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中山杉407ThP5CSThδ-OAT的克隆及干旱胁迫下的表达分析

Cloning and Expression Analysis of ThP5CS and Thδ-OAT Gene in Taxodium Hybrid 'Zhongshanshan 407' under Drought Stress

  • 摘要:
    目的 探究中山杉407(Taxodium mucronatum ♀×T.distichum ♂)(T.hybrid 'Zhongshanshan 407')的ThP5CSThδ-OAT基因与植物耐旱性的关系,为落羽杉属植物抗性育种提供新的基因资源。
    方法 利用RACE技术从中山杉407中分别克隆ThP5CSThδ-OAT基因cDNA全长,通过生物信息学手段分析预测其编码蛋白的结构和功能。基于干旱-复水试验,采用半定量和实时荧光定量技术研究中山杉407 ThP5CSThδ-OAT的表达特性。
    结果 从中山杉407中克隆了编码P5CS和δ-OAT蛋白的基因,分别命名为ThP5CSThδ-OAT,其中,Thδ-OAT包含1个长度为1 494 bp的ORF,编码497个氨基酸,与樟子松(Pinus sylvestris L.)δ-OAT蛋白的序列相似性高达92%;ThP5CS包含1个长度为1 545 bp的ORF,编码514个氨基酸,与花烛属植物(Anthurium amnicola)P5CS蛋白的序列相似性为87%。半定量与定量PCR结果一致,在自然干旱和复水环境下,ThP5CS在中山杉407及其父本落羽杉(T.distichum)、母本墨杉(T.mucronatum)中均表现为先上调再下调的趋势,而Thδ-OAT在中山杉407及其父母本中相对表达量则存在差异。
    结论 干旱胁迫下,Thδ-OATThP5CS基因的正反方向调节是中山杉407及其亲本在干旱胁迫与恢复状态下控制脯氨酸水平的关键机制,其中,ThP5CS在中山杉407及其亲本脯氨酸的合成过程中起重要作用。

     

    Abstract:
    Objective To study the function of ThP5CS and Thδ-OAT in T. hybrid 'Zhongshanshan 407' (Taxodium mucronatum ♀×T. distichum ♂), and to explore the relationships between the genes and drought resistance of T. hybrid 'Zhongshanshan 407'. This research would provide candidate gene resources for resistance breeding in Taxodium.
    Method Rapid-amplification of cDNA ends (RACE) was applied to clone the full-length complementary DNA (cDNA) sequences of P5CS and δ-OAT in T. hybrid 'Zhongshanshan 407'. And then, the structures and functions were predicted with bioinformatics analysis. Finally, a natural drought and rewatering experiment was conducted to investigate the expression patterns of ThP5CS and Thδ-OAT by semi-quantitative RT-PCR (sRT-PCR) and quantitative real-time PCR (qRT-PCR).
    Result The cDNA sequences of P5CS and δ-OAT in T. hybrid 'Zhongshanshan 407' were isolated and then were named as ThP5CS and Thδ-OAT. The full-length sequence of Thδ-OAT and ThP5CS contained open reading frames (ORF) of 1 494 bp and 1 545 bp respectively, and encoded polypeptides of 497 and 514 amino acids residues. The protein sequences of Thδ-OAT exhibited 92% sequence identities with Pinus sylvestris L., and the protein sequences of ThP5CS exhibited 87% sequence identities with Anthurium amnicola. sRT-PCR and qRT-PCR analysis showed that in T. hybrid 'Zhongshanshan 407' and its parents, ThP5CS showed an increasing expression pattern and then decreasing in the period of drought and recovery. While the relative expression of Thδ-OAT showed differentiations in T. hybrid 'Zhongshanshan 407', T. distichum, and T. mucronatum.
    Conclusion ThP5CS might be important in response of Taxodium to drought and could play a leading role in proline synthesis.

     

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