Identification and expression analysis of NHX genes family in grape

Abstract: 【Objective】Grape (Vitis vinifera L.) is widely grown the world. China is the second country for grape production in the world. As the increase of cultivation area, it is very important to study the resistance of grape to the abiotic stresses. NHX has the functions of maintaining Na+ (K+) concentration, regulating pH in cells, controlling cell expansion, and maintaining cell turgor. NHX is a key factor related to drought and salt tolerance in plants. The bioinformatics characteristics of grape NHX genes family and its expression under abiotic stresses were surveyed to screen out family members related to abiotic stresses.【Methods】The test materials were the seedlings propagated in vitro of‘Red Globe'preserved by the College of Horticulture, Gansu Agricultural University. The single-bud stem segment of the‘Red Globe'grape was cultured on the subculture medium (MS+0.2 mg · L^-1 IAA) for 35 d. Then the tissue culture seedlings were removed from the containers, the root agar was washed off, and they were transferred into MS liquid medium without hormones. The seedlings wer cultured in incubator at (28±0.5) ℃with, a photoperiod of 16 h/8 h (light/dark) . After 30 d culture, the seedlings were transfered to the MS media with 10% PEG, 100 mmol · L^-1 ABA and 200 mmol · L^-1 NaCl, respectively. The pure water was used as CK. Three biological replicates were set for each treatment, and the treatment duration was 6 h.And based on the NHX gene sequences of Arabidopsis thaliana, rice and poplar, homologous cloning, chromosome localization, amino acid composition, physicochemical properties, motif, protein secondary structure and gene chip expression of grape NHX gene were analyzed. Quantitative analysis of grape NHX gene family expression by qRT-PCR was performed.【Results】Grape NHXs gene could be divided into two sub families, eight members of the grape NHX gene family were obtained by comparison with the NHX genes of existing plants. According to the localization analysis, eight grape NHX genes were distributed on 6 th chromosomes, VvNHX07 was located on the first chromosome, and VvNHX02 on the 5 th chromosome, VvNHX03 on the 7 th chromosome, VvNHX01 and VvNHX08 on the 14 th chromosome, VvNHX05 and VvNHX06 on the 15 th chromosome, and VvNHX04 on the 19 th chromosome. And the number of exons was 12-23. VvNHX06 gene only had 291 amino acids. VvNHX07 gene had 1 141 amino acids. The full-length protein sequence was predicted by MEME software, and 8 domains were obtained. No conserved amino acid sequence was predicted in VvNHX07. Only motif1 was present in VvNHX05, and all 8 domains were existing in VvNHX01, VvNHX02, VvNHX03, VvNHX04 and VvNHX08. Analysis of each domain revealed that motif1, motif2, motif3, motif4, and motif6 all contained 50 conserved amino acids with high conservation. Motif7 and motif8 contained only 41 conserved amino acids, but they were highly conserved. Gene structure analysis revealed that there were significant differences in the number of exons of VvNHXs gene family members, and 8 VvNHXs genes contained introns and exons proton. There were 14 exons proton in VvNHX01, VvNHX02, VvNHX03, VvNHX05 and VvNHX08 genes, 15 exons in VvNHX04, 12 exons in VvNHX06, and 23 exons in VvNHX07. Subcellular localization predicted that grape NHX genes were mainly distributed in cell membrane, endoplasmic reticulum, mitochondria, vacuole and cytoplasm. VvNHX05 was expressed in both peroxisome and cell matrix; VvNHX07 was only expressed in chloroplast. VvNHX01 was only expressed in cell plasmids; VvNHX02 and VvNHX04 were expressed in cell membrane, endoplasmic reticulum, mitochondria, vacuole and cytoplasm; VvNHX05, VvNHX06, VvNHX07 were expressed in the nucleus. The secondary structure of the protein had Alpha helix, Beta turn and Random coil. Therefore, the secondary structure of VvNHXs was mainly composed of Alpha helix and Random coil. The gene chip analysis showed that as increase of the treatment time of salt, ABA and PEG stress the expression levels, of VvNHX02 and VvNHX06 significantly increased in grape leaves. The highest gene expression was found on VvNHX06 at 24 h after treatment. The expression level of VvNHX08 gene significantly increased under 8 h treatment, and then decreased rapidly. The expression level of the VvNHX03 gene reached a maximum at 4 h. After treatment with ABA, the expression levels of VvNHX03, VvNHX04, and VvNHX08 genes decreased as the increase of the treatment time. The results of qRT-PCR analysis showed that there was a difference in the expression of grape NHXs in the leaves with different treatments. Under different stress treatments, the VvNHXs genes families were expressed in different degrees in the leaves. The relative expressions of VvNHX01, VvNHX02, VvNHX03, VvNHX04, VvNHX05, VvNHX06 and VvNHX08 in the leaves under 200 mmol · L^-1 NaCl were up-regulated to different degrees compared with those in the control group (CK) , among which the up-regulated expressions of VvNHX06 were the most significant, 30 times higher than those in the control group (CK) . After treatment with 100 mmol · L^-1 ABA, the relative expressions of VvNHX02, VvNHX03, VvNHX06, VvNHX07 and VvNHX08 were significantly higher than those of the control group (CK) . VvNHX06 showed the most significant change compared with the control group, which was 60 times higher than those in the control group. VvNHX01 and VvNHX04 were lower than those in the control group (CK) . VvNHX05 did not change significantly compared with those in the control group (CK) . Under 10% PEG treatment, the relative expressions of VvNHX01, VvNHX03, VvNHX05 and VvNHX08 in the leaves were up-regulated to different degrees compared with those in the control group. The upregulation of VvNHX05 was the most obvious, 5 times higher than that of the control group. VvNHX02, VvNHX04, VvNHX06 and VvNHX07 were down-regulated.【Conclusion】VvNHX05 and VvNHX06 genes could be closely related to salt tolerance and drought resistance in the grape plants, and it would provide a reference for the research on abiotic stresses to grape.