| Ascorbic acid or vitamin C, is a small molecule antioxidant with high abundance in plant tissue. It plays an important role in scavenging free radicals, regulating of cell growth and division, and resistance to environmental stress. In addition, it is also a necessary micronutrient for maintain healthy of human. AsA content has become an important indicator to measure the quality of agricultural products, and thus be payed close attention. Isolation, cloning genes which be involved in AsA accumulation and metabolism, and analyzing the functions and expression patterns of these genes, have a great significance for further understanding of AsA accumulation and metabolism in plants, increasing AsA content and enhancing plant stress resistance.In this study, two key enzymes genes Faapx-c and Fadhar, which involved in AsA metabolic pathway were cloned from Fragaria×ananassa cv. Toyonaka rich in vitamin C. And the structures and functions of them were predicted by means of bioinformatics. At the same time, the expression patterns of the two genes be analyzed in different tissues and different developmental stages of fruit in strawberry. The following findings were obtained:1. The primers were respectively designed by comparing homologous genes from GenBank, and the conserved regions of apx and dhar were obtained from Fragaria×ananassa cv. Toyonaka. On this basis, Faapx-c gene sequence including the full of 3' end was obtained by RACE, named Faapx-c. The sequence was 1034 bp in length, containing a ORF of 753 bp, which encodes a polypeptide of 250 amino acid residues a molecular mass of 27.359kDa and a pI of 5.92. The non-coding region of 3'end was 248 bp, containing the potential polyadenylation signal aaataa and poly (A) of 28bp. A full length of open reading frame of dhar gene was isolated by SON-PCR, named Fadhar. The sequence was 1374 bp in length interrupted with three introns, including a full ORF of 887 bp, which encoding a deduced protein of 190 amino acids with a molecular weight of 20.987 kDa and a pI of 6.24.2. Homology analysis of Faapx-c and Fadhar genes showed that they shared high homology with other plants.The nucleotide sequence homology were respectively over 65% and 62%, and amino acid sequence homology were respectively over 79% and 57%. This suggested they are all conservative genes.3. The base compositions of Faapx-c and Fadhar CDS were analyzed by Bioedit. The results showed that the numbers and percents of A, C, G, T in Faapx-c are 171 (22.71%),200(26.56%),201(26.69%) and 181(24.04%), respectively. The content of A +T is (46.75%) lower than G+C(53.25%); The numbers and percents of A, C, G, T in Fadhar are 144(25.13%),125(21.82%),147(25.65%),157(27.40%), respectively. The content of A+T is (52.53%) higher than G+C(47.47%). The analysis of codon preferences of Faapx-c and Fadhar CDS showed that they all preferred to use of T ending codons. Faapx-c contains five E. coli rare codon, and they are scattered in the suquences; and Fadhar contains only one E. coli rare codon.4. The analysis of physicochemical properties of FaAPX-c and FaDHAR indicated that they were all acidic, hydrophilic and structural stable protein by Bioedit and ProtParam tools in ExPASy. Secondary structures analysis showed that FaAPX-c and FaDHAR contained mainly a-helix and random coil predicted by ANTHERWIN5.0 and PredictionProtein.3D structures of FaAPX-c and FaDHAR were predicted using the ESyPred3D and CPH models-3.0. FaAPX-c only contains 12α-helices predicted by ESyPred3D, but it contains 12α-helices and twoβ-sheets predicted by CPH models-3.0. FaDHAR contains sevenα-helix, twoβ-sheet and one turn predicted by ESyPred3D, but it contains eightα-helix, threeβ-sheet and one turn predicted by CPH models-3.0.3D structures of FaAPX-C and FaDHAR were reliable by analysis of corresponding Ramachandran blots using Swiss-Pdb Viewer.5. Function prediction was carried out in net server. The results showed that FaAPX-C was a member of plant peroxidase superfamily, containing peroxidases active site signature (APlmLRLaWHSA) and peroxidases proximal heme-ligand signature (DIVALSGGHTL). And FaDHAR was a member of GST-S-family superfamily, containing two soluble glutathione S-transferase terminal domain. FaAPX-c and FaDHAR had no signal peptides, coils, transmenbrane domains, thus they were not secreted proteins or transmembrane proteins. In addition, they had no N-glycosylation sites, but with some O-glycosylation sites and phosphorylation sites. Electronic expression profiles revealed that apx and dhar expressed in different tissues of plants, which suggested that they played an important function in plant development.6. The expression profile analysis showed that Faapx-c and Fadhar can be expressed in different tissues of Fragaria×ananassa cv. Toyonaka, especially in fruits. Faapx-c may be associated with fruit growth and development, because the amounts of expression were increased gradually from small green; Fadhar is highly expressed during fruit development, which regulates the level of AsA accumulation in strawberry fruits. |
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