Cloning and Expressions of RhMAX2A in Rosa hybrida
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摘要:
目的 克隆月季（ Rosa hybrida L.）RhMAX2A基因cDNA序列，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（ Rosa hybrida ‘Dianhong’）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（ Nicotiana tabacum ）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（ Rosa chinensis ‘Old Blush’）氨基酸序列（XP_024283944.1）相似性最高，其次是同亚科的草莓（ Fragria vesca subsp. vesca ）（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。 Abstract:Objective Biological functions and lateral branching transduction mechanism of RhMAX2A were investigated by cloning the cDNA and determining the after-decapitation expressions in tissues of Rosa hybrida. Methods The cDNA sequence of RhMAX2A was cloned from hybrid tea rose Dianhong by RT-PCR for a bioinformatic analysis. The subcellular location of the gene was determined by transient transformation of PC1300s-RhMAX2A-GFP in tobacco leaves. After decapitating the plant, expressions of the gene in different organs were determined by qRT-PCR. Results The cDNA of RhMAX2A (GeneBank accession number: OP055810) was 1030 bp in length encoding 246 amino acids with the chemical formula of C2910H4793N1029O1244S210, a molecular weight of 27.35 kD, and a total atomic weight of 3909. The instability coefficient of the unstable hydrophilic protein was 53.07, the fat coefficient, 106.30, and the GRAVY value, 0.049. The protein secondary structure was mainly α-helix and random coil of a presumed F-box domain belonging to the α/β hydrolase family. RhMAX2A (OP055810) had the highest homology with that in R. chinensis Old Blush (XP_024283944.1) followed by that in Fragaria vesca subsp. vesca (XP_004287076.1) of the same closely related subfamily. The encoded protein was in the nucleus. The gene expressed most highly in the roots but also in the axillary buds, and nodes among tested organs. Decapitation on the plant significantly upregulated RhMAX2A in the roots and axillary buds. Conclusion RhMAX2A was successfully cloned from R. hybrida Dianhong, which functioned in the nucleus, expressed mainly in the roots and axillary buds, and could be upregulated by plant decapitation. -
Key words:
- Rosa hybrida /
- MAX2A /
- Cloning /
- Subcellular localization /
- Expression analysis
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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. Sequences of RhMAX2A nucleotide and deduced amino acids
图 3 RhMAX2A 蛋白亲/疏水性预测
Figure 3. Predicted hydrophilicity/hydrophobicity 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. Predicted secondary structure 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. Predicted tertiary structure and interaction of RhMAX2A protein
A: Tertiary structure; B: protein interaction network.
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 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. Expressions of RhMAX2A in tissues after decapitation on plant
A: Different tissues; B: roots; C: axillary buds; ns: no significance at P˃0.05; *: significant difference at P＜0.05; **: extremely significant difference at P＜0.01; ***: extremely significant difference at P＜0.001.
表 1 引物序列
Table 1. Primer sequences
引物名称 Primer 引物序列 Primer sequence 引物用途 Primer application RhMAX2A-F 5′-TCTCTCTCGAGCTTTCGCGAGCTCATGCAATCGCGTTCACGTG-3′ 基因克隆 Gene cloning RhMAX2A-R 5′-TCGCCCTTGCTCACCATGGATCCATCAAGGATTGTGCGCCTGT-3′ 基因克隆 Gene cloning 18S rRNA-F 5′-CCTGAGAAACGGCTACCACAT-3′ 内参基因 Endogenous gene 18S rRNA-R 5′-CACCAGACTTGCCCTCCA-3′ 内参基因 Endogenous gene UBC-F 5′-GCCAGAGATTGCCCATATGTGTA-3′ 内参基因 Endogenous gene UBC-R 5′-TCACAGAGTCCTAGCAGCACA-3′ 内参基因 Endogenous gene RhMAX2A-qRT-F 5′-GATCGACTTCTTCTCCGGGCTT-3′ 荧光定量引物 qRT-PCR RhMAX2A-qRT-R 5′-CCAATCCACTGTCCCTCACGTT-3′ 荧光定量引物 qRT-PCR RhMAX2A-GFP-F 5′-TCTCTCTCGAGCTTTCGCGAGCTCATGCAATCGCGTTCACGTG-3′ 载体引物 Vector primer RhMAX2A-GFP-R 5′-TCGCCCTTGCTCACCATGGATCCATCAAGGATTGTGCGCCTGT-3′ 载体引物 Vector primer 
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