Functional identification of 6-phosphogluconate dehydrogenase Md6PGDH6 gene in apple

Abstract：【Objective】The catabolism of saccharides includes glycolysis pathway and pentose phosphate pathway. 6-phosphogluconate dehydrogenase is a key enzyme and rate-limiting enzyme in the pentose phosphate pathway. Previous studies have found that 6-phosphogluconate dehydrogenase could respond to biotic and abiotic stresses, and promoted the growth of straw mushroom (Volvariella volvacea) and needle mushroom (Flammulina velutipes). At present, there is a little research on 6-phosphogluconate dehydrogenase in woody plants, especially in apple (Malus domestica Borkh.). In order to explore the function of 6-phosphogluconate dehydrogenase gene in apple, the 6-phosphogluconate dehydrogenase gene (MDP0000235230) was cloned from‘Gala'apple and was named Md6 PGDH6 by comparing with the phylogenetic tree of 6-phosphogluconate dehydrogenase family in Arabidopsis thaliana. The bioinformatics were analyzed and the biological functions were studied by transforming‘Orin'calli, Arabidopsis thaliana and tobacco.【Methods】The full length of MDP0000235230 gene was obtained by RT-PCR(reverse transcription-PCR) and PCR. A variety of software and online sites were used to analyze its properties. The physical and chemical properties of Md6 PGDH6 protein were analyzed with ExPASyProtParam tool. Arabidopsis thaliana 6-phosphogluconate dehydrogenase gene family was obtained with TAIR website. The phylogenetic tree was structured with software MEGA-X.The conservative functional domains, secondary and tertiary structures of the protein, subcellular localizations and promoter cis-elements were analyzed using the NCBI CDD tool, SOPMA and Phyre2, the WoLF PSORT online website, and PlantCARE online database. The temporal and spatial expression patterns were studied by quantitative real-time PCR(qRT-PCR). Md6 PGDH6 protein was obtained by using the prokaryotic expression system of Escherichia coli. Pfu DNA polymerase was used to amplify the target gene. Purpose fragment was recycled, and connected to pEASY®-Blunt E1. Then, the recombinant was transformed into DH5 competent cells. Positive clones were screened by PCR, and several positive clones were selected and sent for sequencing. A small number of sequenced correct positive clones was dipped and shaken, and extracting plasmids were transformed into Trans BL21 competent cells. Trans BL21 recombinant cells were cloned and screened using PCR, and then the positive clones were used for in vitro protein induction experiments.“A”was added before the forward and reverse primers of the target gene as the forward primer and reverse primer connecting the overexpression vector pCXSN-Myc. High fidelity DNA polymerase HiFi was used to clone target fragments. The product was connected to pCXSN-Myc at 16 ℃ overnight, and then transformed into agrobacterium.‘Orin'calli transgenic with Md6 PGDH6 and Arabidopsis thaliana and tobacco with heterogenously expressed Md6 PGDH6 were obtained by agrobacterium-mediated transformation. The phenotypes of the obtained transgenic calli and wild calli were observed by measuring the fresh weight of different calli. A similar experiment was carried out in Arabidopsis thaliana and transgenic tobacco to verify the conserved function of the gene in different species. Observations were made by measuring the length of the primary root and the fresh weight of the shoot of Arabidopsis thaliana. In addition, the activity of 6-phosphogluconate dehydrogenase in Arabidopsis thaliana was also detected to indicate that Md6 PGDH6 indeed promoted the growth of Arabidopsis thaliana by increasing the activity of 6-phosphogluconate dehydrogenase in Arabidopsis thaliana.【Results】Phylogenetic tree analysis showed that MDP0000235230 gene was classified as At4 g29120.1 in Arabidopsis thaliana, and MDP0000235230 was named Md6 PGDH6 gene. Md6 PGDH6 gene contained a 942 bp complete open reading frame, encoding 313 amino acids.Protein molecular weight was 37.68 kDa and its theoretical isoelectric point was 8.28. Md6 PGDH6 protein had a conserved structural domain of MmsB, belonging to NAD binding 11 superfamily, and its overall performance was hydrophilic. By SOPMA analysis, the secondary structure of Md6 PGDH6 protein in apple was predicted to consist of 42.81% α-helix, 32.59% random coil, 16.29% extended strand and 8.31% β-turn. Therefore, α-helix and random coil were the largest structural elements of Md6 PGDH6 protein in apple, while the extended strand and β-turn were scattered in the whole polypeptide chain. The Phyre2 prediction also proved this outcome. TMHMM analysis of the amino acid sequence of Md6 PGDH6 in apple showed no transmembrane domain. WoLF PSORT was used to predict the location of the protein in chloroplast. The promoter of Md6 PGDH6 gene was found to contain CAT-box, a cis-acting element in meristem. There were also environmental response elements, such as the light response element Box 4, and abiotic stress response elements, such as the low temperature response element LTR and the drought response element MBS. In addition, there were also a variety of regulatory elements related to hormone response, such as ABA response element ABRE, ethylene response element ERE, gibberellin response element P-box and salicylic acid response element TCA-element. The qRTRCR showed that Md6 PGDH6 gene was expressed in different tissues of apple, with higher expression in stem, peel, pulp and seed and lower expression in root, leaf and flower. Md6 PGDH6 protein with correct prokaryotic expression was obtained. Md6 PGDH6 gene promoted the accumulation of biomass in calli of‘Orin'. Md6 PGDH6 gene promoted the primary root length and shoot growth of Arabidopsis thaliana. Md6 PGDH6 gene promoted the growth of primary root length in tobacco.【Conclusion】Md6 PGDH6 was highly expressed in stem, peel, pulp and seed. Md6 PGDH6 contributed to the growth of‘Orin'calli, and such a conservative mechanism also existed in Arabidopsis thaliana and tobacco.