Cloning, expression and bioinformatic analysis of MiFRK genes in mango varieties with high or low sugar content

Abstract:【Objective】Sweetness is one of the key factors for mango fruit quality. In our previous study, we found that the soluble sugar contents of Tainong No.1 (high- sugar-content variety) and Renong No.1 (low-sugar-content variety) had significant differences at different fruit development stages, in which the difference of fructose content was the largest. Moreover, it is well known that fructokinase (FRK), as a key gene of the sugar metabolism pathways, plays an important role in fructose phosphorylation. We also screened out four MiFRK genes from mango genomic data, and there was a significant difference in gene expression levels of the MiFRK1 and MiFRK2 between Tainong No.1 and Renong No.1 based on transcriptome data, suggesting that the MiFRK1 and MiFRK2 would be one of the key genes for mango sugar metabolism. This study intended to further understand the differences of the MiFRK1 and MiFRK2 and their encoded proteins between high- sugar- content mango variety Tainong No.1 and low- sugar- content mango variety Renong No.1.【Methods】Two mango varieties Tainong No.1 and Renong No.1 were selected as materials. Firstly, the DNA sequences of the MiFRK1 and Mi-FRK2 genes in two varieties were cloned respectively and their structural differences between the two varieties were analyzed. The cDNA sequences of the two MiFRK genes were further cloned from two varieties, and the physicochemical properties, structural domain, subcellular localization and phylogenetic trees of their encoded proteins were analyzed by bioinformatic software. Finally, the fruits of each variety in four stages were selected as samples, namely, young fruit stage (about 40 days after anthesis), fruit expansion stage, green maturity stage (harvest maturity stage) and full maturity stage (edible maturity stage, one week after harvest). The gene expression patterns of the two genes in fruits of two varieties at different fruit stages were detected by quantitative real-time PCR (qRT-PCR).【Results】The results showed that there was no difference in the DNA sequences of the MiFRK1 between the two varieties, which contained six exons and encode 334 amino acids. In the promoter region of the MiFRK1, besides the typical core promoter elements, TATA- box and CAAT- box, it also contained cis- acting elements involved in abscisic acid responsiveness, gibberellin responsiveness, salicylic acid responsiveness, and water-deficient, abscisic acid-resistant and antifreeze responsiveness, ABRE, CAT-box, MYC, P-box and TCA-element. However, the MiFRK2 of the two varieties both contained four exons and encode 329 amino acids. And the DNA sequences of the MiFRK2 in two varieties had 69 differential sites, resulting in seven differential positions in the encoded amino acid sequence and the similarity of amino acid sequences being 97.87%. The amino acid sequence analysis showed that both the MiFRK1 and MiFRK2 had the conservative domain of the phosphofructokinase-B (pfkB) family of carbohydrate kinases and contained three substrate- binding regions, six ATP- binding sites and one conservative domain PLN02323. Both proteins encoded by the MiFRK1 and MiFRK2 were stable hydrophobic proteins without transmembrane structure. The subcellular localization analysis revealed that MiFRK1 protein was localized in the chloroplast and cytoplasm, while the subcellular localization analysis result of MiFRK2 in the two varieties was different. The MiFRK2 of Renong No.1 was localized in the chloroplast, the MiFRK2 of Tainong No.1 was localized in the chloroplast and mitochondria. The three-stage structure analysis showed that both MiFRK1 and MiFRK2 proteins existed in the form of a dimer. The phylogenetic tree analysis showed that FRK protein was relatively conservative in evolution, but MiFRK1 and MiFRK2 were clustered in two branches, respectively. The results of the qRT-PCR showed that the MiFRK1 and MiFRK2 genes had different expression patterns at different fruit stages. During the fruit development of the two varieties, the relative expression of the MiFRK1 decreased firstly and then increased. The relative expression of the MIFRK1 in Renong No.1 was larger than that in Tainong No.1, and the change was more significant in Renong No.1. The expression pattern of the MIFRK2 was significantly different between the two varieties. In young fruit stages, the relative expression level of the MIFRK2 in the two varieties was high, and the expression level in Renong No.1 was significantly higher than that in Tainong No.1. Then the expression level of the MIFRK2 in the two varieties decreased in the fruit expansion stage. Subsequently, the relative expression of the MiFRK2 in the green stage of Renong No.1 increased in the green maturity stage and decreased in the fully mature fruit. However, the expression of the MiFRK2 in the green and fully mature stages of Tainong No.1 was opposite to that of Renong No.1. The relative expression of the MiFRK2 continued to decrease in the green maturity stage and increased in the fully mature fruit of Tainong No.1. At last, we calculated the Pearson correlation coefficient between the relative expression of the MiFRK1 and MiFRK2 genes and the fructose content. The results showed that the expression of the MiFRK1 was negatively correlated with the fructose content (-0.798) in Renong No.1, while the relative expression of the MiFRK1 was significantly and positively correlated with the fructose content (0.979* ) in Tainong No.1. However, there was no significant  correlation between the relative expression of the MiFRK2 and the fructose content in the two varieties. 【Conclusion】The MiFRK1 might be regulated by abscisic acid, gibberellin, salicylic acid and stress and functions in chloroplast and cytoplasm in the form of a dimer. The MiFRK2 was different greatly in gene sequence and expression pattern between the two varieties, and the MIFK2 might act in the chloroplast and mitochondria in the form of a dimer. In addition, Both the MiFRK1 and MIFRK2 contributed to the difference in the fructose content between the two mango varieties.