Identification and expression analysis of CHS gene family in grape

Abstract:【Objective】This paper aimed to clarify the expression pattern of the grape chalcone synthase (CHS) gene family in response to exogenous hormones and abiotic stresses.【Methods】The CHS gene family members were identified from grape genome using Blast and HMMER. The CHS gene family was analyzed using bioinformatics, including physicochemical property, gene structure, subcellular location, phylogenetic relationship, and cis-acting element distribution. In vitro propagated plantlets of Vitis vinifera L.‘Pinot Noir’under different treatments were used as the experiment materials. The experimental materials were cultured in a growth chamber under the conditions of 25 ℃, 16 h light / 22 ℃, 8 h dark. After 30 d of culture, these plantlets were transferred to GS liquid media with 0.2 mmol·L^-1 ABA (abscisic acid), 50 mg·L^-1 GA3 (gibberellin), 0.05 mg·mL^-1 NAA (naphthylacetic acid), 5 mmol·L-1 (salicylic acid) SA, or 0.1 mmol·L-1 MeJA (methyl jasmonate). Some plantlets were subjected to the treatments of low temperature (4 ℃), 400 mmol · L^{- 1} NaCl (sodium chloride) or 10% PEG (polyethylene glycol). Meanwhile, the same volume of sterile water and test-tube plantlets with normal growth were used as control. Each treatment had three replicates. qRT-PCR was carried out to detect the expression levels of VvCHS genes in different tissues under different hormone treatments and abiotic stresses. The genes with significant relative expression were selected for cloning and subcellular localization verification.【Results】The isoelectric point of grape CHS gene family members ranged from 5.61 to 8.86, and the instability indexes were between 36.64 and 43.50, five of them were stable proteins, and VvCHS1, VvCHS2, VvCHS3, VvCHS4 were acidic proteins. Phylogenetic tree was constructed from CHS protein sequences of Vitis vinifera, Arabidopsis thaliana, Citrus sinensis and Solanum lycopersicum. The 26 CHS proteins from the four species were divided into three subgroups according to genetic distance. Both CitCHS and SlCHS proteins were clustered in Group Ⅰ, Group Ⅱ and Group Ⅲ were composed of VvCHS1, VvCHS6, VvCHS7 and AtCHS proteins. Phylogenetic analysis showed that the VvCHS and AtCHS had high homology. Except AtCHS2, AtCHS5 and AtCHS8, the exon numbers of the other CHS genes were between 2 to 6, of which 12 genes contained 2 exons and 1 intron. The pressure of gene pair selection showed that purification selection was mainly carried out between the VvCHS gene family and those of the other species. Secondary structure analysis showed that the VvCHS gene family was mainly composed of α-helices and Random coils. Cis-acting element analysis showed that the promoter sequences of CHS genes contained a large number of acting elements related to hormones and stress responses. Tissue specific expression indicated that the expression levels of VvCHS genes were higher in roots and lower in stems. qRT-PCR analysis showed that MeJA treatment induced high expression of VCHS3 gene, and the expression level of VvCHS3 in the stems was the highest, 44.3- fold higher than that in the control group, and the expression level of VvCHS3 in the leaves was 25-fold higher than that in the control group. After NAA treatment, the expression level of VvCHS2 in the roots and the leaves was low, while that in the stems was high. Compared with the control, the expression level of VvCHS3 in roots, stems and leaves was significantly up-regulated under SA treatment. Under the treatment of GA3, the expression levels of VvCHS1 and VvCHS3 in the leaves were significantly higher than those in the roots and the stems. The expression of VvCHS6 was up-regulated only in SA-induced grape root tissue. The results showed that the expression levels of VvCHS genes in different tissues of grape and under different hormone treatments were different. After treatment with NaCl, the expression of VvCHS5 increased significantly, and the expression level of VvCHS5 in the roots, stems and leaves was 30.6, 24.9, and 8.4 times higher than that of the CK, respectively. Under the treatment of 4 ℃ and PEG, the relative expression of VvCHS4 was 53.4 and 24.4 folds in roots, respectively. Under NaCl, 4 ℃ and PEG treatments, VvCHS2 and VvCHS6 genes were up-regulated in the roots, stems and leaves, while the other genes were down-regulated. The above results indicate that there are differences in the expression levels of VvCHS genes in different tissues. VvCHS3 was successfully cloned and transiently transformed into Nicotiana benthamiana by Agrobacterium tumefaciens. Fluorescence transient expression showed that VvCHS3 was localized in the nucleus and cell membrane, which was consistent with the predicted results.【Conclusion】Grape CHS gene family is involved in the regulation of hormones such as SA and MeJA and responses to low temperature, drought and high salt stresses. The study laid a solid basis for further identifying the basic function of grape CHS gene family and understanding the regulatory mechanism of VvCHS in stress.Grape; Chalcone synthase; Bioinformatics; qRT-PCR; Gene cloning【Objective】This paper aimed to clarify the expression pattern of the grape chalcone synthase (CHS) gene family in response to exogenous hormones and abiotic stresses.【Methods】The CHS gene family members were identified from grape genome using Blast and HMMER. The CHS gene family was analyzed using bioinformatics, including physicochemical property, gene structure, subcellular location, phylogenetic relationship, and cis-acting element distribution. In vitro propagated plantlets of Vitis vinifera L.‘Pinot Noir’under different treatments were used as the experiment materials. The experimental materials were cultured in a growth chamber under the conditions of 25 ℃, 16 h light / 22 ℃, 8 h dark. After 30 d of culture, these plantlets were transferred to GS liquid media with 0.2 mmol·L^-1 ABA (abscisic acid), 50 mg·L^-1 GA3 (gibberellin), 0.05 mg·mL^-1 NAA (naphthylacetic acid), 5 mmol·L^-1 (salicylic acid) SA, or 0.1 mmol·L^-1 MeJA (methyl jasmonate). Some plantlets were subjected to the treatments of low temperature (4 ℃), 400 mmol · L^{- 1} NaCl (sodium chloride) or 10% PEG (polyethylene glycol). Meanwhile, the same volume of sterile water and test-tube plantlets with normal