Identification and expression analysis of AeAPX gene family in Actinidia eriantha

Abstract:【Objective】Ascorbate peroxidase (APX) is one of the important antioxidant enzymes in the active oxygen metabolism pathway of plants and animals, especially it is the key enzyme to clear H2O2 in chloroplast and the main enzyme of ascorbate acid (AsA) metabolism. However, knowledge about APX gene family members and their evolutionary and functional characteristics in kiwifruit is limited. Therefore, the objective of this study was to identify and analyze the ascorbate peroxidase (APX) gene family of Actinidia eriantha, so as to provide reference for the molecular mechanism of ascorbate acid (AsA) metabolism regulation in A. eriantha.【Methods】In this study, the APXs gene family members of A. eriantha were identified from Kiwifruit Genome Database according the ascorbate peroxidase gene (APXs) family members protein conserved domain of Arabidopsis thaliana, and their physicochemical properties, evolutionary relationship, gene structure and cis-acting elements of promoter were analyzed, and the expression of the AeAPXs at different fruit developmental stages and after light stress was verified and analyzed by quantitative real-time PCR (qRT-PCR).【Results】The results showed that twentynine AeAPX gene members were identified from the whole genome of kiwifruit, and they were not even-y distributed on the chromosomes. They were named as AeAPX1~AeAPX29 according to their positions on the chromosome from the top to bottom. Among them, the AeAPX3 and AeAPX4 on chromosome 4, the AeAPX6 and AeAPX7 on chromosome 7, the AeAPX11 and AeAPX12 on chromosome 10, the AeAPX18 and AeAPX19 on chromosome 19, the AeAPX26 and AeAPX27 on chromosome 28 were tandem repeats. The results of the physical and chemical properties analysis showed that the CDS of these genes were 840-1515 bp, the protein length of the AeAPXs ranged from 275 to 504 aa, and their isoelectric point were between 4.73-9.12, their protein molecular weights were between 30 019.12-55 354.20 ku; and their instability index ranged from 26.63 to 51.59, instability index more than 40 means unstable, except that AeAPX3, AeAPX5, AeAPX6, AeAPX9, AeAPX12, AeAPX13, AeAPX17, AeAPX21, AeAPX22, AeAPX23, AeAPX24 and AeAPX29 proteins are unstable proteins, other AeAPXs belong to stable proteins; aliphatic index ranged from 72.73-100.09, aliphatic index less than 100 means lipid soluble protein, except for AeAPX23 protein, other AeAPXs proteins belong to lipolytic protein. Subcellular localization prediction analysis showed that the AeAPXs gene family members were 16 APX genes predicted to be located in the chloroplast, 5 genes were located in extracellular matrix, 4 genes were located in cytoplasm, 2 genes were located in vacuole and 2 genes were located in nucleus. The secondary structures of these protein were mainly composed of alpha helix and random coil. There were 13 gene family members belonging to the transmembrane proteins, including AeAPX1, AeAPX3, AeAPX5, AeAPX6, AeAPX8, AeAPX11, AeAPX14, AeAPX18, AeAPX21, AeAPX22, AeAPX23, AeAPX26, AeAPX28. The analysis of gene structure showed that the protein motifs of the AeAPXs family genes had obvious block identity, sequence homology and conservation, and most of them contained four exons. A large number of cis-acting elements related to light, hormone and stress response were found in the upstream promoter region of the AeAPXs. These cis-acting elements included optical response element Box4, G- box, GT1- motif, GATA- motif, MRE, TCT- motif and TCCC- motif; Box4 and G- box were distributed more in the gene family; abscisic acid response elements (ABRE) were involved in the ABA and drought response, drought stress response element (MBS), low temperature stress response element (LTR), anaerobic response element (ARE), defense and stress response elements (TC- rich repeats). Conserved domain analysis showed that Motif 3 and Motif 9 present in all AeAPXs proteins, and seven similar conserved motifs widely present in 26 AeAPXs family members, indicating that the amino acid sequences encoded by these 26 genes are highly conservative. The above results showed that the conserved motifs of each member of the gene family in the conserved domain were different in position and quantity, which was likely to lead to gene functional differentiation. Phylogenetic analysis showed that the AeAPXs gene family could be classified into three subgroups; Subgroup A, Subgroup B, Subgroup C were respectively consisting of seven, ten, twelve members of the AeAPXs gene family with a total of 6 co-linear gene pairs. The results of ascorbic acid content determination showed that ascorbic acid content decreased with fruit ripening. And the content of ascorbic acid decreased significantly after bagging. Gene expression analysis showed that the selected 10 genes could be divided into three categories, of which the AeAPX3 and AeAPX27 were grouped into one category. The AeAPX27 had high expression levels in young fruit period, color turning period and physiological ripening period, which was significantly correlated with the content of AsA in fruit. It was the key gene for the regulation of ascorbic acid in kiwifruit. The expression of the AeAPX3 gene was just opposite to that of the AeAPX27. The AeAPX8, AeAPX13, AeAPX14, AeAPX22 and AeAPX25 were grouped together, and their gene expression levels gradually decreased, which was consistent with the content of AsA. The expression levels of the AeAPX6, AeAPX21 and AeAPX24 genes were up-regulated in the three developmental stages of fruit, which was opposite to the content of AsA. The effect of bagging on the expression of these genes was shown in Figure 8, except for the AeAPX3 and AeAPX13, the expression of other genes decreased after bagging, which was consistent with the content of AsA.【Conclusion】A total of twentynine AeAPXs family genes in A. eriantha were identified in this paper, gene expression of fruits in different growth stages showed that the AeAPX27 had high expression levels in three growth stage, which was significantly correlated with the content of ascorbic acid in the fruit. We can speculate that it would be the key gene related to fruit growth and development and regulation of ascorbic acid.