Abstract:

【Objective】The aim of this study was to explore the role of VviHKT1;7 in the salt tolerance mechanism of grapes, so as to provide a theoretical reference for the subsequent cultivation of new salt-tolerant varieties. 【Method】DANMAN and MEGA software were used to analyze the biological information of VviHKT. The strongly salt resistant rootstocks SA15, SA17 and the commonly used rootstock 1103P tissue cultured seedlings were used as materials. Seedlings were treated under 100 mmol·L^-1 NaCl for 0, 3, 6, 12, 24, 48 h, and the corresponding time of water treatment were taken as control. Real-time quantitative PCR (qRT-PCR) was used to detect the relative expression of HKT1 in the roots of grapes. VviHKT1;7 was cloned from SA17 cDNA and then linked with pRI101-AN-GFP, and the inflorescence of Arabidopsis thaliana was infected by Agrobacterium tumefaciens. Subsequently, T₃ homozygous lines were screened out from resistant MS plates. Wild-type and transgenic Arabidopsis seeds were sowed on MS plates and MS plates (150 mmol·L^-1NaCl added), their germination and growth were observed, and the root length and fresh weight were counted. The SA17 transgenic grape roots were obtained by Agrobacterium rhizogenes technology. After being treated with 100 mmol·L^-1NaCl for 24 hours, the NMT in vivo physiological detector based on non-damaging micro-measurement technology was used to detect the net flow of Na⁺ and K⁺ instantaneous flow under salt stress in the roots of wild-type and transgenic grapes. 【Result】Multiple sequence alignment and phylogenetic tree analysis showed that VviHKT had high homology, among which the VviHKT1;7 open reading frame sequence length was 1 380 bp and it was the closest to VviHKT1;6. Salt stress significantly induced the expression of HKT1 gene in three varieties of grapes. Among them, the relative expression of VviHKT1;7 was up-regulated, which was still increased after long-term stress. The relative expression of VviHKT1;7 reached the peak at 6 or 12 h under salt stress, and its relative expression in SA17 and SA15 was significantly higher than 1103P. Results of germination and growth experiments in Arabidopsis showed that there was no significant difference between wild-type and transgenic Arabidopsis under normal conditions, but the germination rate, root length and fresh weight of transgenic Arabidopsis were significantly higher than those of wild type under salt stress. Fluorescence detection experiments showed that green fluorescence could be seen in the transgenic grape roots under fluorescence, rather than in the wild-type roots. Further, qRT-PCR results also showed that the relative expression of VviHKT1;7 in the transgenic grape roots was 20-folds higher than that in the wild-type roots. The results of ion flow rate detection showed that the net flow of Na ⁺ both in wild-type and transgenic roots showed efflux under normal conditions. Besides, no significant difference was found between wild-type and transgenic roots (208 and 205 pmol·cm^-2·s^-1) and the fluctuation range of ion flow rate in each time period was small. After salt stress, the Na⁺ net fluxes of them increased significantly, and the fluctuations in each time period also increased; the average net fluxes of wild-type and transgenic roots were 1 053 and 1 340 pmol·cm^-2·s^-1, respectively. Under normal conditions, the K⁺ absorption and efflux of the two roots were in a dynamic equilibrium. Salt stress significantly induced K⁺ efflux, and the efflux of K⁺in transgenic roots was significantly smaller than that in the wild type, which were 406 and 952 pmol·cm^-2·s^-1, respectively. The results indicated that the ability of removing Na⁺ and keeping K⁺ of transgenic roots was significantly greater than that of wild type. 【Conclusion】VviHKT1;7 played an important role in the response of grapes to salt stress, and the overexpression of this gene could improve the adaptability of Arabidopsis and grape roots under salt stress.

Key words: grape, VviHKT1, 7, salt stress, transgene, functional identification