Identification of CsCuAO Gene Family in Cucumber and Their Regulatory Roles in Adventitious Root Formation

doi:10.16420/j.issn.0513-353x.2023-0903

Acta Horticulturae Sinica ›› 2025, Vol. 52 ›› Issue (2): 309-321.doi: 10.16420/j.issn.0513-353x.2023-0903

• Genetic & Breeding·Germplasm Resources·Molecular Biology • Previous Articles Next Articles

Identification of CsCuAO Gene Family in Cucumber and Their Regulatory Roles in Adventitious Root Formation

WEN Zhengyang, SUN Jingbo, ZHANG Mengxia, ZHANG Feng, DONG Chunjuan^*()

State Key Laboratory of Vegetable Biobreeding，Institute of Vegetables and Flowers，Chinese Academy of Agricultural Sciences，Beijing 100081，China

Received:2024-09-21 Revised:2024-12-26 Online:2025-02-25 Published:2025-02-23
Contact: DONG Chunjuan

Abstract

Abstract:

Seven copper amine oxidase genes（CsCuAO）were identified from the genome of cucumber（Cucumis sativus L.）using bioinformatic techniques. The amino acid lengths of these deduced CsCuAO proteins ranged from 652 to 710 aa，with the isoelectric points（pI）ranging from 5.41 to 8.31. Phylogenetic analysis indicated that CsCuAOs were divided into three subfamily groups，and the genetic structure of the same subfamily had a high similarity. Treatment with putrescine，the CuAO enzymatic substrate，could promote adventitious root formation in cucumber hypocotyls，while application with aminoguanidine，a CuAO specific inhibitor，could inhibit adventitious root formation，with less adventitious root number and shorter adventitious root length. H₂O₂，but not the other CuAO’s catalytic products，could reverse the effect of aminoguanidine on adventitious root formation. The results of qRT-PCR suggested that the expression of CsCuAOβ and CsCuAOζ were dominantly induced at the early stage of adventitious root formation（24 h），while CsCuAOγ1 was significantly induced at the late stage（72-120 h）. Further analysis indicated that the expression of CsCuAOβ and CsCuAOζ was inhibited by IAA，but induced by ACC，the synthesis precursor of ethylene. Both IAA and ACC did not exert a significant influence on the expression of CsCuAOγ1. H₂O₂ could induce the expression of CsCuAOβ，CsCuAOζ and CsCuAOγ1 in different degrees. Transient overexpression of CsCuAOβ，CsCuAOζ and CsCuAOγ1 in cucumber cotyledons could increase the CuAO enzymatic activity and H₂O₂ contents，however，only CsCuAOβ overexpression could promote adventitious root formation at the base of the cotyledon petioles，while CsCuAOγ1 and CsCuAOζ overexpression had no significant effect on adventitious root formation. The results showed that CsCuAOβ gene responds to ethylene signal，and generates H₂O₂ and thereby promotes adventitious root formation in cucumber.

Key words: cucumber, copper amine oxidase, adventitious root, hydrogen peroxide, gene identification

WEN Zhengyang, SUN Jingbo, ZHANG Mengxia, ZHANG Feng, DONG Chunjuan. Identification of CsCuAO Gene Family in Cucumber and Their Regulatory Roles in Adventitious Root Formation[J]. Acta Horticulturae Sinica, 2025, 52(2): 309-321.

Figures/Tables 9

References 43

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用途Use	基因Gene	引物序列（5′-3′） Primer sequence
基因克隆	CsCuAOα1	F：CATTTGTTTTCAATATATGATTAATGTG；R：ACAAACTCTCAAATCAAATGACCG
Gene cloning	CsCuAOα2	F：TTCTCTTCTCCCTCTCACTCCT；R：GACACAACTCATTTAGCTTGTTATTC
	CsCuAOα3	F：GCCACACCCCTAACATATACATC；R：CGCAAGAGTTGTGGATGAATATC
	CsCuAOβ	F：ATGAATCCTCTTCTGTTTCTTCTTG；R：TCATTTAAGTAAAGGGTTTCTATCAAAG
	CsCuAOγ1	F：CTTACCTCGTCCTTCACTACC；R：TTGTCAATCAAGCCGAGGC
	CsCuAOγ2	F：CCACCCAATAAGACAAAGAAAGC；R：CGAGCATTACACACAGGCA
	CsCuAOζ	F：CGGAGGATAAGAGGAGAAGTGTG；R：GAGCAGTGATGAAGAAGAGAGAGT
qRT-PCR	CsCuAOα1	F：GCGGGTGGAACTGAGTACAG；R：TTTGCCCAACTAACGGAGTGA
	CsCuAOα2	F：TGGACTCTCAGGAACAGGAAGA；R：GGTCTAAGCTCAAAGCCACG
	CsCuAOα3	F：GACACACTGTCAGTTGGGCA；R：CCGAGATTTGGCCTCGGTAT
	CsCuAOβ	F：TGATTAGACGTGGGGAAGCAG；R：CCCGTTAAAGCAACCCCAAC
	CsCuAOγ1	F：ATGGAAAGCCGTATGTGCGA；R：CTCAGTGATATCCATGCCGGT
	CsCuAOγ2	F：ACACTTGTAGCTCGCATGGT；R：ATAGCCCAACCTCAACACGA
	CsCuAOζ	F：TCCAAAGGTGTTGTTCCTCCAA；R：TATCTCTGACCTCTGGCGTG
	CsActin-7	F：CCCATCTATGAGGGTTACGCC；R：TGAGAGCATCAGTAAGGTCACGA
瞬时表达载体构建 Construction of transient expression vector	CsCuAOβ	F：GGACAGGGTACCCGGGGATCCATGAATCCTCTTCTGTTTCTTCTTGTAC； R：AGTGTCGACTCAGAGGATCCTCATTTAAGTAAAGGGTTTCTATCAAAGT
	CsCuAOγ1	F：GGACAGGGTACCCGGGGATCCATGGCTGCCAGAAAATATCTCCTC； R：AGTGTCGACTCAGAGGATCCTCAAGCCGAGGCAGCAGGCTTGC
	CsCuAOζ	F：GGACAGGGTACCCGGGGATCCATGGCCCCAGCTTCGAAA； R：AGTGTCGACTCAGAGGATCCTTAGAGCTTAGCGATGATGGGAG

用途Use	基因Gene	引物序列（5′-3′） Primer sequence
基因克隆	CsCuAOα1	F：CATTTGTTTTCAATATATGATTAATGTG；R：ACAAACTCTCAAATCAAATGACCG
Gene cloning	CsCuAOα2	F：TTCTCTTCTCCCTCTCACTCCT；R：GACACAACTCATTTAGCTTGTTATTC
	CsCuAOα3	F：GCCACACCCCTAACATATACATC；R：CGCAAGAGTTGTGGATGAATATC
	CsCuAOβ	F：ATGAATCCTCTTCTGTTTCTTCTTG；R：TCATTTAAGTAAAGGGTTTCTATCAAAG
	CsCuAOγ1	F：CTTACCTCGTCCTTCACTACC；R：TTGTCAATCAAGCCGAGGC
	CsCuAOγ2	F：CCACCCAATAAGACAAAGAAAGC；R：CGAGCATTACACACAGGCA
	CsCuAOζ	F：CGGAGGATAAGAGGAGAAGTGTG；R：GAGCAGTGATGAAGAAGAGAGAGT
qRT-PCR	CsCuAOα1	F：GCGGGTGGAACTGAGTACAG；R：TTTGCCCAACTAACGGAGTGA
	CsCuAOα2	F：TGGACTCTCAGGAACAGGAAGA；R：GGTCTAAGCTCAAAGCCACG
	CsCuAOα3	F：GACACACTGTCAGTTGGGCA；R：CCGAGATTTGGCCTCGGTAT
	CsCuAOβ	F：TGATTAGACGTGGGGAAGCAG；R：CCCGTTAAAGCAACCCCAAC
	CsCuAOγ1	F：ATGGAAAGCCGTATGTGCGA；R：CTCAGTGATATCCATGCCGGT
	CsCuAOγ2	F：ACACTTGTAGCTCGCATGGT；R：ATAGCCCAACCTCAACACGA
	CsCuAOζ	F：TCCAAAGGTGTTGTTCCTCCAA；R：TATCTCTGACCTCTGGCGTG
	CsActin-7	F：CCCATCTATGAGGGTTACGCC；R：TGAGAGCATCAGTAAGGTCACGA
瞬时表达载体构建 Construction of transient expression vector	CsCuAOβ	F：GGACAGGGTACCCGGGGATCCATGAATCCTCTTCTGTTTCTTCTTGTAC； R：AGTGTCGACTCAGAGGATCCTCATTTAAGTAAAGGGTTTCTATCAAAGT
	CsCuAOγ1	F：GGACAGGGTACCCGGGGATCCATGGCTGCCAGAAAATATCTCCTC； R：AGTGTCGACTCAGAGGATCCTCAAGCCGAGGCAGCAGGCTTGC
	CsCuAOζ	F：GGACAGGGTACCCGGGGATCCATGGCCCCAGCTTCGAAA； R：AGTGTCGACTCAGAGGATCCTTAGAGCTTAGCGATGATGGGAG

基因名称 Gene name	序列号 Accession No.	基因位置 Gene locus	基因长/bp Gene length	外显子数 Exon number	内含子数 Intron number	ORF/bp	蛋白长/aa Protein length	分子量/kD MW	等电点 pI
CsCuAOα1	CsaV3_7G022600	Chr7:11469830 ~ 11476230	6 401	5	4	2 037	678	77.27	6.49
CsCuAOα2	CsaV3_6G038130	Chr6:21656887 ~ 21661068	4 182	5	4	2 007	668	75.33	6.51
CsCuAOα3	CsaV3_6G038150	Chr6: 21670803 ~ 21666955	3 849	6	5	1 983	660	75.01	5.88
CsCuAOβ	CsaV3_6G038140	Chr6:21661851 ~ 21664612	2 762	4	3	1 959	652	74.23	8.73
CsCuAOγ1	CsaV3_5G003800	Chr5:2454206 ~ 2458762	4 557	4	3	2 178	725	81.37	5.79
CsCuAOγ2	CsaV3_5G003790	Chr5:2448731 ~ 2453203	4 473	6	5	2 133	710	80.45	5.41
CsCuAOζ	CsaV3_1G001170	Chr1:659948 ~ 667885	7 938	12	11	2 115	704	79.27	6.29

基因名称 Gene name	序列号 Accession No.	基因位置 Gene locus	基因长/bp Gene length	外显子数 Exon number	内含子数 Intron number	ORF/bp	蛋白长/aa Protein length	分子量/kD MW	等电点 pI
CsCuAOα1	CsaV3_7G022600	Chr7:11469830 ~ 11476230	6 401	5	4	2 037	678	77.27	6.49
CsCuAOα2	CsaV3_6G038130	Chr6:21656887 ~ 21661068	4 182	5	4	2 007	668	75.33	6.51
CsCuAOα3	CsaV3_6G038150	Chr6: 21670803 ~ 21666955	3 849	6	5	1 983	660	75.01	5.88
CsCuAOβ	CsaV3_6G038140	Chr6:21661851 ~ 21664612	2 762	4	3	1 959	652	74.23	8.73
CsCuAOγ1	CsaV3_5G003800	Chr5:2454206 ~ 2458762	4 557	4	3	2 178	725	81.37	5.79
CsCuAOγ2	CsaV3_5G003790	Chr5:2448731 ~ 2453203	4 473	6	5	2 133	710	80.45	5.41
CsCuAOζ	CsaV3_1G001170	Chr1:659948 ~ 667885	7 938	12	11	2 115	704	79.27	6.29

Identification of CsCuAO Gene Family in Cucumber and Their Regulatory Roles in Adventitious Root Formation

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