葡萄钾离子转运基因VviHKT1;7在盐胁迫下的功能鉴定

doi:10.3864/j.issn.0578-1752.2021.09.012

摘要/Abstract

摘要：

【目的】探讨VviHKT1;7在葡萄抗盐机制中的作用,为后续培育抗盐品种提供理论参考。【方法】利用DANMAN和MEGA软件对葡萄HKT进行生物学信息分析。以抗盐性较强的砧木SA15和SA17以及生产上常用砧木1103P组培苗为材料,用100 mmol·L^-1NaCl分别处理0、3、6、12、24和48 h,以清水处理相应时间为对照,荧光定量PCR（qRT-PCR）检测HKT1在葡萄根部的相对表达量;以SA17的cDNA为模板克隆基因,连接表达载体pRI101-AN-GFP,利用农杆菌侵染法侵染拟南芥花序,在抗性MS板上筛选直到获得T₃纯合株系;将野生型与转基因拟南芥种子播种于MS板和含有150 mmol·L^-1NaCl的MS板上,观察其发芽和生长情况并统计根长及鲜重;利用发根农杆菌技术获得SA17转基因葡萄根系,100 mmol·L^-1NaCl处理24 h后,利用基于非损伤微测技术的NMT活体生理检测仪检测野生型和转基因葡萄根系Na⁺的净流量以及盐胁迫下K⁺瞬时流量。【结果】多序列比对和系统进化树分析表明,葡萄HKT之间同源性较高,其中VviHKT1;7开放阅读框序列长度为1 380 bp,与VviHKT1;6的亲缘关系最近。盐胁迫显著诱导了葡萄HKT1在3个品种中的表达,其中VviHKT1;7的相对表达量上调较高,长时间胁迫后表达量仍有上升趋势,胁迫6或12 h时表达量达到峰值,且在耐盐性强的SA17、SA15中表达量明显高于1103P。拟南芥的发芽与生长结果表明,正常情况下野生型和转基因拟南芥的发芽和生长情况无显著差异,但盐胁迫下转基因拟南芥的发芽率、根长、鲜重明显高于野生型。荧光检测结果表明,转基因葡萄根系在荧光下可以明显看到绿色荧光,而野生型根系检测不到荧光;进一步qRT-PCR检测结果表明,转基因葡萄根系中VviHKT1;7的表达量是野生型根系的20多倍。离子流速检测结果表明,正常情况下野生型和转基因根系Na⁺净流量显示出外排,各个时间段的波动幅度较小且无显著差异,平均净流量分别为208和205 pmol·cm^-2·s^-1;盐胁迫后,两者Na⁺净流量明显增大,各个时间段的波动幅度增大,平均净流量分别为1 053和1 340 pmol·cm^-2·s^-1。正常情况下两种根系K⁺吸收与外排处于动态平衡状态,盐胁迫显著诱导K⁺外排,转基因根系的外排量明显小于野生型,分别为406和952 pmol·cm^-2·s^-1,表明转基因植株根系的Na⁺外排、K⁺保持能力明显大于野生型。【结论】VviHKT1;7在葡萄响应盐胁迫中发挥着重要作用,过表达该基因可以提高拟南芥和葡萄根系在盐胁迫下的适应能力。

关键词: 葡萄, VviHKT1, 7, 盐胁迫, 转基因, 功能鉴定

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

刘闯,高振,姚玉新,杜远鹏. 葡萄钾离子转运基因VviHKT1;7在盐胁迫下的功能鉴定[J]. 中国农业科学, 2021, 54(9): 1952-1963.

LIU Chuang,GAO Zhen,YAO YuXin,DU YuanPeng. Functional Identification of Grape Potassium Ion Transporter VviHKT1;7 Under Salt Stress[J]. Scientia Agricultura Sinica, 2021, 54(9): 1952-1963.

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基因名称 Gene name	引物序列 Primer sequence	用途 Purpose
VvActin	F: 5′-TCTCAACCCAAAGGCTAATC-3′ R: 5′-GCATAGAGGGAAAGAACAGC-3′	qRT-PCR
VviHKT1;1	F: 5′-ATAGATGGTGGTGAAAAGGCT-3′ R: 5′-GTTGTGATAACAGTAGATGCTCC-3′	qRT-PCR
VviHKT1;2	F:5′-TGAGAGAGACAAGATGAGGGAAG-3′ R:5′-ACAAATCCATACCAGGCGTC-3′	qRT-PCR
VviHKT1;3	F:5′-GAGCATCGCCCTGGAAGTC-3′ R:5′-TGCCGAGAACAGTGATACCC-3′	qRT-PCR
VviHKT1;6	F:5′-AAATGGAGTGACGAGGGGA-3′ R:5′-TAGCCTTCAGACTTTGCCTC-3′	qRT-PCR
VviHKT1;7	F:5′-CCATCTTCATCATCCTTATCTGC-3′ R:5′-CTCTACCCAACTATTAATCCCGG-3′	qRT-PCR
VviHKT1;7	F:5′-ATGATGAACTCTGCTAGTTTCACC-3′ R:5′-AAAGCCCCTGATTACTTCTATCAC-3′	扩增基因 Amplified gene
VviHKT1;8	F:5′-CTTGTAAGTTGTAACCGAGGCA-3′ R:5′-CATGCATTGTGGATAGCTAAGG-3′	qRT-PCR