RLVPSGPNPLHN
CLE22175 (PKU68119)	RLSPGGPDPEHH	CLE15729	RTVYTGPNPLHN	CLE01792 (PKU85961)	RFVPCGPNPLHH
		(PKU75207/PKU86730)
CLE11641 (PKU78021)	RLSPGGPDPQHH	CLE02038 (PKU86207)	RIIYAGPNPLHN	CLE5097 (PKU64567)	RFVPQGPNPLHN

铁皮石斛CLE基因家族鉴定与功能分析

Identification and functional analysis of CLE gene family in Dendrobium officinale Kimura et Migo

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