Isolation and Expression of Peroxidase POD1 Gene from Cucurbita pepo
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摘要: 通过西葫芦转录组测序，筛选得到1条功能注释为过氧化物酶（peroxidase，POD）的基因全长序列（c47620.graph_c0）。分析发现，该序列全长1 245 bp，包含1个996 bp的开放读码框（open reading frame，ORF），预测编码331个氨基酸，理论分子量（molecular weight，Mw）为35.70 kDa，等电点（isoelectric point，pI）为5.40，编码的蛋白质与黄瓜、甜瓜和苦瓜等常见瓜果类蔬菜同源蛋白质的相似性在84%、84%和81%，显示其高度的保守性，基因命名为CpPOD1，GenBank登录号为MF988285。荧光定量PCR分析结果表明，CpPOD1基因具有组织表达特异性，在叶片中表达丰度最高，其次为根，在花中的表达最低。CpPOD1基因在低温弱光条件下，其表达量随时间增加而显著上调。初步推测，CpPOD1基因在西葫芦低温弱光胁迫下存在一定上调表达，猜测可能参与调控下游基因对低温弱光胁迫的响应有关。Abstract: A functional annotation as peroxidase (POD) gene (i.e., c47620.graph_c0) was screened from the transcriptomic sequencing of Cucurbita pepo. It was found that thesequence length was 1 245 bp, including a 996bp open reading frame (ORF) encoded a protein of 331 amino acids. The protein had a predicted molecular weight of 35.70 kDa and a hypothetical isoelectric point of 5.40. The similarity of the proteins to the homologous proteins of cucumber, melon and balsam pear were 84%, 84% and 81%, respectively, witha highly conservative nature. It was designated as CpPOD1 gene with a GenBank accession code of MF988285. The RTqPCR revealed the geneto betissue-specific, andits expression in C. pepo highest in the leaves, followed by roots and flowers. Under low temperature and dim light, the CpPOD1 expression was up-regulated over time. It suggested that the gene expression correlatedwith the low temperature/dim light stress as C. pepo regulated the downstream responses. This study could lead to further understanding of the function of CpPOD1 gene inzucchinion cold-and-low-lighttolerance and the regulation mechanism providing evidence and theoretical basis for improved breeding of C. pepo.
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Key words:
- Cucurbita pepo /
- POD /
- RNA-Seq /
- qRT-PCR
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图 1 西葫芦低温弱光处理后植株
注：A为正常处理；B为弱光处理；C为低温处理；D为低温弱光处理。
Figure 1. C. pepo plant after low temperature and dim light treatment
图 2 CpPOD1全长cDNA序列及氨基酸序列
Figure 2. cDNA full sequence and corresponding amino acid sequence of CpPOD1
图 3 西葫芦CpPOD1与其他物种中的同源蛋白的多序列比对
注：XP_011653567.1为甜瓜; NP_001295845.1为黄瓜; XP_022153752.1为苦瓜; ABF68751.1为西葫芦; AKN08989.1为丝瓜。
Figure 3. Multiple sequence pairs of CpPOD1 in C. pepo and homologous proteins in other species
图 4 西葫芦CpPOD1与其他同源蛋白序列的系统进化树
Figure 4. Phylogenetic tree of CpPOD1 in C. pepo and other homologous sequences
图 6 西葫芦在不同处理条件下CpPOD1表达模式
Figure 6. CpPOD1 of C. pepo under treatments as analyzed by qPCR
图 7 在不同处理时间下的西葫芦酶活性含量变化
注：图中不同小写字母表示差异达显著水平(P＜0.05)。
Figure 7. Enzyme activities of C. pepo under treatments for varied time periods
表 1 西葫芦不同光照和温度处理组合
Table 1. Combined light and temperature treatments on C. pepo
处理 温度(白天/黑夜)/℃ 光强/lx 1(对照) 25/15 21000 2(弱光) 25/15 4200 3(低温) 15/3 21000 4(低温弱光) 15/3 4200 
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表 2 不同植物来源POD的氨基酸组成成分及理化性质分析
Table 2. The comparison of composition and physical and chemical characterization of amino acid of POD from different plant species
植物 氨基酸个数 相对分子质量/kDa 等电点 氨基酸比例/% 总平均疏水性 酸性 碱性 脂肪族 芳香族 西葫芦Cucurbita pepo 331 35.7 5.40 33 27 288 28 0.039 甜瓜Cucurbita melo 331 35.6 5.71 28 24 305 26 0.088 黄瓜Cucumis sativus 330 35.5 5.51 28 23 294 28 0.102 丝瓜Luffa aegyptiaca 331 36.3 5.54 31 26 307 61 0.057 笋瓜Cucumis maxima 332 35.8 5.27 33 27 300 27 0.067 苦瓜moschata charatia 328 35.2 6.06 28 25 278 28 0.052 南瓜Cucurbit moschata 320 34.6 5.29 33 27 278 28 0.052 毛茛Aquilegia coerulea 327 34.7 5.89 35 28 288 28 0.065
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