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月季RhMAX2A基因的克隆与表达分析

李莎莎 杜高齐 李雪娇 关文灵 孟静

李莎莎,杜高齐,李雪娇,等. 月季RhMAX2A基因的克隆与表达分析 [J]. 福建农业学报,2024,39(2):1−11
引用本文: 李莎莎,杜高齐,李雪娇,等. 月季RhMAX2A基因的克隆与表达分析 [J]. 福建农业学报,2024,39(2):1−11
LI S S, DU G Q, LI X J, et al. Cloning and expression analysis of RhMAX2A gene in Rosa hybrid [J]. Fujian Journal of Agricultural Sciences,2024,39(2):1−11
Citation: LI S S, DU G Q, LI X J, et al. Cloning and expression analysis of RhMAX2A gene in Rosa hybrid [J]. Fujian Journal of Agricultural Sciences,2024,39(2):1−11

月季RhMAX2A基因的克隆与表达分析

基金项目: 云南省科技厅农业联合专项-面上项目(202301BD070001-032);云南省科技厅基础研究专项-面上项目(202201AT070256)
详细信息
    作者简介:

    李莎莎(1999 —),女,硕士,主要从事观赏植物资源收集与评价研究,E-mail:3143966131@qq.com

    通讯作者:

    孟静(1981 —),女,博士,副教授,主要从事园林植物资源开发与创新利用研究,E-mail:mengjing2514@163.com

  • 中图分类号: S685

Cloning and expression analysis of RhMAX2A gene in Rosa hybrid

  • 摘要:   目的  克隆月季RhMAX2A基因cDNA序列,并分析其序列特征及其在不同组织中和去顶后的表达情况,为探究该基因在月季中的生物学功能及调控侧枝发生的转导机制提供理论支持。  方法  以月季品种滇红为材料,通过 RT-PCR 技术克隆RhMAX2基因的cDNA序列,利用生物信息学方法对其序列和所编码的蛋白质进行分析,利用烟草瞬时转化技术分析蛋白的亚细胞定位,同时采用实时荧光定量PCR(qRT-PCR)检测其在不同组织中及去顶后的表达情况。  结果  RhMAX2A基因(GeneBank登录号为OP055810)cDNA序列长1030 bp,编码246 个氨基酸,该蛋白分子式为C2910H4793N1029O1244S210,相对分子质量为27.35 kD,总原子量为3909;该蛋白不稳定系数为53.07,脂肪系数为106.30,GRAVY值为0.049,是一类不稳定亲水性蛋白;RhMAX2A蛋白的二级结构主要由α-螺旋和无规则卷曲构成,且RhMAX2A为推定的F-box结构域,属于α/β水解酶家族;同源序列比对和系统进化树关系分析结果表明,RhMAX2A 氨基酸序列(OP055810)与同属的古老月季品种月月粉氨基酸序列(XP_024283944.1)相似性最高,其次是同亚科的草莓(XP_004287076.1),三者亲缘关系较近。亚细胞定位结果显示,RhMAX2A编码蛋白位于细胞核。qRT-PCR检测结果显示,RhMAX2A基因在根、腋芽和节中表达,根中表达量最高,去顶处理显著上调RhMAX2A基因在根和腋芽中的表达。  结论  成功克隆了滇红RhMAX2A基因,其编码蛋白在细胞核上发挥作用,主要在根和腋芽中表达,且受去顶诱导上调表达。
  • 图  1  RhMAX2A PCR 扩增电泳图

    M:DL2000 marker;1:PCR 产物。

    Figure  1.  Gel electrophoresis of PCR amplified products of RhMAX2A

    M: DL2000 marker; 1: PCR amplification product.

    图  2  RhMAX2A基因核苷酸序列及推导的氨基酸序列

    Figure  2.  Nucleotide sequence of the RhMAX2A gene and deduced amino acid sequence

    图  3  RhMAX2A 蛋白亲/疏水性预测

    Figure  3.  Hydrophilicity/hydrophobicity prediction of RhMAX2A protein

    图  4  RhMAX2A 蛋白保守结构域预测

    Figure  4.  Conserved domain prediction of RhMAX2A protein

    图  5  RhMAX2A蛋白二级结构预测

    蓝色:α-螺旋;红色:延伸链;紫色:无规则卷曲;绿色:β-转角。

    Figure  5.  Secondary structure prediction of RhMAX2A protein

    Blue: α-helix; red: Extended strand; orange: Random coil; green: β-turn.

    图  6  RhMAX2A蛋白三级结构及蛋白互作预测结果

    A :三级结构; B: 蛋白互作网络。

    Figure  6.  Tertiary structure and protein interaction prediction of RhMAX2A protein

    A: Tertiary structure; B: Protein interaction network.

    图  7  RhMAX2A 蛋白信号肽预测

    Figure  7.  Predicted signal peptide of RhMAX2A protein

    图  8  RhMAX2A蛋白跨膜区域预测

    Figure  8.  Prediction of the transmembrane region of RhMAX2A protein

    图  9  RhMAX2A蛋白磷酸化位点预测

    Figure  9.  Phosphorylation sites prediction of RhMAX2A protein

    图  10  基于不同物种MAX2A蛋白氨基酸序列多重比对

    Figure  10.  Multiple alignment based on MAX2A amino acid sequences in different species

    图  11  基于不同物种MAX2A蛋白氨基酸序列构建的系统发育进化树

    Figure  11.  Phylogenetic tree based on amino acid sequence homology of MAX2A in different species

    图  12  RhMAX2A蛋白亚细胞定位

    Figure  12.  Subcellular localization of RhMAX2A protein

    图  13  RhMAX2A基因在不同组织及去顶处理下的表达模式

    A. 不同组织,B. 根,C. 腋芽; ns表示无显著差异(P > 0.05),*表示差异显著(P < 0.05),**表示差异极显著(P < 0.01),***表示差异极显著(P < 0.001)。

    Figure  13.  Expression patterns of RhMAX2A in different tissues and under decapitation

    A. Different tissues, B. Roots, C. Axillary buds; ns indicates no significant (P > 0.05), * indicates significant difference (P < 0.05), ** indicates extremely significant difference (P < 0.01), *** indicates extremely significant difference (P < 0.001).

    表  1  引物序列

    Table  1.   Primer sequences

    引物名称 Primer 引物序列 Primer sequence
    RhMAX2A-F 5′-TCTCTCTCGAGCTTTCGCGAGCTCATGCAATCGCGTTCACGTG-3′
    RhMAX2A-R 5′-TCGCCCTTGCTCACCATGGATCCATCAAGGATTGTGCGCCTGT-3′
    18S rRNA-F 5′-CCTGAGAAACGGCTACCACAT-3′
    18S rRNA-R 5′-CACCAGACTTGCCCTCCA-3′
    UBC-F 5′-GCCAGAGATTGCCCATATGTGTA-3′
    UBC-R 5′-TCACAGAGTCCTAGCAGCACA-3′
    RhMAX2A-qRT-F 5′-GATCGACTTCTTCTCCGGGCTT-3′
    RhMAX2A-qRT-R 5′-CCAATCCACTGTCCCTCACGTT-3′
    RhMAX2A-GFP-F 5′-TCTCTCTCGAGCTTTCGCGAGCTCATGCAATCGCGTTCACGTG-3′
    RhMAX2A-GFP-R 5′-TCGCCCTTGCTCACCATGGATCCATCAAGGATTGTGCGCCTGT-3′
    下载: 导出CSV
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出版历程
  • 收稿日期:  2023-08-21
  • 录用日期:  2024-02-27
  • 修回日期:  2024-01-26
  • 网络出版日期:  2024-03-28

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