Cloning And Expression Analysis Of Phenylalanine Ammonialyase Gene (PAL) From Cucurbita Pepo And The Study On Varieties Resistance To Botrytis Cinerea

Taken the hulled and hull-less Cucurbita pepo as materials，cloned the full-length sequence ofPAL gene respectively from seed coat and analysed the bioinformatics and expression characteristics,studied the variations in PAL activities and PAL gene expression in different tissues of the plant duringthe different development, maked clear the changes of several defense enzyme activities in differentparts of the plant and the relationship with PAL gene expression level after inoculation of Botrytiscinerea. The experimental results are as follows:1. Using RT-PCR technique, the cDNA segment of PAL gene were cloned respectively from seedcoat in hulled (P3, P5) and hull-less (P13, P15) C. pepo. Homology analysis showed that the PALnucleotide sequences and deduced amino acids of P3, P5, P13, P15were highly homologous to otherPAL nucleotide sequences and amino acids from different plant species, the sequences of amino acidshad a PAL-HAL functional domain and PAL enzyme activity centre region (GTITASGDLVPLSYIA),and they were one member of Lyase_I_Like superfamily. Comparing the PAL sequences of hulled andhull-less C. pepo, the result indicated that25nucleotide sites and15amino acid sites of P3and P13were changed;25nucleotide sites of P5and P15were varied, the intermediate amino acid sequences inP15were in full accord with deduced amino acid sequences in P5.2. The PAL gene sequence from seed coat in hulled C. pepo (P3) were obtained by RT-PCR andRACE technique. The full-length sequence of PAL (designated as CP-PAL) was1720bp, andcontained a complete open reading frame (ORF) of1359bp, which encoded452amino acid residues,the molecular weight of this protein was48.86KDa, pI was6.55. Homology analysis showed that thePAL nucleotide sequences and deduced amino acids of P3were highly homologous to Cucumis sativusPAL nucleotide sequences and amino acids, the number were85%and94%respectively. The aminoacid sequences of CP-PAL contained three functional domains of PAL-HAL, PLN02457,phe_am_lyase and the region of PAL enzyme activity centre (GTITASGDLVPLSYIA), and it was onemember of Lyase_I_Like superfamily. The CP-PAL was most likely to be located in the cytoplasm andendoplasmic reticulum, without signal peptide and leader peptide. And it was non-transmembrane andsoluble protein. The neighbor-joining phylogenetic tree indicated that PAL gene in seed coat of P3hada very close relationship with PAL gene in C. sativus. The main structural element in CP-PAL proteintertiary structure was α-Helix, less in β-Turn and random coil.3. The PAL gene sequence from seed coat in hull-less C. pepo (P13) were obtained. The resultsshowed that CPL-PAL cDNA completed sequence contained2340bp and the ORF contained2115bpand coded704amino acids, the molecular weight and pI were predicted as77.38KDa,6.34separately. The PAL nucleotide sequences and deduced amino acids of P13showed the highest homology of80%and85%with C. sativus, respectively. The amino acid sequences of CPL-PAL contained threefunctional domains of PAL-HAL, PLN02457, phe_am_lyase, and was one member of Lyase_I_Likesuperfamily. The CPL-PAL was most likely to be located in the cytoplasm, endoplasmic reticulum andchloroplast, without signal peptide, leader peptide and transmembrane region. And the protein washydrophilic. The neighbor-joining phylogenetic tree indicated that PAL gene in seed coat of P13hadthe most of familiar genetic relationship with PAL gene in C. sativus. The main structural element inCPL-PAL protein tertiary structure was α-Helix, less in β-Turn and random coil.4. In the whole process of seed coat development, the relative expression of PAL gene in hull-lessC. pepo (P13, P15) was lower than the relative expression in hulled C. pepo (P3, P5). Real-time PCRanalysis revealed that PAL gene in seed coat of P13showed the opposite tendency to P3, the relativeexpression of PAL gene in seed coat of P3was increased, but the relative expression in P13wasdecreased after20d of self-pollination. The PAL gene in hulled and hull-less C. pepo were expressed inleaves, stems, roots, petals, the relative expression in the leaves and petals were higher. During thewhole growth period, the activity of PAL and PAL gene expression in different tissues of hulled C. pepowere higher than that of hull-less C. pepo. The PAL activity and PAL gene expression in the leaves,stems and roots had synergistic increase trend, and reached its peak during the flowering period.5. After inoculation of Botrytis cinerea, the disease index of resistant and susceptible varieties ofhull-less C. pepo were increased, the chlorophyll content of leaves, petioles and stems were decreasedand the activities of POD, PPO and PAL were increased with the extension of onset time. Thechlorophyll content, PAL, POD and PPO activity in leaves, petioles and stems of resistant variety werehigher than that of susceptible variety. Real-time PCR results displayed PAL gene expression in leaves,petioles and stems of resistant cultivar were significantly higher than that susceptible cultivar, and thepeak times of PAL gene expression in leaves and stems were earlier than that of susceptible cultivar.There were differences between PAL gene expression level and trend of PAL activity in different tissuesof hull-less C. pepo.