Expression of NRAT1 Gene in Hydrangea macrophylla Family
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摘要:
目的 铝胁迫是酸性土壤中限制植物生长的重要元素，八仙花是一种富集铝的园林观赏植物；本研究对八仙花NRAT1基因进行生物信息学分析及表达分析，以期揭示NRAT1基因在耐铝生理中的作用。 方法 以八仙花品种Hydrangea macrophyllacv laybla为材料，提取RNA进行RT-PCR，检测实时表达情况，克隆NRAT1基因全长并进行生物信息学分析。 结果 试验克隆得到了巨噬基因家族的NRAT1基因全长，该基因有1个1 881 bp的开放阅读框，编码548个氨基酸。多重序列分析表明其具有相当高的保守性，该蛋白有12个跨膜结构，预测位于质膜或液泡膜上，预测结果显示该蛋白是一个疏水性蛋白，试验还分析得到了蛋白质的二、三级结构。RT-PCR试验表明，NRAT1基因在八仙花的根、茎、叶中均有表达，3个组织中NRAT1基因随着处理时间的增加表达量先升后降，在处理2 h表达量达到顶峰，之后维持低水平表达，在12 h之后基因表达被抑制，在根中的平均表达量最高且表达量变化幅度最大。 结论 NRAT1基因在八仙花感受铝胁迫时，表达量立即上调，NRAT1基因参与了八仙花铝离子的吸收和转运，在八仙花铝富集作用中扮演了重要角色。 Abstract:Objective Bioinformatics and expression of NRAT1 gene of the ornamental Hydrangea macrophylla plants that tend to accumulate aluminum (Al), a growth-limiting heavy metal in acidic soil, were studied to understand the physiology of the plant on Al-tolerance. Method The RNA of H. medalensis cv. Laybla was extracted for RT-PCR to detect the real-time expression and cloned for bioinformatic analysis to determine the molecular structure of NRAT1. Result The cloned macrophage gene obtained in the lab revealed that the gene had a 1 881 bp open reading frame and encoded 548 amino acids. A multiple sequence analysis showed it to be highly conservative with 12 transmembrane structures located on the plasma or vacuole membrane. The secondary and tertiary structures of the hydrophobic protein were obtained. RT-PCR indicated that NRAT1 was expressed in the roots, stems and leaves of H. macrophylla, and the expressions increased initially to peak in 2h followed by a decline with time to a levered low level. After 12h under Al-stress, the expression was completely inhibited. Among the three issues, the roots had the highest expression and were affected the most by the stress. Conclusion The NRAT1 gene expression was up-regulated in Hydrangea macrophylla immediately upon the exposure to Al. The gene was positively confirmed to participate in the absorption and transport of Al ions playing an important role in the heavy metal accumulation in the plants. -
Key words:
- Hydrangea macrophylla /
- NRAT1 gene /
- Aluminum stress /
- expression analysis
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图 1 NRAT1基因电泳
注：A为RNA电泳图，B为RACE全长电泳图。
Figure 1. Electrophoresis of NRAT1
Note: A:RNA electrophoresis map, B:RACE full-length electrophoresis map.
图 2 NRAT1编码蛋白的跨膜结构预测
Figure 2. Predicted transmembrane domain of protein encoded by NRAT1
图 3 NRAT1编码蛋白的亲水性和疏水性预测
Figure 3. Hydrophobicity/hydrophilicity prediction on protein encoded by NRAT1
图 4 NRAT1编码蛋白二级结构预测
Figure 4. Predicted secondary structure of protein encoded by NRAT1
图 5 NRAT1编码蛋白的三级结构分析
注：A为八仙花(NRAT1)，B为粳稻(XP_015647626.1)，C为栓皮栎(XP_023908030.1)，D为高粱(XP_002459640.1)。
Figure 5. Predicted tertiary structure of proteins encoded by NRAT1
Note: A:Hydrangea macrophylla(NRAT1), B:Oryza sativa (XP_015647626.1), C:Quercus suber(XP_023908030.1), D:Sorghum bicolor(XP_002459640.1).
图 6 NRAT1基因在根、茎、叶中不同时间的实时表达量
Figure 6. Expression of NRAT1 in roots, stems and leaves of H. macrophylla plant at different time
表 1 八仙花NRAT1基因扩增所用引物
Table 1. Primers for amplification of NRAT1 in H. macrophylla
引物名称
Primers引物序列
primer sequenceQPCR-F AGCAACTTTCGAAGGGACTA QPCR-R GATACTGAGATCACTGCCACA PCR-F ATGGCCAGTCTGCAGCAACAAC PCR-R CTATTCGGGTAATGGTATATCCGCC RACE-F GGCAAAGAAACGCCCATCGAGAAG RACE-R GACTACGCTGACAAGCCTGGATTGG 
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