葡萄类钙调磷酸酶B亚基互作蛋白激酶VvCIPK10的特性与表达

【目的】在葡萄中克隆丝苏氨酸蛋白激酶Vv CIPK10,分析其激酶特性和在逆境胁迫下的表达模式,为进一步研究该基因参与逆境胁迫的分子功能,探讨葡萄抗逆分子机制提供理论依据。【方法】利用电子克隆技术获得Vv CIPK10序列,设计特异引物进行RT-PCR反应,对克隆到的序列进行开放阅读框和保守结构域分析;构建原核表达载体,转化表达菌株后用IPTG进行诱导表达,收集菌体后裂解细胞,制备蛋白上样液,SDS-PAGE电泳对表达产物进行分析,同时对融合蛋白进行可溶性分析;IPTG大量诱导表达融合蛋白,收集菌体后进行超声破碎细胞,用麦芽糖结合蛋白纯化柱纯化MBP-Vv CIPK10融合蛋白,SDS-PAGE电泳进行分析;纯化后的融合蛋白与体外自磷酸化缓冲液进行自磷酸化反应,反应后SDS-PAGE电泳,压磷屏检测体外自磷酸化反应;构建重组瞬时表达载体p BI221-GFP/Vv CIPK10;分离拟南芥原生质体,通过PEG介导的瞬时转化方法将重组表达载体p BI221-GFP/Vv CIPK10转化至原生质体;通过基因枪介导的转化方法将重组表达载体p BI221-GFP/Vv CIPK10转化至洋葱表皮细胞,培养16 h后用激光共聚焦显微镜进行荧光信号检测;选择生长相对一致且健壮的葡萄植株,于干旱、低温和盐胁迫处理后不同时间取样,同时在田间取葡萄不同组织样品,实时荧光定量PCR检测Vv CIPK10在葡萄不同组织中的表达以及在不同逆境胁迫下的表达模式。【结果】PCR克隆获得葡萄Vv CIPK10全长为1 357 bp,5′端非编码区为30 bp,3′端非编码区为156 bp,开放阅读框为1 171 bp,编码436个氨基酸,理论等电点为8.59,分子量为48.7 k Da。保守结构域预测分析显示该蛋白5′端具有一个激酶结构域,3′末端具有一个PPI结构域和一个NAF结构域。BLSATP分析表明葡萄Vv CIPK10与桃树CIPK(XP_007205151)一致性最高(74%)。重组表达载体p MAL-C5X/Vv CIPK10在大肠杆菌中经诱导表达获得与理论分子量(43 k Da+48.7 k Da)相一致的融合蛋白。MBP-Vv CIPK10融合蛋白经柱纯化后获得单一的蛋白条带,Vv CIPK10的自磷酸化活性依赖于Mn~(2+),不依赖于Mg~(2+)和Ca~(2+),EDTA可以抑制Vv CIPK10的自磷酸化活性。亚细胞定位结果显示,Vv CIPK10定位在细胞核、细胞膜和细胞质。Vv CIPK10在葡萄各个组织中均有表达,主要在葡萄根和叶片中大量表达,葡萄茎、花序、果实和卷须中的表达量较低。在干旱、低温和盐胁迫处理后,Vv CIPK10呈现受诱导表达模式。Vv CIPK10的表达在低温胁迫后6 h达到峰值,干旱和盐胁迫后2 h即达到峰值。【结论】葡萄Vv CIPK10能够响应干旱、低温和盐胁迫,推测Vv CIPK10在葡萄抗非生物逆境胁迫中具有重要作用。     

24-表油菜素内酯对葡萄叶片抵御霜霉菌侵染的影响

【目的】研究外源24-表油菜素内酯(24-epibrassinolide,EBR)处理对霜霉菌侵染葡萄叶片的影响,为探究葡萄霜霉病的致病机理提供参考。【方法】试验以欧亚种酿酒葡萄(Vitis vinifera.L)赤霞珠(Cabernet Sauvignon)叶片为材料,在霜霉菌侵染离体葡萄叶片的初期,研究不同浓度的EBR处理(0.1、0.5和1.0 mg·L~(-1) EBR)对葡萄叶片霜霉病发病率和病情指数、霜霉菌菌丝生长、孢囊梗的形成、气孔周围孢子数量、葡萄叶片气孔开度和内源激素含量的影响及相互关系。【结果】EBR各处理均显著抑制了接种霜霉菌0.5 h后葡萄叶片气孔开度,以及接种后1 d和2 d病菌菌丝的发育。0.5和1.0 mg·L~(-1) EBR处理均显著抑制了接种霜霉菌0.5 h后叶片气孔周围的游动孢子数量和3 d后菌丝体在侵染区域的覆盖面积;接种4 d后,EBR各处理均显著抑制了霜霉菌孢子囊数量以及叶片发病率与病情指数,且0.5 mg·L~(-1)和1.0 mg·L~(-1) EBR处理降低发病率和病情指数的幅度最大,发病率分别比CK降低51.4%和45.0%,病情指数分别降低71.2%和62.9%。总体而言,0.5 mg·L~(-1)和1.0 mg·L~(-1) EBR处理抑制霜霉菌生长发育较为显著,且二者之间无显著性差异。0.5 mg·L~(-1) EBR处理的叶片脱落酸(ABA)、茉莉酸(JA)和水杨酸(SA)含量与CK之间均存在显著差异,气孔孔径与SA含量,ABA含量与JA含量呈显著正相关。【结论】EBR处理提高了葡萄叶片抵御霜霉菌侵染的能力,可能与其抑制病菌发育,改变寄主内源激素含量,从而促进气孔关闭等因素有关。     

Flavescence dorée phytoplasma titre in field‐infected Barbera and Nebbiolo grapevines

Flavescence dorée phytoplasma (FDP) titre in two red grapevine cultivars, Barbera and Nebbiolo, was measured over the vegetative seasons of two consecutive years in two vineyards of the Piemonte Region (northwestern Italy), with a double absolute quantification of FDP cells and grapevine DNA in real‐time PCR. The relationships of pathogen concentration to cultivar susceptibility and symptom severity were investigated. FDP titre was always higher in cv. Barbera than in cv. Nebbiolo infected vines, and this difference was significant at early and late summer samplings of 2008 and at early summer sampling of 2009. A seasonal trend in FDP concentration (low in spring, high in early summer and intermediate in late summer) was conserved for cvs Barbera and Nebbiolo in both years and vineyards. Considering both cultivars and years from both vineyards, a significant positive correlation between FDP concentration and symptom severity was found in the spring samples. Regarding the FDP strains (‐C or ‐D), no differences in pathogen titres were detected for either cultivar. Similarly, the presence of another grapevine yellows phytoplasma, bois noir, a subgroup 16SrXII‐A phytoplasma, in mixed infection with FDP strains had no effect on FDP concentration. These results demonstrate for the first time that grapevine cultivars with different susceptibility to FDP support different pathogen titres.     

葡萄SA 和JA 信号转导重要基因克隆及其对外源信号应答分析

 以‘藤稔’葡萄（Vitis vinifera × V. labrusca‘Fujiminori’）的叶片为试材,克隆了水杨酸（SA）和茉莉酸（JA）信号转导途径中重要基因NPR1、PR1、COI1和LOX2,利用定量和半定量PCR法研究其在SA与JA处理后的表达情况,发现PR1特异地受到SA诱导,而施加JA后抑制其表达;LOX2特异地受到JA的诱导,SA处理抑制其表达。上、下游基因的表达情况说明,PR1和LOX2可作为葡萄SA和JA信号转导途径的标记基因,同时研究发现葡萄中NPR1、PR1、COI1和LOX2表达量的快速上升出现在SA和JA处理后6 ~ 24 h;SA与JA信号转导途径存在着协同或拮抗作用,它们之间的相对浓度决定着这种相互关系的变化。     

15%石灰氮催芽后巨峰葡萄芽内激素以及有机物质变化研究

用石灰氮对巨峰葡萄(Kyoho grapevine)进行催芽试验,对休眠解除过程中芽体内源激素和有机物质含量以及淀粉酶活性的变化进行了测定。结果表明:随着ABA含量降低IAA含量升高,冬芽逐渐解除休眠而最终萌发;在药剂处理后淀粉酶活性加强,淀粉降解为可溶性糖供的速度加快,蛋白质含量则先下降后升高,最后诱导大部分的芽都能萌发。该试验进一步证明了GA3、ZRI、AA和ABA含量可能是引起巨峰葡萄休眠的关键原因,而ABA含量水平则应该是引起巨峰葡萄冬芽休眠的最关键因素。ABA含量低则有利于冬芽解除休眠。     

‘巨峰’葡萄不同生育期植株矿质元素需求规律

【目的】矿质元素的均衡供应是实现葡萄优质高效栽培的前提,研究‘巨峰’葡萄的矿质营养需求规律,为葡萄的合理施肥与精准施肥提供理论依据。【方法】本研究于2012—2018年连续7年在‘巨峰’葡萄的关键生育期进行整株取样,测定树体的各矿质元素含量,计算不同生育阶段各矿质元素的需求量与比例,研究葡萄的矿质营养需求规律。【结果】‘巨峰’葡萄在整个生长季不同生育阶段各矿质元素的需求规律有所差异。萌芽期至始花期对氮、钾、铁、锰、锌、钼的需求量均超过了全年总需求量的15%,磷、钙、镁、铜、硼的需求比率也均超过了10%。始花期至末花期,氮、铁、钼的需求比率均超过15%,磷、钾、钙、镁、锌的需求比率均超过10%。末花期至转色期,各矿质元素的需求比率分别为氮41.7%、磷47.44%、钾44.83%、钙45.88%、镁44.92%、铁39.75%、锰27.40%、锌30.28%、铜60.20%、硼38.72%、钼41.59%。转色期至采收期对钾、锰和硼的需求量较大,分别占比21.76%、22.19%和20.17%,其次磷、镁、钙、锌的需求比率分别为17.12%、16.76%、16.34%、14.72%,氮、铁、铜和钼的需求比率均低于10%。采收期至落叶期,锰和锌的需求比率分别为28.71%和23.57%,铁和硼的需求比率均超过15%,氮、钙、镁和钼的占比也均超过了10%,磷、钾和铜占比分别为9.72%、4.78%和8.69%。【结论】‘巨峰’葡萄施肥需要重视各生育阶段各种矿质养分的供给。生产1 000 kg果实各矿质元素的需求量为氮5.67 kg、磷2.37 kg、钾5.66 kg、钙5.70 kg、镁1.02 kg、铁153.45 g、锰53.14 g、锌36.25 g、铜7.28 g、硼41.84 g、钼0.47 g。‘巨峰’葡萄各矿质元素占干物质重的平均含量分别为氮0.92%、磷0.36%、钾0.66%、钙0.84%、镁0.15%、铁269.27 mg·kg ^-1、锰57.24 mg·kg ^-1、锌49.64 mg·kg ^-1、铜12.66 mg·kg ^-1、硼66.35 mg·kg ^-1、钼1.09 mg·kg ^-1。     

Electronic identification systems for reducing diagnostic workloads after disease outbreak

Diagnostic tests for grapevine viruses subjected to phytosanitary rules involve a heavy workload for plant protection services and laboratories. Propagation schemes enable nurseries, where mother plants (MPs) are cultivated, to be linked to batches of certified plants (CPs). This approach entails post‐production checks of MPs once infection occurs in CPs. However, this traceability system is not tight and follow ups are demanding. This study assessed radio frequency identification (RFID) tagging of plants in terms of its ability to reduce laboratory workloads for nursery health checks. RFID‐tagged plants (RFID‐CPs) were produced from individually tagged MPs (RFID‐MPs) or row‐tagged MPs (RFID‐ROW, a less expensive approach). In a 10‐year case study, the health status of CPs and RFID‐CPs were assessed and the occurrence of infections then led to health checks in MPs, RFID‐MPs or RFID‐ROWs. Laboratory workloads were evaluated by considering two sampling methods (single or pool sampling). Using single sampling, the workload was reduced by 93–98% in RFID‐ROW or RFID‐MP checks compared to the conventional approach. Considerable reductions in workload due to the tagging system (93–96%) were also observed using pool sampling. Traceability of CPs and MPs using RFID reduces laboratory workloads, and supports emergency measures that can be taken to stop any unsafe sales of plants after a virus outbreak.     

Mapping and managing vineyard homogeneous zones through proximal geoelectrical sensing

The objective was to delimitate homogeneous soil zones that correspond to differentiated areas of growth and yield of a vineyard, using soil electrical resistivity. The soil electrical resistivity (ρ) at three depths was measured with an automatic on-the-go recording resistivity meter, the data was georeferenced, and zones of homogeneous soil characteristics were drawn. A grid of equally spaced sampling points was superimposed to the area where vegetative growth and yield data of the grapevines were recorded. ρ showed a marked spatial distribution and two homogeneous areas were delineated. ρ ranged from 48.6 to 54.3 ohm-metre for cluster A and from 90.2 to 208.5 ohm-metre for cluster B. Negative significant correlations were found between ρ and plant growth and yield variables. Vegetative growth and crop yield were significantly different between homogeneous soil ρ areas. Plants growing on cluster A had an average shoot length of 1.4 m and yielded 3.1 kg per plant; on cluster B, shoot length was 0.9 m and yield was 0.9 kg per plant. The procedure of measuring the soil electrical resistivity and georeferencing methodology proved fast and reliable, albeit expensive, and might be a useful tool in precision viticulture for delineating homogeneous soil zones.