延边苹果梨苯丙氨酸解氨酶基因克隆及植物表达载体的构建

Clone and Construction of Plant Expression Vector of Phenylalanine Ammonia Lyase Gene of Pyrus pyrifolia cv. Pingguoli in Yanbian Area

以苹果梨果皮为试材,根据Genbank上已经登录的蔷薇科植物的PAL核酸序列,利用DNAStar-MegAligan软件进行多序列比对确定它们的保守区,参照西洋梨PAL序列(GenBank:DQ230992.2)设计了1对苹果梨特异的PAL引物,运用RT-PCR技术对苹果梨的苯丙氨酸解氨酶(PAL)基因进行了克隆、序列分析及分子进化方面的研究。结果表明:试验成功获得了苹果梨PAL基因的全长cDNA序列,命名为PyPAL,GenBank登录号为JQ247318.1。序列分析表明,苹果梨PALcDNA全长2 160bp,为完整的开放阅读框,推测其编码719个氨基酸,与鸭梨和西洋梨PAL基因的同源性均高达95%以上,所编码氨基酸序列同源性达95%以上。成功构建了含有PAL基因的重组质粒PBI121-PyPAL的植物表达载体,转化到根瘤农杆菌LBA4404菌种中形成工程菌株,为进一步研究苹果梨果实着色调控基因及其遗传改良奠定了基础。

Taking Pyrus pyrifolia cv. Pingguoli in Yanbian as material, based on the nucleic acid sequences of Rosaceae plants logged in Genbank, a pair of PAL primers specific for Pyrus pyrifolia cv. Pingguoli was designed through determination of their conserved region by the alignment of multiple sequences using DNAStar-MegAligan software with the reference to Pyrus communis sequence （GenBank. DQ230992. 2）, and the PAL gene cloning, sequence analysis and molecular evolution were studied. The results showed that the full-length eDNA sequence of Pyrus pyrifolia ev. Pingguoli PAL gene named PyPAL with registry number of JQ247318. 1 was obtained by RT-PCtL Sequence analysis showed that the full-length eDNA of Pyrus pyrifolia ev. Pinguoli PAL was a complete open reading frame of 2 160 bp and speculated to encode 719 amino acids, and it had a homology over 95% with those of Pyrus bretschneideri and Pyrus communis while the homology of the encoded amino acid sequences reached over 95%. And a plant expression vector contained PAL gene recombinant plasmid PBI121- PyPAL was successfully constructed and transformed into agrobacterium tumefaciens LBA4404 forming an engineering strain, laying the foundations for further study on coloring regulatory genes and genetic improvement for fruit coloring of Pyrus pyrifolia cv. Pingguoli.