Identification and expression analysis of APX gene family in grape

Abstract：【Objective】Grape is one of the oldest fruit trees in the world, native to Europe and Asia. Although the cultivation area of grape is very large and wide, grape cultivation is now facing many abiotic stresses, such as drought, low temperature, salt damage and so on. APX gene plays an important role in removing ROS (reaction oxygen species) producing under stress in plants. This experiment aims to identify APX gene family members in grape, and predict the characteristics of this gene family, so as to provide a basis for the analysis of VvAPX genes’function and the mining and utilization of resources of resistant genes to the stresses in grape.【Methods】Plantlets of‘Pinot noir’(Vitis vinifera) cultured in vitro, were stored in the laboratory and stem-segments with single bud were subcultured on the medium (GS+0.2 mg·L^-1 IAA) in a growth chamber under 16 h of light/8 h of dark conditions at 25 ℃/20 ℃for 30 d. The subcultures, which had consistent growth state and were healthy, were treated with abiotic stresses, including 10% PEG, 500 μmol · L^-1 ABA, 200 mmol · L^-1 NaCl and 4 ℃ low temperature and pure water as a CK. Each treatment was repeated three times. APX gene family members were searchedfrom the grape genome in the website. The physicochemical property, phylogenetic relationship, cis-acting element distribution in the 2 kb upstream region of the promoter, gene chip expression were analyzed. The expression of genes under different abiotic stresses was analyzed using qRT-PCR.【Results】 Seven APX gene members in the grape had been homogenously searched, and the gene family could be divided into two sub families (Ⅰ, Ⅱ). The physicochemical property analysis showed that VvAPX02 had the longest protein sequence, CDS sequence and the largest molecular weight in all genes. In addition, VvAPX07 had the shortest protein sequence and the largest full length of genome. We find that all genes were located on 5 chromosomes of the grape, namely the 3th, the 4th, the 6th, the 8th and the 18th chromosomes, respectively. Among them, both the 4th and the 18th chromosomes had two genes, the former had VvAPX06 and VvAPX07, and the latter had VvAPX01 and VvAPX02. Analysis of gene structure showed that VvAPX07 had the longest intron region beyond 14 kb. Genes from the same subgroup had similar structures, such as VvAPX03 and VvAPX04 in group Ⅰ, both of them had 8 exons. VvAPX02 had 12 exons, VvAPX01 had 11 exons, VvAPX06 had 10 exons, VvAPX05 had 9 exons, and VvAPX07 had 3 exons. Besides, only VvAPX01, VvAPX02, VvAPX03 and VvAPX05 had integrated structure. VvAPX04 had no downstream (3’UTR), and VvAPX06 and VvAPX07 had no upstream (5’ UTR) and downstream. Multiple sequence alignment revealed that the seven APX genes had high homology because all of them had active site A and binding site H. The conservative motif analysis showed that all APX genes in the grape had motif1, among them VvAPX01 and VvAPX07 only had motif1. Secondary structure analysis showed that the 7 proteins encoded by VvAPX gene family were mainly composed of alpha helix and random coil. The subcellular localization indicated that APX gene family was mainly expressed in cytoplasm, chloroplast, nucleus, mitochondria, vacuole and plasma membrane. VvAPX03, VvAPX04, VvAPX05, VvAPX06 and VvAPX07 were expressed in cytoplasm. VvAPX01, VvAPX02, VvAPX05, VvAPX06 and VvAPX07 were expressed in chloroplast. VvAPX03 and VvAPX06 were expressed in nucleus. VvAPX02, VvAPX03, VvAPX04, VvAPX05 and VvAPX07 were expressed in mitochondria. VvAPX05 was the only one expressed in the vacuole. Both VvAPX02 and VvAPX06 were expressed in plasma membrane. The cis-acting element analysis showed that all VvAPX genes contained MYB and MYC, which were concerned with drought resistance. Gene chip expression analysis showed that the expression level of VvAPX01, VvAPX03 and VvAPX07 decreased significantly under ABA stress. Compared with the control group 3 (CK3), all of genes were up-regulated under the treatments of salt, PEG and 4 ℃, among which VvAPX01, VvAPX04 and VvAPX05 were more obvious than the others. Besides, the genes family expressed differently under different abiotic stresses at different time, such as VvAPX03 was up- regulated significantly higher than those of the control group 3 (CK3), salt and PEG treatment for 24 h was up- regulated significantly higher than them under other time period. Real- time fluorescence quantitative analysis showed that the VvAPX genes family expressed distinguishingly under different abiotic stresses, including 500 μmol · L^-1 ABA，200 mmol · L^-1 NaCl，10% PEG and 4 ℃ low temperature, and there were significant differences in the expression levels between the treatments. It was found that the expression pattern of VvAPX02, VvAPX03, VvAPX06 and VvAPX07 were analogous, the expression of all of them was lower than those of the control group (CK), 500 μmol· L^-1 ABA, 200 mmol· L^-1 NaCl, 10%PEG and 4 ℃ stresses. The expression pattern of VvAPX01 and VvAPX05 were analogous, the expression of both of them was higher in the leaves under 10% PEG than those of the control group (CK). VvAPX04 gene was distinctly up expressed, which was 14.5 times higher than that of the control group (CK).【Conclusion】The experiment preliminarily identified the APX gene family members in the grape and predicted the functions of this gene family, whichwould provide references for further studies on APX gene functions in the grape.