超量表达响应β-氨基丁酸诱导的StWRKY8基因提高马铃薯晚疫病抗性

晚疫病是马铃薯(Solanum tuberosum)生产中最为严重的病害,抗晚疫病是马铃薯育种的主要目标之一。前期研究中,在具有田间抗性的马铃薯材料中筛选到一个受广谱诱抗剂β-氨基丁酸(β-aminobutyric acid,BABA)诱导表达的转录因子StWRKY8基因片段。为了进一步研究该基因在马铃薯晚疫病抗性中的功能,本研究利用qRT-PCR技术进一步分析了该基因受β-氨基丁酸和晚疫病菌—卵菌致病疫霉(Phytophthora infestans)诱导表达的模式;从马铃薯中克隆了该基因cDNA,构建了35S启动子驱动的植物表达载体,通过农杆菌(Agrobacterium tumefaciens)介导的方法进行了马铃薯的遗传转化;对获得的超量表达转基因株系进行了离体叶片晚疫病接种抗性鉴定,同时利用qRT-PCR技术检测了StWRKY8下游植保素合成相关基因—3-羟-3-甲戊二酰辅酶A还原酶基因2(3-hydroxy-3-methylglutaryl Co A reductase 2,HMGR2)和NADP-苹果酸酶基因(NADP-malic enzyme,NADP-ME)的表达。研究结果表明,BABA和P.infestans都能诱导StWRKY8基因上调表达,BABA诱导24 h达到表达高峰,而P.infestans诱导36 h达到表达高峰。本研究克隆的StWRKY8基因编码区长1 605 bp,编码534个氨基酸,与已报道马铃薯StWRKY8(NP_001274836.1)蛋白序列相似性达到98%,含有两个WRKYGQK保守结构域。获得了StWRKY8超量表达转基因株系,晚疫病接种抗性鉴定表明,4个表达量高的转基因株系晚疫病抗性显著提高。另外,本研究结果表明,受StWRKY8调控的HMGR2和NADP-ME基因的表达量在超量表达转基因株系中并未明显提高。但是,当转基因株系受到晚疫病菌液体培养物(culture filtrate,CF)诱导时,这两个基因在转基因株系中显著上调表达。本研究结果为进一步探讨StWRKY8抗性调控机理以及利用该基因提高马铃薯晚疫病抗性提供了重要信息。     

TRV介导的葡萄叶片VvANR基因瞬时表达分析

为建立葡萄叶片TRV-VIGS系统,分析验证VvANR基因功能,本研究以烟草脆裂病毒(Tobacco rattle virus,TRV)介导基因沉默表达载体pTRV2-ANR,采用真空侵染和主叶脉针孔注射法分别侵染葡萄幼嫩、成熟叶片,观察表型并测定原花青素含量,实时荧光定量PCR测定被侵染叶片中VvANR及其相关基因表达。结果表明,真空侵染的幼嫩、成熟叶片分别于第6、第7天出现漂白,侵染15 d后,幼嫩叶片几乎全部漂白,成熟叶片大面积漂白。主叶脉针孔注射侵染的幼嫩、成熟叶片分别于第17、第19天出现漂白;侵染25 d后,幼嫩和成熟叶片发生全叶漂白,且漂白均沿主叶脉逐渐扩展至整个叶面;对照均未发生漂白现象。pTRV2-ANR侵染后叶片中原花青素含量极显著降低,VvANR基因表达量极显著降低,且原花青素生物合成相关基因VvLAR1、VvLAR2、VvMYBPA1、VvMYBPA2、VvUFGT表达量显著降低,除VvUFGT表达量呈显著差异外,其余均呈极显著差异;而VvANS、VvDFR表达量轻微上调,且与对照差异不显著。本研究建立了葡萄叶片VIGS体系,为基因功能快速验证提供了新方法;同时,揭示了葡萄叶片中原花青素含量、VvANR表达及与相关基因的关系,本研究为完善葡萄原花青素积累机制奠定了一定的理论基础。     

利用RNA沉默抑制子HC-Pro提高基因表达的策略

利用植物生物反应器生产药用蛋白具有独特的发展优势,但外源基因在受体内会发生基因沉默现象而影响基因的表达。由于基因沉默抑制子能有效抑制外源基因的沉默反应,本研究利用沉默抑制子蚜传辅助因子(helper component-proteinase,HC-Pro)转基因烟草有效提高了外源基因的表达。采用叶片注射法将携带表达载体p35S-30B-GFP的农杆菌(Agrobacterium tumefaciens)重悬液分别侵染野生型烟草(Nicotiana bentamiana)和HC-Pro转基因烟草(N.bentamiana),通过蛋白电泳检测和Western blot检测技术对绿色荧光蛋白(GFP)表达进行比较。研究结果表明,与野生型烟草Nb相比,HC-Pro转基因烟草中GFP的表达水平显著增加。本研究为基因沉默抑制子在植物生物反应器中的应用提供了理论依据。     

RNA沉默介导的转基因烟草抗中国番茄黄化曲叶病毒研究

近年来,中国番茄黄化曲叶病毒(Tomato yellow leaf curl China virus,TYLCCNV)病在我国自南向北不断蔓延,发病地块减产甚至绝产,造成了巨大的经济损失.本研究应用RNA沉默(RNA silencing)防治病毒病原理构建TYLCCNV外壳蛋白(coat protein,CP)部分基因片段dsRNA(double-strand RNA)导入本氏烟(Nicotiana benthamiana)获得了抗TYLCCNV的转基因烟草.实验结果表明,目的基因已整合到转基因烟草植株的基因组中.用TYLCCNV侵染性克隆接种转基因烟草,症状观察和PCR检测发现,对TYLCCNV表现为免疫的转基因烟草占14.6％.Northern blot分析表明,在不同的抗病类型转基因植株中,目的基因mRNA的积累量存在明显的差异,其积累量与抗病型呈负相关.研究结果对利用RNA沉默防治TYLCCNV有一定的理论指导意义.     

瓜类细菌性果斑病菌hpaP基因的功能分析

瓜类细菌性果斑病(bacterial fruit blotch of watermelon,BFB)是近年来发生在西瓜、甜瓜等葫芦科(Cucurbitaceae)植物上的重要细菌病害,由西瓜噬酸菌(Acidovorax citrulli,Ac)引起.本研究以xjL12菌株为背景构建了转座子(Tn5)插入文库.通过注射接种哈密瓜(Cucumis melo var.saccharinus)子叶和烟草叶片进行突变体的筛选,得到l株致病性完全丧失并失去激发烟草(Nicotiana tabacum)过敏反应的突变体△xj48.对△xj48中转座子插入基因的克隆和测序表明,其突变基因为西瓜噬酸菌Ⅲ型分泌系统(T3SS)中的保守基因hpaP.利用基因重组技术构建了hpaP基因的突变体,发现hpaP突变后影响了xjL12菌株的游动性、生物膜的形成能力及hrcV基因的表达.hpaP基因的互补菌株部分恢复其致病力.本研究证实了果斑病菌中的hpaP基因与该细菌的致病力相关,在侵染瓜类作物的过程中起着不可缺失的作用.     

马铃薯Y病毒CP基因RNAi载体构建与干涉效果测定

RNA沉默（RNAi）介导的植物抗病毒研究是近年来引起广泛关注的一项植物抗病毒基因工程策略。马铃薯Y病毒（PVY）在世界范围内引起马铃薯、烟草等重要经济作物的病害,并且造成严重的经济损失。本文以PVY的外壳蛋白（CP）基因为模板扩增300bp和354bp两个片段,正向和反向分别插入植物表达载体pROKⅡ的35S启动子下游,从而构建了以PVY的CP基因为靶标的RNAi植物表达载体pROKY300,转入农杆菌EHA105中,以农杆菌渗入法在本氏烟中瞬时表达与wyCP同源的hairpin RNA。结果表明,瞬时表达的hairpin RNA有效干涉了PVY侵染。     

玉米TRV-VIGS的优化与顶腐病抗病基因的鉴定

烟草脆裂病毒(TRV)介导的基因沉默方法简便易行,但在常规试验条件下按照报道的方法操作沉默玉米基因的效率较低,难以实际应用。为了优化TRV-VIGS体系,并鉴定顶腐病抗病基因,本研究以玉米八氢番茄红素脱氢酶(PDS)基因为靶基因,构建沉默载体pTRV2-ZmPDS,从浸种处理、农杆菌菌株、真空辅助渗入和共培养时间4个试验因素优化玉米TRV-VIGS体系,提高基因沉默效率。结果表明,无菌水浸泡种子24 h,剥去种皮后用LB4404农杆菌菌株真空渗入处理60 min,再共培养10 h时,全株白化苗占出苗植株总数的25.1%~29.3%,较优化前沉默比率(3.0%)提高了8~10倍。半定量RT-PCR分析表明,与对照和未白化植株相比,白化植株的ZmPDS基因转录水平显著降低。应用优化的TRV-VIGS体系沉默ZmMAC3基因,高抗顶腐病的玉米杂交种先玉335在接种亚粘团镰孢菌后表现出典型的顶腐病病症,表明,与双子叶植物相似,MAC3蛋白是玉米内生免疫和抗病所必需的关键调控因子。综上所述,优化后的TRV-VIGS体系可以应用于玉米基因功能的鉴定。     

瞬时表达葡萄病毒A外壳蛋白基因对烟草中病毒转录的影响

为验证葡萄病毒基因在模式植株上瞬时表达对接种宿主中病毒复制与感病症状的影响,本研究以一株鲜食葡萄无核王子混合感染葡萄病毒A(GVA)的不同变异株为试验材料,克隆其中一个变异株的外壳蛋白基因的部分核酸序列。在p BI121质粒35S启动子后插入具有发夹结构的外壳蛋白基因片段并转化农杆菌EHA105。农杆菌瞬时浸润本氏烟叶片5 d后,以摩擦接种GVA侵染性克隆的体外转录物作为处理组,只单独接种病毒侵染性克隆体外转录物的烟草作为对照组。结果表明,GVA体外转录产物接种30 d后,对照组植株系统叶表现黄化症状,而瞬时表达具有发夹结构的病毒外壳蛋白基因的处理组植株症状不明显,且系统叶上病毒基因的转录量相对较低,表明宿主叶片上瞬时表达病毒结构基因,可诱导植物产生抗病性,该结果为抗病葡萄种质的创制提供了一定的理论依据。     

VIGS沉默番茄SlDCL2和SlDCL4破坏Ty-1/Ty-3对TYLCV的抗性

番茄黄化曲叶病毒(TYLCV)严重威胁茄科蔬菜作物的生产。抗性标记Ty-1和Ty-3是一对等位基因,在番茄抗TYLCV育种中应用较为广泛。为了探究Ty-1/Ty-3抗病毒分子机制,本研究以携带Ty-1/Ty-3抗性标记的番茄Y19为材料,利用病毒诱导基因沉默(VIGS)技术,沉默RNA干扰(RNAi)机制关键基因,即番茄核糖核酸内切酶编码基因2a/b/c/d(SlDCL2)和番茄核糖核酸内切酶编码基因4(SlDCL4),初步分析其在Ty-1/Ty-3抗TYLCV中的功能。PCR与测序结果发现SlDCL2、SlDCL4的VIGS沉默载体pTRV2∶SlDCL2和pTRV2∶SlDCL4构建成功。以沉默番茄八氢番茄红素脱氢酶(SlPDS)基因为标记植株,定量PCR检测SlDCL2、SlDCL4沉默株系中SlDCL2、SlDCL4基因的沉默效率,结果显示SlDCL2、SlDCL4沉默株系中对应基因的表达均小于对照植株的50%,表明SlDCL2、SlDCL4沉默载体确实可以降低对应基因的表达,且不影响其他DCL基因的表达。VIGS沉默植株SlDCL2、SlDCL4基因后接种TYLCV,接种TYLCV 30dpi结果显示,Y9材料表现出明显的TYLCV病毒症状,通过比较植株发病严重度发现,SlDCL2、SlDCL4沉默株系发病严重度分别为1.97、2.35,显著高于空载体注射株系(0.14)和对照株系(0.07)。本研究结果表明RNAi通路关键基因SlDCL2、SlDCL4在Ty-1/Ty-3抗TYLCV中发挥着重要作用,这为利用Ty-1/3抗性标记基因育种奠定了基础。     

基于机器视觉的马铃薯晚疫病快速识别

晚疫病是马铃薯的一种严重病害,可造成减产甚至绝收。因此马铃薯晚疫病的识别与控制对提高其产量有非常重要的意义。该文基于机器视觉技术对马铃薯叶部晚疫病进行检测,根据马铃薯叶片上晚疫病斑的颜色、纹理和形状特征参数的不同,提取叶片表面的特征参数,并建立数学模型对病害程度做出评价。在RGB、HSV颜色空间中,根据马铃薯叶片在患病早期叶片颜色发生变化且与健康叶片不同,利用颜色特征,建立马铃薯晚疫病的无病和患病模型,该模型对马铃薯患病早期的识别率为67.5%。利用灰度共生矩阵,采用纹理统计参数进行病害等级评价,用熵值和能量值描述晚疫病的严重程度,纹理特征对患病程度的识别率比较稳定,对患病中期与后期的识别率分别为72.5%与80%。利用形状特征的相对特征,根据病斑面积比进行晚疫病诊断,该方法对马铃薯叶片晚疫病患病后期的诊断取得较好效果,识别率为90%,但由于叶片患病早期的病斑面积小且分散,识别难度大,识别率仅为50%。针对颜色、纹理及形状特征在识别马铃薯叶片晚疫病时的优势与局限性,提出颜色纹理形状特征结合的识别方法,对患病中期与后期的识别率分别为90%和92.5%。通常马铃薯晚疫病的理化值检测法耗时数天,但利用机器视觉识别马铃薯晚疫病患病情况非常快速,根据颜色特征进行病害识别的时间约为4 s,纹理特征识别的时间为7 s,形状特征特征识别的时间为3 s,综合颜色纹理形状特征的识别由于计算量较大,识别时间为9 s。该研究可为基于机器视觉的马铃薯晚疫病的快速检测提供理论依据。     

Foliar resistance to the late blight pathogen Phytophthora infestans (Mont.) de Bary in a backcross of potato (Solanum tuberosum L.) cultivar Ando

ABSTRACT

Regardless of variable environmental conditions and changing pathogen populations, the Estonian potato cultivar (cv.) Ando has shown elevated field resistance to Phytophthora infestans for over 40 years. One of cv. Ando’s parents, the Latvian cultivar Agra, is considered a cultivar of medium to low resistance, the other parent, line 382-48, has a well-known source of resistance, Solanum demissum, in its pedigree. Segregation of late blight disease severity in a population of 95 hybrid plants from cv. Ando’s backcross to cv. Agra (cv. Ando x cv. Agra BC1) in field trials of three consecutive years (2013?2015) and in a test of a single pathogen isolate fits a model of additive action of multiple unlinked race-specific resistance genes responsible for the field resistance of cv. Ando. At least some of the resistance genes in cv. Ando’s genome do not function in juvenile plants and are developmentally upregulated.     

临夏州马铃薯晚疫病始发期的预测预报

马铃薯是临夏州主要种植作物,随着种植区域的扩展,马铃薯晚疫病危害程度呈上升趋势,已成为制约马铃薯产业发展的主要因素之一。传统的晚疫病预测预报技术参数主要来源于干旱山区,在川塬区和二阴区预报准确率较低。本文依据多个试验点连续3a的观测资料,研究得出2个较好的预测模型：临夏州干旱山区马铃薯晚疫病始发期（Y）与8月上旬降水量（X）之间呈显著负相关关系,其线性回归方程为Y=9.1699-0.0153X（F=35.8〉F0.01）,预测准确率达到100%;二阴区马铃薯晚疫病始发期与7月上旬温度（X1）、空气湿度（X2）呈显著负相关,其线性回归方程为Y=13.8180-0.1970X1-0.0403X2,预测准确率达到83%。     

Early blight destroys potato foliage in the northern Baltic region

In recent years, potato early blight [Alternaria solani (Ellis & G. Martin) L.R. Jones, A lternaria alternata (Fr.) Keissl.] has occurred with increasing frequency in European potato fields, including those in northern regions. In our study, early blight was evaluated during the two years 2010–2011 on potato plants in a conventional farming experiment. Both growing seasons were very favourable for early blight development and evaluation. Our study indicates that, despite chemical treatments, potato early blight is a great problem for potato growers in conventional farming systems in the North-East region in years when temperatures are higher than average. In 2011, early blight damage was particularly high on the untreated (N0P0K0) treatment plots where severity increased abruptly from an initial 2% to 91%, and killed the potato haulms within five weeks. In 2010, the most severely infected plants were also recorded on the untreated (N0P0K0) plots, where 70% of foliage was destroyed by the end of the growing season. Results showed that a more efficient chemical strategy is needed against this disease on susceptible cultivars. Breeding and growing more resistant cultivars of potato is considered the most efficient, cost effective and environmentally friendly control method for sustainable cultivation in conventional growing systems. As an increase in the frequency of warmer summers is predicted, greater problems from this pathogen are to be expected in Northern Europe.     

2016 — 2020年西北部分地区马铃薯致病疫霉交配型分析

为合理选用抗性品种防治马铃薯晚疫病，采用对峙培养的方法对 2016 — 2020年分离自甘肃、青海和宁夏等3省(区)25个县(区)的694株马铃薯致病疫霉[Phytophthora infestans(Mont.) de Bary]进行了交配型测定。结果供试菌株中只检测到了A1交配型和A2交配型，未发现A1A2和SF(自育型)，其中A2交配型菌株为优势菌株，2016 — 2020年占所测菌株的比例分别为86.4%、96.3%、86.4%、80.9%、96.5%。5年间A2交配型菌株占比呈先升后降再升趋势。A2交配型比例的增大给马铃薯晚疫病的防控和抗病育种带来新的挑战。     

马铃薯晚疫病气象等级预报方法研究

通过对1990-2008年围场县马铃薯晚疫病的发生面积与气象条件进行相关分析,表明马铃薯晚疫病的发生是生物因子在适宜的气象条件下发生作用的结果。气温、降水、相对湿度、日照时数等气象条件是马铃薯晚疫病发生的决定性因素。在温度适宜的情况下,阴雨连绵或多雾、多露最有利于晚疫病的发生和流行。以受灾率作为马铃薯晚疫病气象等级的划分标准,选取与晚疫病相关的前期气象要素作为预报因子,采用综合聚类法建立了马铃薯晚疫病气象等级的预报模型。回代结果表明,用气象综合聚类法类做马铃薯晚疫病的气象等级预报,可以为控制晚疫病的蔓延,赢得宝贵时间,提高气象决策服务水平。     

Late blight and virus host-plant resistances,crossing ability and glycoalkaloids in Nordic potato germplasm*

Old potato cultivars from the Nordic Genetic Resource Center (NordGen) and advanced breeding clones from the Swedish University of Agricultural Sciences (SLU) were evaluated for susceptibility to Potato Virus Y (PVY) and Potato Leafroll Virus (PLRV), foliar and tuber resistance to late blight – caused by Phytophthora infestans – as well as for glycoalkaloid content and crossing ability. Enzyme-Linked Immunosorbant Assay (ELISA) tests were used for PVY and PLRV screening during two years that were characterized by intensive virus incidence and severity in the field, while foliar and tuber resistances to late blight were assessed under artificial inoculation with an aggressive Swedish isolate of P. infestans. Hybrid seeds were obtained by crossing cultivars such as ‘Kiva’, ‘Sarpo Mira’, ‘Rosamunda’ and ‘Superb’ with SLU advanced breeding clones and a selected clone of Solanum tuberosum Group Andigena. Some cultivars (‘Hårek’, ‘Sarpo Mira’) and breeding clones (04-2662, 04-2085) with late blight resistance did not show virus infection. The α-solanin and α-chaconin contents of some of the old Nordic potato cultivars and breeding clones were similar to the known Dutch table cultivar ‘Bintje’ after 3 years of testing. This research allows identification of promising Nordic potato cultivars and SLU breeding clones for further use in developing germplasm aiming at organic and conventional farming systems.     

Nematicidal and antifungal activities of annonaceous acetogenins from Annona squamosa against various plant pathogens

The methanol extract of Annona squamosa seeds was highly active against two phytoparasitic nematodes, Bursaphelenchus xylophilus and Meloidogyne incognita. It efficiently suppressed plant diseases, caused by Phytophthora infestans and Puccinia recondita. Ten annonaceous acetogenins (AAs) were isolated, and their chemical structures were identified by mass and nuclear magnetic resonance spectral data. Out of 10 substances, eight displayed strong in vitro nematicidal activity against B. xylophilus with LD(50) values ranging 0.006 to 0.048 μg/mL. Squamocin-G showed potent nematicidal activity against M. incognita. Squamocin, squamocin-G, and squamostatin-A also displayed potent in vitro and in vivo antifungal activities against P. infestans causing tomato late blight. In addition, squamostatin-A effectively controlled the development of wheat leaf rust caused by P. recondita. Our findings suggested that A. squamosa seeds and its bioactive AAs can be an alternative resource of a promising botanical nematicide and fungicide to control various plant diseases.     

甘薯抗疮痂病多胺与吲哚乙酸代谢的研究

研究不同抗性甘薯品种受疮痂病菌侵染前后多胺和吲哚乙酸代谢变化结果表明 ,感染疮痂病菌后抗病与感病品种甘薯叶片中吲哚乙酸含量和多胺氧化酶活性降低 ,多胺总量及腐胺含量增加 ,腐胺 / (精胺 亚精胺 )比值升高 ,感病品种变化幅度高于抗病品种。疮痂病菌侵入后感病品种甘薯叶片中绿原酸含量下降 ,吲哚乙酸氧化酶和过氧化物酶活性升高 ,而抗病品种则相反。