Identification and bioinformatics analysis of PPO gene family in Chinese chestnut (Castanea mollissima)

Abstract:【Objective】Polyphenol oxidases (PPOs) are oxidative enzymes that convert monophenols and o-diphenols to o-quinones using molecular oxygen. The quinone products are highly reactive fol- lowing tissue damage and can interact with cellular constituents and cause oxidative browning and cross-linking. The browning of kernel occurs in chestnut processing, the metabolism of browning substances is closely related to PPO. However, little is known about the molecular mechanisms regulating this process, by the identification of PPOs gene family in Chinese chestnut and bioinformatics analysis, the present study attempts to establish some basic features of the function of PPO protein that have not been previously described【. Methods】Three PPO genes, CmPPO1 (BUA.CMHBY222345), CmPPO2(BUA. CMHBY216654), CmPPO3 (BUA.CMHBY216989), were collected from the latest published database of Chinese chestnut genome. The bioinformatic analysis of these three genes was carried out for further understanding of their putative functions.ORF regions of PPO gene sequences were founded by ORF finder program in NCBI. The basic physicochemical properties of the PPO proteins were ana- lyzed by ExPASy ProtParam. The secondary structure analysis about the content of α-helix, random coil, extended strand, beta turn in PPO proteins were fulfilled by SOPMA online software. The distribu- tion of curl and the homology modeling of the tertinary structure of PPO genes were performed using Swiss-Model online software. Neighbor-joining phylogenetic tree of BUA, MDP and PGSC based on these edited alignments were generated using MEGA version 6.0 software, pairwise deletion, distance matrix, and 1 000 bootstrap replicates were applied to the tree. Furthermore, expression patterns ofCmPPO1 in three chestnut cultivars with different enzymatic activity were analyzed by Quantitative Re- al-time PCR. The total RNA of chestnut fruit was extracted using EASYspin Plus Plant RNA Kit (Aid- lab, China) according to manufacturer’s instructions. First-strand cDNA was synthesized from 200ng of total RNA using the Prime Script RT reagent kit with gDNA Eraser (TaKaRa, China). The qRT-PCR was performed using the SuperReal PreMix Plus (SYBR Green) (TianGen, China) in a total volume of 20 μL in each well containing 10 μL of 2×SuperReal PreMix Plus, 1 μL of cDNA, and 0.6 μL 10 μmol·L-1primers. The qPCR conditions were 15 min at 95 °C, followed by 40 cycles of 15s at 95 °C, 30 s at 60 °C . Actin gene (ev253704) was used as the reference【. Results】The full- length CDs of the three genes was 1 794-1 809 bp in length and they encoded 509-539 amino acid sequences with an apparent molecular weight of 66.4 -67.4 ku and a theoretical pI of 6.47-6.94 , which contained the conservative domains of tyrosinase (PF00264), PPO1_DWL (PF12142), PPO1_KFDV (PF12143). The CmPPOs belonged to hydrophilic acid protein without transmembrane region and were not secretory protein. The secondary structure of the proteins included the form of α-helix, random coil, extended strand and beta turn mainly. It showed the highest ratio of random coil, which has been reported to be affected by the chain interaction and closely related to the composition of the active part of the protein. And analysis of the tertiary structure of the deduced amino acid sequence showed that it had the same template of the se- quence of MdPPO1 (Malus pumila Mill.) in Rosaceae. The homology of the deduced amino acid se- quence to the PPO sequence of apple was over 70%. The location signals of CmPPO1 in mitochondria and cytoplasm were 39.1% and 34.8% respectively, which were much higher than that in endoplasmic reticulum and other regions. The CmPPO2 and CmPPO3 were mainly located in the nucleus, and the signal proportion was 69.6%. Twenty-three PPO gene coding proteins of apple, potato and chestnut, were analyzed by multiple sequence alignment and the conserved domains among these PPOs were ana- lyzed, the searched motifs were annotater on Pfam with regard to phylogenetic relationship and domain analysis of PPO family encoding proteins, with 6 motifs being founded and being located in conserva- tion domain. Phylogenetic tree showed that genetic relationship was not related to species, but mainly related to the structure and the function of PPO enzymes. The expression patterns of PPO genes in different varieties of chestnut were analyzed, only CmPPO1 was detected. The results showed that PPO activity in chestnut kernel could not directly affect the browning degree, and the browning difference of different varieties would be possibly induced by the PPO expression.【Conclusion】In Chinese chestnut, there were three PPO genes. Their expression was enhanced in three chestnut cultivars with different enzymatic activity.