Cloning and Expression of Premnaspirodiene Oxygenase Gene from Dendrobium officinale
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摘要:
目的 克隆铁皮石斛（Dendrobium officinale）豆腐柴螺二烯加氧酶（premnaspirodiene oxygenase, PO）基因，分析该基因在铁皮石斛开花期不同器官及营养生长期叶片中的表达模式，以期为进一步鉴定基因功能及探讨铁皮石斛植保素的生物合成及抗病机制奠定基础。 方法 利用RT-PCR和RACE-PCR技术克隆DoPO基因全长cDNA序列和开放阅读框（open reading frame, ORF），利用ProtParam进行理化性质分析，使用BLAST P在线搜索氨基酸同源性，采用MEGA 6.0构建系统进化树。利用qPCR方法分析DoPO基因在铁皮石斛开花期不同器官及不同营养生长期叶片中的表达模式。 结果 DoPO基因cDNA序列全长1704 bp，含1个编码498个氨基酸的完整阅读框。DoPO蛋白属于p450超级家族，分子量为56.914 kD，属不稳定蛋白。系统进化分析表明，DoPO与兰科植物姬蝴蝶兰（XP 020571355）和深圳拟兰（PKA52400）PO聚为一类，与姬蝴蝶兰的PO亲缘关系最近。qPCR分析结果表明，铁皮石斛开花期，DoPO基因在叶片的相对表达量极显著高于茎、根和花；叶片DoPO基因的相对表达量10月份极显著高于8月和12月，12月显著高于8月。 结论 克隆了铁皮石斛DoPO基因cDNA全长，该基因在叶片中的相对表达量极显著高于其他器官。叶片DoPO基因的相对表达量10月份最高。 Abstract:Objective To decipher the gene function, phytoalexin biosynthesis, and disease resistance mechanism of Dendrobium officinale (Do), the premnaspirodiene oxygenase gene (PO) was cloned and its expressions in the plant in various organs at different growth stages studied. Method RACE-PCR and RT-PCR were employed to clone the full-length cDNA and ORF of DoPO. ProtParam was used for the physiochemical determination, BLAST P on the amino acid homology, and MEGA 6.0 for the phylogenetic tree construction. The expressions of DoPO in different organs at flowering stage as well as in leaves at growth stages were examined by qPCR. Result The DoPO encoded 498 amino acids, 1 704 bp in length with an 1 497 bp ORF. The unstable protein had a molecular weight of 56.914 kDa belonging to the P450 superfamily. It was phylogenetically clustered in the same branch with Phalaenopsis equestris (XP 020571355) and Apostasia shenzhenica (PKA52400) and most closely related to P. equestris. The relative DoPO expression in the leaves was significantly higher than that in the stems, roots, or flowers at flowering stage (P＜0.01); and during a year, that in October significantly higher than that in either August or December (P＜0.01) and that in December significantly higher than in August (P＜0.05). Conclusion The full-length cDNA of DoPO was successfully cloned. Its relative expression in leaf peaked in October and was highest in leaves among the organs at flowering stage. -
Key words:
- Dendrobium officinale /
- premnaspirodiene oxygenase /
- gene cloning /
- expression analysis
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图 1 PCR电泳结果
注：M：分子量标准；A：3′RACE；B：开放阅读框。
Figure 1. PCR products by electrophoresis
Note: M: DNA marker; A: 3’RACE; B: ORF.
图 2 铁皮石斛DoPO基因cDNA序列及其推导的氨基酸序列
Figure 2. Sequences of cDNA and its deduced amino acids of DoPO from D. officinale
图 3 不同物种豆腐柴螺二烯加氧酶的系统进化树
Figure 3. Phylogenetic tree of premnaspirodiene oxygenase from different species
图 4 DoPO基因在不同器官的表达
注：柱上不同大写字母表示差异极显著（P＜0.01）。
Figure 4. Expression of DoPO in various organs
Note: Data with different capital letters on same column indicate significant difference at 0.01 level.
图 5 DoPO基因在叶中的相对表达量
注：柱上不同大、小写字母分别表示差异极显著（ P ＜0.01）或显著（P＜0.05）。
Figure 5. Relative expression of DoPO in leaves
Note: Data with different uppercase letters and lowercase letters on same column indicate significant difference at 0.01 level and 0.05 level, respectively.
表 1 PCR引物及其序列
Table 1. PCR primers and sequences
引物名称
Primer name碱基序列（5'-3'）
Primer sequence（5’-3'）3PO-1 GCTGGAACTGACACCTCATCTG 3PO-2 CCAGCAGGAAGTCGGATTGTGA Adpt CTGATCTAGAGGTACCGGATCC dT-Adpt CTGATCTAGAG dGTACCGGATCC
TTTTTTTTTTTTTTTTTDoPO-F CCGGGATCCATGGAGCCACCAACCTTC DoPO-R CCGCTCGAGACGTAGGCAATGGAGGAT PO-F GCTCTTGTCGGGATTCAG PO-R GATTCTTTGCTCGATGCTC DoACT-F AGGAAGGCGGCTTTGAATC DoACT-R CCATGCCAACCATGACACC 注：下划线碱基为酶切位点。
Note: The underlined bases were the enzyme site.
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