Cloning and expression analysis of RhMAX2A gene in Rosa hybrid
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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。 Abstract: :Objective To study the biological function and the transduction mechanism that regulates lateral branching of the RhMAX2A gene in Rosa, the cDNA sequence of RhMAX2A was cloned and was analysed for sequence characteristics and its expression in different tissues and after decapitation. Methods The cDNA sequence of RhMAX2A gene was cloned from Rosa hybrida ‘Dianhong’ by RT-PCR and the bioinformatics analysis was conducted. The subcellular location of RhMAX2A was analyzed by transient transformation of PC1300s-RhMAX2A-GFP in tobacco leaves. The expression levels of RhMAX2A in different tissues and the treatment of decapitation was detected by real-time quantitative PCR (qRT-PCR). Results The cDNA sequence of RhMAX2A gene (GenBank accession number: OP055810 ) was 1030 bp in length, encoding 246 amino acids. The calculated chemical molecular formula of RhMAX2A protein is C2910H4793N1029O1244S210, with 27.35 kD in molecular mass and the total atomic weight is 3909. The instability coefficient of this protein is 53.07, with fat coefficient-106.30 and GRAVY value 0.049, indicating that it is an unstable hydrophilic protein. The secondary structure of RhMAX2A protein is mainly composed of α-helix and random coil, and RhMAX2A is a presumed F-box domain, belonging to the α/β hydrolase family. Homologous sequence alignment and phylogenetic tree analysis showed that the amino acid sequence of RhMAX2A (OP055810) had the highest similarity with that of Rosa chinensis Old Blush (XP_024283944.1), followed by Fragaria vesca subsp. vesca (XP_004287076.1) of the same subfamily, which were closely related. The subcellular localization results showed that the encoded protein of RhMAX2A was located in the nucleus. The results of qRT-PCR indicated that RhMAX2A gene was expressed in roots, axillary buds and nodes, with the highest expression level in roots. The decapitation treatment significantly upregulated the expression of RhMAX2A in roots and axillary buds. Conclusion The RhMAX2A gene was successfully cloned from Rosa hybrida ‘Dianhong’, which encodes a protein that plays a role in the nucleus, mainly expressed in roots and axillary buds, and could be upregulated by decapitation. -
Key words:
- Rosa hybrida /
- MAX2A gene /
- gene 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. 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 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 8 RhMAX2A蛋白跨膜区域预测
Figure 8. Prediction of the transmembrane region 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 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′ 
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