黄瓜CsCuAO家族基因鉴定及其在不定根形成中的作用研究

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

摘要/Abstract

摘要：

利用生物信息方法从黄瓜（Cucumis sativus L.）基因组中克隆到7个铜胺氧化酶（copper amine oxidase，CuAO）基因，其编码蛋白的氨基酸长度在652 ~ 710 aa之间，等电点介于5.41 ~ 8.31之间，系统进化树分析将其分为3个亚族。采用CuAO底物腐胺处理后，黄瓜下胚轴不定根数量增加，而CuAO特异抑制剂氨基胍处理后，不定根数量显著减少；将CuAO的催化产物H₂O₂与氨基胍复配处理可有效消除氨基胍的抑制效应。qRT-PCR分析发现CsCuAOβ和CsCuAOζ在不定根形成的早期（24 h）高表达，CsCuAOγ1在不定根形成的后期（72 ~ 120 h）高表达；进一步分析不同植物生长调节剂和H₂O₂信号对CsCuAOβ、CsCuAOζ和CsCuAOγ1的表达调控，发现生长素（IAA）抑制CsCuAOβ和CsCuAOζ的表达，而乙烯合成前体氨基环丙烷羧酸（ACC）促进二者的表达，IAA和ACC对CsCuAOγ1的表达无显著影响，H₂O₂对三者的表达均有不同程度的诱导作用。将CsCuAOβ、CsCuAOζ、CsCuAOγ1在黄瓜子叶中瞬时表达，发现3个基因均可使子叶中CuAO活性升高，H₂O₂积累量增加，CsCuAOβ过量表达可促进子叶柄部位不定根形成，而CsCuAOγ1和CsCuAOζ过量表达对不定根形成无显著影响。试验结果表明，黄瓜CsCuAOβ响应乙烯信号，通过产生H₂O₂信号促进黄瓜不定根形成。

关键词: 黄瓜, 铜胺氧化酶, 不定根, 过氧化氢, 基因鉴定

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

温正阳, 孙靖博, 张梦夏, 张锋, 董春娟. 黄瓜CsCuAO家族基因鉴定及其在不定根形成中的作用研究[J]. 园艺学报, 2025, 52(2): 309-321.

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.

图/表 9

表1 本研究中所用的引物

Table 1 Primers used in this study

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

图1 腐胺（Put）处理对黄瓜不定根形成的影响采用SPSS 26.0软件进行one-way ANOVA分析，不同小写字母表示不同处理间差异显著（P < 0.05）

Fig. 1 Effect of putrescine（Put）application on adventitious root formation of cucumber seedlings Statistical analysis was carried out with SPSS 26.0 software，using one-way ANOVA analysis. Different lowercases indicate significant differences among different treatments（P < 0.05）

图2 CuAO抑制剂氨基胍（AG）对黄瓜不定根形成的影响采用SPSS 26.0软件进行one-way ANOVA分析，不同小写字母表示处理间差异显著（P < 0.05）

Fig. 2 Effect of aminoguanidine（AG）application on adventitious root formation of cucumber seedlings Statistical analysis was carried out with SPSS 26.0 software，using one-way ANOVA analysis. Different lowercases indicate significant differences among different treatments（P < 0.05）

图3 氨基胍（AG）与H2O2、γ-氨基丁酸（GABA）、琥珀酸（Succ）和NH3复配处理对黄瓜不定根形成的影响采用SPSS 26.0软件进行one-way ANOVA分析，不同小写字母表示处理间差异显著（P < 0.05）

Fig. 3 Effect of co-application of aminoguanidine（AG）with H2O2，γ-aminobutyric acid（GABA），succinic acid（Succ） and NH3 on adventitious root formation of cucumber seedlings Statistical analysis was carried out with SPSS 26.0 software，using one-way ANOVA analysis. Different lowercases indicate significant differences among different treatments（P < 0.05）

表2 黄瓜CsCuAO基因家族成员的序列特征

Table 2 Sequence characteristics of CsCuAO gene family members

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

图4 黄瓜CsCuAO和拟南芥AtCuAO的系统进化树

Fig. 4 Phylogenetic tree of CsCuAO and AtCuAO proteins

图5 黄瓜不定根形成过程中CsCuAO的表达量变化采用SPSS 26.0软件采用t检验进行统计学分析，* P < 0.05，** P < 0.01

Fig. 5 Expression patterns of CsCuAO genes during adventitious root formation of cucumber Statistical analysis was carried out using SPSS 26.0 software by t-test. * P < 0.05，** P < 0.01

图6 黄瓜不定根形成过程中CsCuAOβ、CsCuAOγ1和CsCuAOζ对激素和H2O2信号的表达响应采用SPSS 26.0软件进行one-way ANOVA分析，不同小写字母表示处理间差异显著（P < 0.05）

Fig. 6 Expression levels of CsCuAOβ, CsCuAOγ1 and CsCuAOζ during adventitious root formation in response to plant hormones and H2O2 Statistical analysis was carried out with SPSS 26.0 software，using one-way ANOVA analysis. Different lowercases indicate significant differences among different treatments（P < 0.05）

图7 CsCuAOβ、CsCuAOγ1和CsCuAOζ在黄瓜子叶中瞬时表达量及其对CuAO活性、H2O2含量和不定根形成的影响采用SPSS 26.0软件采用t检验进行统计学分析，* P < 0.05，** P < 0.01

Fig. 7 Effects of transient expression of CsCuAOβ，CsCuAOγ1 and CsCuAOζ on adventitious root formation in cucumber cotyledon Statistical analysis was carried out using SPSS 26.0 software by t-test. * P < 0.05，** P < 0.01

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