关键词: 葡萄, K ⁺/H ⁺逆向转运体, 生物信息学, 胁迫处理

Abstract:

【Objective】 Isolation and characterization of KEA family genes from grape. Analysis of the tissue-specific expression patterns of KEA family genes and response to K ⁺ depletion, ABA, NaCl and sorbitol treatments. Screen the potential major KEA genes in grape. 【Method】 By carrying out homology-based cloning, putative KEA family genes were isolated and characterized from grape. A phylogenetic tree was constructed by multiple alignment of KEA family proteins from 9 known plants (grape, Arabidopsis, rice, maize, sorghum, slender false brome, polar, pear, and apple) using the neighbor-joining method via MEGA7.0 software. Details of grape KEA family genes and encoded proteins were analyzed with the help of bioinformatical analysis softwares. By screening the EST database, electrical expression profiles of grape KEA genes were determined. Quantitative real-time PCR (qRT-PCR) was carried out to analyze the expression patterns of KEA family genes and response to K ⁺ depletion, ABA, NaCl, and sorbitol treatments, and obtained the major genes. 【Result】 Four KEA family genes were isolated from grape, entitled by VvKEA1—VvKEA4, which were all containing the K/H exchanger and TrkA-N functional domains that belonging to the classic plant KEA family antiporters. The amino acid sequences of KEA proteins from 9 plants shared an overall identity of 33.10%. These KEA members were classified into 2 major groups (Groups Ⅰ and Ⅱ), and VvKEA1and VvKEA2 belong to Group Ⅰ that containing 7 Motifs, while VvKEA3 and VvKEA4 belong to GroupⅡ that just containing 4 Motifs. Phylogenetic tree analysis showed that VvKEA1, VvKEA 2 and VvKEA 4 of grape were closely clustered with AtKEA2, AtKEA 3 and AtKEA 5 of Arabidopsis, respectively, and VvKEA3 was clustered with PbrKEA5 of pear and MdoKEA7 of apple. KEA members of 4 grass family plants (rice, maize, sorghum and slender false brome) were prone to clustered together, while three woody plants (polar, apple and pear) KEA members were prone to clustered together. Mainly localized in plasma membrane, all predicted VvKEA proteins possessed similar tertiary structures, contained 12 or 13 transmembrane domains (TMs), and the theoretical isoelectric point (pI) were all less than 7.0. In particular, only VvKEA3 possessed the signal peptide. Fifteen cis-acting regulatory elements, including the stress response, nutrition and development, hormone response and circadian rhythm regulations, et al., were identified in the promoter region of VvKEA genes. Expression profile analysis showed that VvKEA family genes were expressed in different tissues or organs in grape, and the highest percentage was predicted in fruit, followed by leaf, seed, root and pistil. qRT-PCR analysis showed that VvKEA3 was the most abundant expressed gene during different parts of 8-year-old ‘Rosario Bianco’ on the whole, especially in fruitlet, and the other 3 genes were less expressed with similar amount. In grape seedlings, VvKEA1—VvKEA4 genes were more sensitive to ABA treatment, whose expression were all induced in both tested shoots and roots, but had no response to NaCl treatment. The expression of VvKEA3 in both shoots and roots and VvKEA1 in shoots were up-regulated by K ⁺ depletion treatment, and the expression of VvKEA3 in both shoots and roots and VvKEA4 in roots were increased by sorbitol treatment. 【Conclusion】 Four predicted KEA family genes were cloned and characterized from grape, which were majorly expressed in fruit, leaf and seed. Notably, VvKEA3 was the most abundant gene in 8-year old grape tree, especially in fruitlet, whose expression was prone to be regulated by K ⁺ depletion, ABA, and sorbitol osmotic stress. VvKEA3 may be a crucial K ⁺/H ⁺ antiporter during grape fruit development.

Key words: grape, K ⁺/H ⁺ antiporter, bioinformatics analysis, stress treatment