刘闯^,, 高振, 姚玉新, 杜远鹏^,山东农业大学园艺科学与工程学院/作物生物学国家重点实验室,山东泰安 271018

Functional Identification of Grape Potassium Ion Transporter VviHKT1;7 Under Salt Stress

LIU Chuang^,, GAO Zhen, YAO YuXin, DU YuanPeng^,College of Horticultural Science and Engineering, Shandong Agricultural University/State Key Laboratory of Crop Biology, Tai’an 271018, Shandong

通讯作者: 杜远鹏,E-mail：duyuanpeng001@163.com

责任编辑: 赵伶俐
收稿日期:2020-07-30接受日期:2020-10-14网络出版日期:2021-05-01

基金资助:

山东省重大科技创新工程.2019JZZY010727 国家现代农业产业技术体系建设专项.CARS-29-zp-2 山东省重大科技创新工程.2018CXG0306

Received:2020-07-30Accepted:2020-10-14Online:2021-05-01
作者简介 About authors
刘闯,E-mail：18364030521@163.com。






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.
Keywords：grape;VviHKT1;7;salt stress;transgene;functional identification


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本文引用格式
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 doi:10.3864/j.issn.0578-1752.2021.09.012
LIU Chuang, GAO Zhen, YAO YuXin, DU YuanPeng. Functional Identification of Grape Potassium Ion Transporter VviHKT1;7 Under Salt Stress[J]. Scientia Acricultura Sinica, 2021, 54(9): 1952-1963 doi:10.3864/j.issn.0578-1752.2021.09.012


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0 引言

【研究意义】土壤盐碱化一直是世界各国面临的严峻问题,制约着农业的可持续发展^[1]。葡萄是一种世界分布较广的植物,广泛分布在五大洲,我国葡萄产量居世界第一位^[2],土壤盐渍化是制约我国西北干旱、半干旱产区葡萄产业发展的重要问题^[3]。土壤盐分对植物既有渗透胁迫又有离子毒害^[4,5]。根区的高盐会对葡萄产生渗透胁迫,从而降低植物的水分吸收和蒸腾作用^[6]。盐胁迫会使葡萄叶片栅栏组织的细胞由排列整齐的长柱状变成无规则且有大量沉积物,海绵组织的细胞也减少,叶绿体变得肿胀,出现淀粉粒,液泡膜遭到破坏,从而使细胞出现大大小小的囊泡^[7]。植物组织中过量的Na⁺和Cl^-积累可引起光合作用降低、叶片坏死、果树和浆果发育不均等离子毒性症状^[8,9]。可见盐胁迫直接破坏了叶片内在结构,使气孔开度减小,从而损坏叶片光系统Ⅱ（PSII）,降低叶片净光合速率^[10]。高浓度盐胁迫还会使葡萄果实汁液中含有较高浓度盐分,并提高其pH^[11,12]。【前人研究进展】为了应对盐胁迫,植物需要进行渗透调节和离子分配,以尽量减少Na⁺对植物的危害。HKT转运蛋白是一种高亲和K⁺转运蛋白,但同样能转运Na⁺,帮助植物更好地完成吸钾排钠,维持正常钠钾比^[13]。HKT蛋白包含多个跨膜区和孔环（P-Loop）,其中第一个P-Loop决定转运底物类型,分为两个亚家族,第一个亚家族只负责运输Na⁺,因为第一个孔环中含有丝氨酸,丝氨酸是钠离子特异性载体,主要存在于双子叶植物^[14],如拟南芥、桉树、杨树等。第二种存在于单子叶植物,是K⁺选择性载体,第一个孔环含有甘氨酸,是Na⁺-K⁺的协同运输体或Na⁺/K⁺的单一运载体^[15,16,17]。HKT在小麦中首次被发现并分离^[18],在提高植物抗盐方面发挥着重要作用^[19]。例如,大麦HvHKT1;5有助于幼苗钠离子的排出,对于维持盐胁迫下大麦正常的Na⁺/K⁺至关重要^[20]。水稻OsHKT1;4可以更好地阻止钠离子在茎叶中的积累,可释放木质部Na⁺,尤其在水稻生殖生长阶段,可以提高其耐盐性^[21]。将拟南芥中AtHKT1在马铃薯中过表达,减少了马铃薯叶片中的Na⁺积累,并促进了K⁺/Na⁺稳态,从而最大程度地减少了渗透失衡,维持了光合作用和气孔导度,并提高了植物的生产力^[22]。【本研究切入点】关于VviHKT家族基因在异源和同源转基因材料中的功能鲜有报道;笔者课题组前期研究发现,NaCl胁迫明显上调了葡萄根系VviHKT家族基因相对表达量^[23];本研究进一步对VviHKT1;7在盐胁迫后的表达量及其功能展开研究。【拟解决的关键问题】通过获得VviHKT1;7相关的转基因材料,对其抗盐性做进一步的验证,为葡萄抗盐基因筛选及分子育种提供理论基础。

1 材料与方法

试验于2018—2019年在山东农业大学园艺科学与工程学院葡萄抗逆与栽培实验室进行。

1.1 植物材料及生长条件

葡萄材料选用‘左山一’（V. amurensis Rupr.）×SO4（V. berlandieri×V. riparia）杂种F₁代中的耐盐株系SA17、SA15组培苗以及生产中常用砧木1103P（V. berlandieri×V. rupestris）的组培苗,所有植物均在MS固体培养基上进行体外培养,辅以30 g·L^-1蔗糖、7.0—7.5 g·L^-1琼脂粉、0.2 mg·L^-1的植物激素IBA及少量活性炭。使植物在25℃/20℃下以16 h光照/8 h黑暗的光周期生长,每月将至少有一个芽和叶的枝条用于继代培养。拟南芥种子用75%乙醇表面消毒1 min,4%次氯酸钠表面消毒10 min,再用无菌蒸馏水冲洗5—6次,将其放在含有MS培养基的平板上,4℃春化3 d后放入光照培养箱,待长出2—3片真叶时移入含有50%营养土和50%蛭石的混合基质中,覆膜两周,在22℃、16 h光照/8 h黑暗的光照培养箱中生长。

1.2 基因扩增以及表达载体的构建

基因号为VIT_211s0103g00140,根据基因CDS区域设计特异性扩增引物,上、下游引物5′端分别添加合适的酶切位点,将扩增出的目的基因与克隆载体（pEASY-Simple-Blunt）连接并转化大肠杆菌,菌落PCR筛选阳性克隆。将测序正确的目的基因酶切下来与表达载体pRI101-AN-GFP成功连接,双酶切位点分别为Sal I和Sma I,转化大肠杆菌并摇菌提取质粒。将构建好的表达载体采用冻融热激法转化农杆菌GV3101和K599。

1.3 葡萄转基因根系和转基因拟南芥的获得

参考WANG等^[24]的方法,准备好在无菌条件下进行侵染的葡萄组培苗的枝条,枝条最少有两个芽和叶。取K599农杆菌菌液500 μL加入到20 mL含有卡那霉素（50 mg·L^-1）和利福平（50 mg·L^-1）的液体LB培养基中,并在28℃下摇动孵育过夜,二次活化菌液至金黄色,离心收集菌体,并使用含有100 μmol·L^-1乙酰丁香酮的无菌1/2 MS液体培养基重悬至A₆₀₀=1.0。在250 mL的锥形瓶中进行发根农杆菌的侵染,使剪下来的枝条浸没在液面下,将锥形瓶在25℃下避光振摇15 min。用无菌滤纸将嫩枝插条吸干以除去多余的农杆菌,然后插入固体生根培养基（1/2 MS,20 g·L^-1蔗糖,7.0 g·L^-1琼脂粉,200 mg·L^-1头孢霉素）。感染后4—7周,在芽插条的伤口周围诱导了独立的再生根。当根长约3 cm时,剪取须根在480 nm的荧光下进行检测,根据绿色荧光识别转基因和非转基因的根,每条根系至少取3个须根进行观测。

用已转化的农杆菌GV3101采用花序侵染法侵染拟南芥,在含有卡那霉素（100 mg·L^-1）的MS板上筛选拟南芥种子,直到获得第三代纯合体。

1.4 RNA的提取与实时荧光定量PCR（qRT-PCR）

RNA提取试剂盒、反转录试剂盒及定量SYBR染料均来自康为世纪生物科技有限公司。20 μL反应体系为：2×UltraSYBR Mixture 10 μL,上、下游引物（10 μmol·L^-1）各1.0 μL（表1）,cDNA 1.0 μL,RNase Free ddH₂O 7.0 μL。每个样本至少做3个重复。反应条件：95℃预变性10 min,95℃变性30 s,56℃退火30 s,65℃延伸10 s,40次循环,溶解温度从65℃到95℃,每升高0.5℃保持5 s;停止反应。

Table 1
表1
表1基因扩增及定量所用的引物
Table 1Primers used for gene amplification and quantification

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

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1.5 葡萄根系离子流速测定

根系Na⁺和K⁺净流量的检测仪器为NMT活体生理检测仪（Younger,美国）。参照高海波等^[25]的方法制作电极,Na⁺和K⁺电极前端分别灌充45和180 μm的液态交换剂液柱,电极尖端垂直于根系,显微镜调节电极尖端与根尖的距离,在600 μm左右最佳,测定过程中电极尖端尽可能靠近,但不接触材料表面。

Na⁺电极不适合在含高Na⁺浓度的溶液中进行测定^[23],因此本研究比较盐胁迫解除后野生型和转基因根系根尖分生区Na⁺流速的变化。测定过程参考付晴晴^[23]的方法,挑选生长状态相对一致的对照或100 mmol·L^-1 NaCl胁迫24 h后的葡萄SA17组培苗根系,去离子水冲洗后剪取2 cm左右根尖并放入测试液中平衡30 min后进行测定,待离子流稳定后测定15 min,每个处理测定6条根尖。

测定瞬时K⁺流的动态变化时,先在缓冲液中测定8 min左右,再加入一定体积的NaCl母液（pH 6.0）,使测定液中的NaCl浓度为100 mmol·L^-1,定点测量20 min左右,每个处理测定6条根尖。

测试耗材和试剂均由北京旭月科技有限公司提供。通过Fick扩散定律公式：J=-D×（dc/dx）,可获得该离子的流动速率（pmol·cm^-2·s^-1）,式中的J为离子流速,D是离子/分子特异的扩散常数（cm^-2·s^-1）,dc/dx为离子浓度梯度。

1.6 数据分析

所有试验都至少重复3次,利用Excel和SPSS24进行数据处理和差异显著性分析。

2 结果

2.1 葡萄HKT生物信息学分析

用DANMAN软件对葡萄HKT家族蛋白序列进行比对发现,6个葡萄HKT蛋白相似度为60.25%,利用Pfam软件对6个蛋白序列进行预测,发现都含有HKT蛋白家族特有的结构功能域TrkH（图1）。

图1

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图1葡萄HKT蛋白序列比对

Fig. 1Alignment of grape HKT protein sequence

2.2 葡萄HKT系统进化树分析

利用MEGA软件将葡萄HKT蛋白序列与其他物种蛋白序列构建系统进化树,发现VviHKT1;7、VviHKT1;6和VviHKT1;8的亲缘关系较近;VviHKT1;1和VviHKT1;3亲缘关系最近;而VviHKT1;2则与大豆中的GmHKT1亲缘关系最近（图2）。

图2

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图2葡萄HKT与其他物种HKT系统进化树分析

Fig. 2Phylogenetic tree analysis of VviHKT and other species HKT

2.3 不同葡萄品种中HKT家族成员的盐胁迫响应分析

如图3所示,在100 mmol·L^-1 NaCl胁迫下,所有HKT在SA17、SA15、1103P根系中的表达量都有升高的趋势,各个基因在1103P中的表达量普遍低于SA17、SA15。胁迫12 h后,VviHKT1;1、VviHKT1;2、VviHKT1;3、VviHKT1;6、VviHKT1;8的表达量普遍呈下降趋势,而VviHKT1;7的表达量呈现下降后又上升的趋势,且在3个品种中的表达量也普遍较高,在胁迫6或12 h时达到峰值,分别上升了14.73、16.8、10.32倍。

图3

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图3盐胁迫后不同葡萄株系根部HKT的表达分析

Fig. 3Expression analysis of HKT in roots of different lines after salt stress

2.4 VviHKT1;7的克隆

以SA17的cDNA为模板克隆基因,经PCR扩增获得了一条约1 400 bp的条带（图4）,测序后发现与葡萄基因组数据库中的序列一致。

图4

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图4VviHKT1;7编码区全长的PCR扩增

Fig. 4PCR amplification of the full length of the coding region of VviHKT1;7

2.5 盐胁迫对野生型和转基因拟南芥种子的发芽情况影响

如图5-A、B所示,在正常生长条件下,野生型和转基因种子发芽率无明显差异,盐胁迫抑制了种子的发芽,且对野生型种子抑制更明显。野生型与3个转基因株系种子的发芽率分别为46%、85%、90.3%、95%（图5-C）,可见盐胁迫下转基因种子的发芽情况明显好于野生型。

图5

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图5野生型和转基因拟南芥种子发芽情况

A：拟南芥种子在MS板上一周后的发芽状况;B：拟南芥种子在添加150 mmol·L^-1 NaCl的MS板上一周后的发芽情况;C：NaCl胁迫后发芽率统计。**表示差异极显著（P<0.01）;WT代表野生型,OE-1、OE-2、OE-3代表不同的转基因株系。下同
Fig. 5Germination of wild-type and transgenic Arabidopsis seeds

A: Germination status of Arabidopsis seeds after one week on MS plate; B: Germination of Arabidopsis seeds on MS plate with 150 mmol·L^-1 NaCl added for one week; C: Statistics of germination rate after NaCl stress. ** indicates extremely significant difference (P<0.01); WT stands for wild type, OE-1, OE-2, and OE-3 stand for different transgenic lines. The same as below

2.6 盐胁迫对野生型和转基因拟南芥的生长影响

将野生型和转基因拟南芥种子在MS板上生长一周后,挑选根长相对一致的拟南芥转移到MS板和添加150 mmol·L^-1 NaCl的MS板上,观察8 d后的生长情况（图6-A、B）。MS板上的拟南芥各株系间根长、鲜重都无显著差异,盐胁迫下野生型及转基因株系主根平均长度分别为19.3、30.2、30.6和31.4 mm（图6-C）,平均鲜重分别为0.027、0.057、0.057、0.053 g（图6-D）,可见转基因拟南芥在盐胁迫下的生长状况明显好于野生型。

图6

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图6盐胁迫后野生型和转基因拟南芥的生长分析

*表示差异显著（P<0.05）
Fig. 6Growth analysis of wild-type and transgenic Arabidopsis after salt stress

* indicates significant difference (P<0.05)

2.7 转基因葡萄根系的获得

如图7-A所示,剪取须根进行检测,因为基因表达载体带有GFP标签,所以在荧光下检测时转基因根系会观察到绿色荧光,而非转基因根系无绿色荧光。剪取少量侧根而不损坏主根,以便后续进行离子流速试验,利用qRT-PCR对筛选出的根系进一步进行鉴定,结果显示转基因根系目的基因的表达量显著高于野生型（图7-B）。

图7

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图7葡萄转基因根系的检测

A：转基因根系的荧光检测;B：转基因根系的定量检测
Fig. 7Detection of grape transgenic roots

A: Fluorescence detection of transgenic roots; B: Quantitative detection of transgenic roots

2.8 野生型和转基因葡萄根系Na⁺净流量的检测

正常情况下,野生型和转基因葡萄根系Na⁺外排较低,约200 pmol·cm^-2·s^-1;100 mmol·L^-1 NaCl处理24 h后,Na⁺外流的净流量明显增大,转基因根系外排能力明显大于野生型（图8-B）。如图8-C所示,在15 min测量过程中,正常条件下根系Na⁺平均净流量无明显差异,盐胁迫后转基因根系平均净流量明显高于野生型,进一步说明转基因根系可以更有效地调控Na⁺外排。

图8

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图8Na⁺净流量的检测

A：根系离子流测试位点;B：野生型和转基因葡萄根系的Na⁺净流量的检测;C：Na⁺平均净流量
Fig. 8Net Na⁺ flux detection

A: The test site of root ion flow; B: Net Na⁺ flux detection of wild-type and transgenic grape roots; C: Na⁺ average net flux

2.9 野生型和转基因葡萄根系K⁺净流量的检测

如图9所示,在加入NaCl前,野生型和转基因根系K⁺流呈现吸收与外排的动态平衡中,且流速相对比较平稳。当施加100 mmol·L^-1 NaCl后,迅速增加了葡萄根系K⁺的外流,但转基因根系的增加幅度明显小于野生型。

图9

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图9野生型和转基因葡萄根系的K⁺净流量的检测

Fig. 9Net K⁺ flux detection of wild-type and transgenic grape roots

3 讨论

在盐胁迫条件下,离子稳态受到严格的调控,使必需离子积累而毒性离子保持在较低水平^[26,27]。HKT作为K⁺和Na⁺转运蛋白,在维持植物细胞离子稳态方面扮演着不可或缺的角色。

葡萄含有6个高亲和钾离子转运蛋白基因（HKT）,包括HKT1;1、HKT1;2、HKT1;3、HKT1;6、HKT1;7和HKT1;8,其中HKT1;1被认为是转运钠离子的主要候选基因,在根中表达量较高,而HKT1;3是非功能性的^[28]。VviHKT1;2的终止密码子出现在第一个成孔结构域的上游,因此其编码功能性蛋白的可能性较小^[29]。VviHKT1;6、VviHKT1;7和VviHKT1;8在葡萄根尖、花、种子中的表达水平较低,在根中表达更低^[30],但这并不能否定它们的功能。SA15、SA17的耐盐性较强,而1103P的耐盐性相对较弱^[23]。本研究中,在不同的盐处理时间下,VviHKT1;7在SA15、SA17中的表达量都显著高于1103P,初步表明盐胁迫可以诱导VviHKT1;7的表达,表达量高低与葡萄的抗盐能力有关。所以在特定的生长阶段,相关刺激物诱导,以及植物不同的耐盐能力都可能改变基因表达量。

在胡杨中,过表达PeHKT1;1可以通过提高抗氧化系统的效率来增强耐盐性^[31];在大麦中,过表达HvHKT2;1通过增强Na⁺和K⁺的转运能力提高其耐盐性^[32],在棉花中过表达SbHKT1可以通过增加K⁺吸收、K⁺/Na⁺动态平衡和清除活性氧的能力来提高其耐盐性^[33]。在番茄中,沉默HKT1;2增加了盐胁迫下花中Na⁺的积累,从而降低了果实产量^[34]。WU等^[30]用VviHKT1;7转化酵母,在含有50 mmol·L^-1NaCl的培养基上培养,半乳糖诱导转基因表达并观察其生长状况,结果初步表明VviHKT1;7在酵母系统中起着Na⁺转运体的作用。用双电极电压钳（TEVC）电生理试验检测Na⁺和K⁺电导性,结果表明VviHKT1;6、VviHKT1;7和VviHKT1;8是强Na⁺转运体,亚细胞定位表明VviHKT1;6和VviHKT1;7定位于质膜,VviHKT1;8定位于细胞内细胞器。本试验中利用花序侵染法获得转基因拟南芥并进行抗性试验,发现转基因拟南芥在盐胁迫下发芽与根的生长能力明显高于野生型,表明过表达VviHKT1;7可以提高拟南芥的耐盐性,与其他植物中HKT的功能相似^{[31,32,33,34]}。利用发根农杆菌技术获得转基因葡萄根系,并且对其盐胁迫后Na⁺和胁迫下K⁺净流量进行检测,发现盐胁迫后转基因葡萄根系的Na⁺排出能力明显强于野生型,盐胁迫下转基因葡萄根系能更好地防止K⁺流失,进一步验证了VviHKT1;7的Na⁺转运能力,与WU等^[30]研究结果一致,也说明了过表达VviHKT1;7可以更好地帮助葡萄维持细胞离子渗透势的稳态,从而减轻盐胁迫对植物的离子毒害。VviHKT1;8作为细胞器定位基因,可能在调节细胞内Na⁺含量方面发挥一定作用。有关葡萄HKT1家族的相关信号通路及分子机制依然值得进一步研究。

4 结论

盐胁迫显著诱导葡萄HKT家族成员表达量上调,其中VviHKT1;7上调最显著;异源过表达VviHKT1;7可以提高拟南芥在盐胁迫下的适应能力,同源过表达该基因可以提高葡萄根系在盐胁迫下的Na⁺排出和K⁺保持能力。

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