山楂过氧化物酶基因的克隆及在烟草中异位表达分析

Cloning of peroxidase gene of hawthorn and its ectopic expression in tobacco

【目的】从山楂(Crataegus pinnatifida)中克隆过氧化物酶(POD)编码基因,通过转基因验证其在木质素合成中的作用,为揭示山楂内果皮形成分子机制奠定基础。【方法】利用RT-PCR技术从山楂克隆基因,以p RI 101-AN为构建基因过量表达载体的基础载体,通过农杆菌介导的遗传转化方法将目的基因导入烟草。【结果】从山楂中克隆出编码序列长度为996 bp的POD72基因,命名为Cp POD72。山楂POD72与木本和草本植物POD72的氨基酸序列一致性均较高。构建了Cp POD72基因的过量表达载体,通过农杆菌介导的转化获得了66个烟草转化植株。RT-PCR检测结果显示Cp POD72基因在烟草转基因植物中异位表达,组织化学染色分析表明转Cp POD72基因烟草植株中木质素含量增加。【结论】Cp POD72基因可能参与木质素合成。

[Objective] The objective of this study was to clone peroxidase （POD） gene from hawthorn （Crataegus pinnatifida）, to investigate its role in lignin biosynthesis, and to lay the foundation for elucidating the mechanism of soft-endocarp formation in hawthorn. [Methods]POD gene was cloned from hawthorn by RT-PCR. Its over-expression vector was constructed base on the plant expression vector pRI 101-AN. The target gene was introduced transfered into tobacco by Agrobacterium-mediated transformation. [Results] The POD72 gene with 996 bp coding sequence was cloned from hawthorn, and it was named CpPOD72. The identity of amino acid sequence of POD72 gene between hawthorn and other woody and herbaceous plants was high. The over-expression vector for CpPOD72 was constructed, and 66 transformed plants of tobacco （Nicotiana tabacum） were obtained by Agrobacterium-mediated transformation. The expression of CpPOD72 in transgenic tobacco plants was detected by RT-PCR, and the result of histochemistry showed that the content of lignin was increased in transgenic tobacco plants. [Conclusion] The CpPOD72 maybe be involved in lignin biosynthesis pathway.