Cloning and Expression Analysis of PAL Genes of Impatiens uliginosa
LI Lin-Ju1, FENG Zhi-Xi1, LI Xin-Yi1, WEI Chun-Mei1, HUANG Hai-Quan1,2, HUANG Mei-Juan1,*
1 College of Landscape Architecture and Horticulture Sciences, Southwest Forestry University, Kunming, 650224, China; 2 Southwest Research Center for Engineering Technology of Landscape Architecture, National Forestry and Grassland Administration / Yunnan Engineering Research Center for Functional Flower Resources and Industrialization/Research and Development Center of Landscape Plants and Horticulture Flowers, Kunming 650224, China
Abstract：Phenylalanine ammonia lyase (PAL), the first key enzyme in the secondary metabolic pathway of plants, which plays an important role in the biosynthesis of anthocyanin and lignin, and the stress and disease-resistance in the plants, at present, there is no related report on PAL gene of Impatiens uliginosa. In this study, the flower organs of I. uliginosa were utilized as the experimental materials, IuPAL were cloned by rapid-amplification of cDNA ends (RACE) and reverse transcription PCR (RT-PCR) technologies and the gene sequence analysis and qRT-PCR expression analysis were performed. The results showed that 3 fragments of PAL genes were amplified from I. uliginosa, named as IuPAL1, IuPAL2 and IuPAL3 respectively. Their full lengths of cDNA sequences were 2 154, 2 145 and 2 136 bp, encoding 717, 714 and 711 aa, respectively. Both IuPAL1 and IuPAL2 genes contain 1 intron, while IuPAL3 without intron. It was found that all 3 IuPAL proteins were stable hydrophilic proteins with α helix as the main secondary structure by using the bioinformatics software. The result of gene homology comparison showed that IuPAL proteins of I. uliginosa had high homology with those of other plants, which reached more than 80%. Phylogenetic analysis showed that IuPAL1 and IuPAL2 were clustered into one branch, while IuPAL3, IuPAL1 and IuPAL2 were co-polymerized into a large branch, which suggested that 3 genes were orthologous. On the basis of the qRT-PCR results, it showed that 3 IuPAL genes were expressed in 4 different flower colors and 4 different periods flowers of I. uliginosa, among which IuPAL1 and IuPAL2 were the highest in deep red flowers , while IuPAL3 was the highest in pink flowers. Meanwhile, IuPAL1, IuPAL2 and IuPAL3 were the lowest in whiteflowers. It's speculated that the 3 copies of PAL genes were involved in the anthocyanins synthesis of I. uliginosa. In addition, there existed redundancy among them, in which PAL1 and PAL2 were the major genes in the anthocyanin biosynthesis pathway of deep red flowers. The above results provide some basic data and theoretical basis for further understanding the flower color variation mechanism and new variety cultivationof PAL gene in I. uliginosa.
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