Clone And Analysis Of PAL And CHS Genes In Pingguoli,and The Construction Of PAL Plant Expression Vector

Pingguoli,which belongs to Rosaceae,pyrus,is popular among consumers due to its big size,cold-resistance,high yield,along with red coloration on sunny side and the juicy and crispy flesh.The genes(PAL、CHS)coding enzymes related to fruit coloring were cloned by RT-PCR and the plant expression vector of PAL gene was constructed in this experiment,laying the foundations for further investigations on the molecular mechanism of fruit coloring as well as the functions of genes.The results are as followings:1、The full-length cDNA sequence of PAL gene was cloned from Pingguoli for the first time and named as PyPAL which had a length of2160bp.It was presumed that coding719amino acids which had a MV of77.91KDa and a PI of6.18.And it is highly homologous with the sequences of Rosaceae plants by homology comparison.2、Bioinformatic analysis showed that the whole polypeptide chain of PyPAL contains416hydrophilic and295hydrophobic amino acids,presenting a hydrophilic character and that it was located in cytoplasmic matrix,without transit peptides or transmembrane domains.The prediction of the secondary structure of PyPAL protein showed that the percentages of a-helix(H), P-sheet(E) and random coil(C) were50.49%,2.36%and47.15%respectively. Therefore,the secondary structure of PyPAL protein mainly consists of a-helixs and random coils.3、The plant expression vector named as PBI121-PyPAL containing PAL gene was constructed and transformed into agrobacterium tumefaciens LBA4404by freeze-thaw transformation method,establishing the bases for transformations from PyPAL into model plants or other plants to further explore its functions.4、The full-length cDNA sequence of CHS gene(named as PyCHSl) obtained by cloning was1170bp, of which A+T accounts for47.26%,while C+G52.74%.And it presumably encodes389amino acids,with a MV of42.5KDa and a PI of5.77,and it is highly homologous with those of Rosaceae plants displayed by homology comparison. 5、Bioinformatic analysis showed that the whole polypeptide chain of PyCHSl contained215hydrophilic and166hydrophobic amino acids,reveling a hydrophilic nature. It was located in cytoplsma matrix,without transit peptides or transmembrane domains.The prediction of the secondary structure of PyCHSl protein showed that the percentages of a-helix(H),random coil(C) and β-sheet(E) were44.22%,32.39%and23.39%respectively.Thus,a-helix and random coil are predominant in the secondary structure of PyCHS1protein.