Isolation and Identification of Virus dsRNA from Strawberry Plants

The analysis of virus genome is based on nucleic acid isolation. The aims of this study were to develop a method for isolation and identification of virus double-stranded ribonucleic acid （dsRNA） and to elucidate the nucleotide sequences of strawberry virus. Using the modified method, virus dsRNA was extracted from strawberry virus indicator plants and cultivated strawberry plants and detected using agarose gel electrophoresis with ethidium bromide staining and reverse transcription-polymerase chain reaction （RT-PCR）. The quantity of virus dsRNA varied among strawberry cultivars. The quantity of dsRNA from in vitro plantlets was higher than that from the young leaves of field plants. For the field-grown plants, there was more dsRNA in the young leaves. Virus dsRNA extracted from strawberry plants was resistant to deoxyribonuclease Ⅰ （DNase Ⅰ）, but evidently, it became resistant to ribonuclease A （RNase A） only in the presence of 0.5 M NaCl. Its bands in agarose gel could be readily recycled using an agarose gel DNA purification kit. With RT-PCR, the segments of both strawberry mottle virus and Strawberry mild yellow edge virus genomes were amplified by using the virus dsRNA recycled from gel or treated with DNase Ⅰ /RNase A as templates. The system developed for dsRNA isolation and identification in strawberry plants laid a sound foundation for the work on genome analysis of strawberry virus isolates in China.

Study on the Molecular Variation and PCR Detection of Strawberry mottle virus

Strawberry mottle virus （SMoV） is an important viral pathogen infecting strawberry （Fragaria spp.）. The study was conducted to analyze the characterization of the molecular variation of SMoV and develop the methods for detection of SMoV by nested PCR and transcriptional enhancement techniques. The 3 non-coding region （NCR） and large coat protein （LCP） gene of SMoV genome were amplified by reverse transcription-polymerase chain reaction （RT-PCR）. The specific segments were cloned and sequenced. The characterization of the molecular variation for some isolates of SMoV and phylogenetic analysis were studied. Based on the primers located in the conserved region of genome of SMoV, SMoV could be steadily detected using semi-nested PCR and transcriptional enhancement techniques. Both semi-nested PCR and transcriptional enhancement techniques were considerably more sensitive than the standard RT-PCR. The nucleotide sequences of NCR and partial LCP gene of Chinese isolates were obtained, and sequence analysis of the partial LCP gene of various SMoV isolates showed nucleotide identities ranging from 76.8 to 99.7%. There was a slight tendency for isolates to group according to their geographical origin. All 3 Polish isolates, 4 isolates of 7 Dutch isolates, and 3 isolates of 4 Chinese isolates formed a small separate branch, respectively. Two Germanic isolates had a far relationship with other isolates, and formed a separate clade. A high level of sequence variability was found among SMoV isolates, and the Germanic isolates were likely to a special strain group.

P1 of strawberry vein banding virus, a multilocalized protein, functions as a movement protein and interacts with the coat protein

Although the complete nucleotide sequence of strawberry vein banding virus(SVBV) has been determined and bioinformatic analysis has revealed that the SVBV genome could encode seven proteins, the precise function of each protein is unclear. This study provided evidence that the P1 protein of SVBV(SVBV-P1) possesses the following features. Bioinformatic and subcellular localization analyses showed that SVBV-P1 is localized in the cytoplasm and cell walls of epidermal cells in Nicotiana benthamiana, and it forms inclusion bodies associated with microtubules and the endoplasmic reticulum. Dilution experiments demonstrated that SVBV-P1 could move from the original agro-infiltrated cells to adjacent cells in N. benthamiana leaves. Further trans-complementation experiments demonstrated that SVBV-P1 could facilitate the intercellular movement of a movement-deficient potato virus X mutant in N. benthamiana leaves. Finally, yeast twohybrid and bimolecular fluorescence complementation assays revealed that SVBV-P1 could interact with the SVBV coat protein, which is a major component of Caulimovirus virions. Results of the electrophoretic mobility shift assay indicated that SVBV-P1 lacks DNA-binding capability. In summary, the results suggest that SVBV-P1 is probably a movement protein of SVBV, providing new insights into the function of movement proteins of the Caulimovirus genus.

Complete genome sequence of two strawberry vein banding virus isolates from China

It was rarely reported about strawberry vein banding virus(SVBV)genome sequence in China and most countries worldwide.In this work,we determined the complete genome sequences of two SVBV isolates in China,designated SVBV-AH and SVBV-BJ,that were obtained from naturally infected strawberry samples from Anhui province and Beijing city of China,respectively.The complete genomes of SVBV-AH and SVBV-BJ were 7,862 nucleotides(nts)and 7,863 nts long,respectively,and both constituted with seven genes typical of the caulimoviruses.Alignment of complete nucleotide sequences showed that SVBV-AH and SVBV-BJ shared a significant nucleotide sequence identity of 97.7%of each other and had 85.7%and 86.0%sequence identity related to SVBV from the United States(SVBV-US),respectively.Phylogenetic trees,based on the alignment of complete nucleotide sequences and amino acid sequences of Coat Protein(CP),both showed that SVBV-AH and SVBV-BJ clustered into one branch with all the other SVBV isolates,and other species of caulimoviruses clustered into another tree branch.It illustrated that all the SVBV isolates had an extremely high relationship but had a distant relationship with other species of caulimoviruses.We further confirmed that SVBV-AH infectious clone could cause similar symptoms to SVBVinfected in strawberry under natural conditions.Taken together,our study provided valuable information to elucidate the origin and dissemination of SVBV Chinese isolates,meanwhile providing the necessary vector for studying the gene functions of strawberry.

“粉玉1号”草莓茎尖组织培养体系及其脱毒效果研究

“粉玉1号”是通过杂交育种方式选育的早熟抗病粉果草莓新品种。从大棚栽培的“粉玉1号”草莓植株上取匍匐茎芽为试料,通过外植体消毒、茎尖剥取、不定芽诱导和生根培养建立了“粉玉1号”茎尖组织培养脱毒技术体系,对茎尖培养苗和大棚栽培植株进行了草莓皱缩病毒(SCV)、草莓斑驳病毒(SMoV)、草莓轻型黄边病毒(SMYEV)和草莓镶脉病毒(SVBV)PCR检测。结果表明,“粉玉1号”草莓外植体灭菌方法为75%乙醇处理30 s,再用0.1%升汞溶液处理10 min;匍匐茎芽一般需剥去1片嫩叶和7片幼叶才能剥出茎尖;茎尖初代培养基宜采用MS+0.5 mg/L 6-BA,继代培养基宜采用MS+0.1 mg/L 6-BA,生根培养基宜采用不加植物生长调节剂的1/2 MS。大棚栽培植株样品存在SVBV感染,通过茎尖组培脱除了该病毒,SCV、SMoV和SMYEV在所有样品中均未检出。建立的茎尖脱毒技术体系可为“粉玉1号”草莓工厂化育苗提供参考。

4个品种草莓茎尖组培快繁体系的构建

以近年来生产上应用较广的草莓优良品种红颜、章姬、香野和粉玉为研究对象,系统开展不同品种脱毒组培快繁技术研究。以各品种的茎尖为外植体,研究了4个品种草莓茎尖的不定芽诱导、增殖及生根情况、移栽成活情况等。研究表明章姬和香野最适宜的茎尖不定芽诱导和继代增殖培养基为MS+0.5 mg/L 6-BA+0.1 mg/L IBA;红颜和粉玉最适宜的茎尖不定芽诱导培养基为MS+0.3 mg/L 6-BA+0.1 mg/L IBA,不定芽继代增殖培养基为MS+0.2 mg/L 6-BA+0.1 mg/L IBA。4个品种最佳生根培养基为1/2 MS培养基。用蛭石进行移栽,成活率均超过95%。

叶片保存方法对RT-PCR检测草莓轻型黄边病毒的影响

【目的】探究通过RT-PCR检测草莓轻型黄边病毒最好的叶片保存方法。【方法】经自然风干法、硅胶干燥法以及氧化钙干燥法保存后,采用RNA小量提取试剂盒法提取被病毒侵染草莓叶片总RNA,利用Thermo NanoDrop 2000测定核酸质量浓度和质量并使用RT-PCR方法对SMYEV进行检测。【结果】经氧化钙干燥保存后可提取高质量的RNA,30 d后仍可通过RT-PCR获得扩增产物。【结论】氧化钙干燥保存效果最好,操作简单,成本低,可广泛应用于大量草莓叶片样本的长距离运输,提高草莓病毒检测效率,为草莓无毒栽培提供保障。

遮光对西藏林芝市草莓脱毒原种苗光合与叶绿素荧光特性的影响

为探究遮光处理对宁玉草莓脱毒原种苗光合与叶绿素荧光特性的影响,以宁玉草莓脱毒原种苗为材料,研究不同遮光率(75%、50%、25%、0%)对其蒸腾速率(T_(r))、气孔导度(G_(s))、胞间CO_(2)浓度(C_(i))、净光合速率(P_(n))、F_(v)/F_(m)、F_(v)/F_(o)的影响。结果表明,草莓叶片的净光合速率(P_(n))、气孔导度(G_(s))、蒸腾速率(T_(r))在3种遮光率处理下均显著高于对照,随着时间的增长,遮光处理的F_(v)/F_(m)、F_(v)/F_(o)均呈逐渐下降的变化趋势,ETR呈先上升后下降的变化趋势。随着遮光率的增大,F_(v)/F_(m)、F_(v)/F_(o)均呈现先下降再升高后下降的变化趋势,在50%遮光率时达到波谷,25%遮光率时达到波峰,可为草莓苗在高原地区生长的光照调控提供参考。

通过玻璃化超低温处理脱除草莓轻型黄边病毒(SMYEV)研究

以草莓品种明宝为材料,取茎尖做初代培养,继代扩繁5次后,取2mm左右的茎尖,利用玻璃化超低温技术对SMYEV进行脱除,并利用结合内标的多重RT-PCR技术对再生植株进行病毒检测。结果表明,在病毒脱除过程中,预培养蔗糖浓度为0.5mol/L,处理3d;装载处理为25℃,60min;玻璃化处理为0℃,120min;液氮处理60min后,进行40℃水浴2min,草莓茎尖的存活率为76%,而草莓轻型黄边病毒的脱除率为95%。只有通过液氮处理才可以脱除该病毒,液氮处理前的玻璃化处理脱毒率为0。利用超低温脱毒法可以简便有效地脱除SMYEV。

草莓病毒病及其脱毒与检测技术研究进展

介绍了在草莓病毒病为害特点及其作用机理。综述了热处理、茎尖培养、花药培养等草莓脱毒技术和运用指示植物、血清学和分子生物学等检测技术。介绍了草莓无毒苗的栽培效果。

草莓轻型黄边病毒的RT-PCR检测及其3'端序列分析

利用RTPCR技术对草莓植株中草莓轻型黄边病毒(SMYEV)的外壳蛋白基因片段进行特异扩增,扩增产物经测序证明为SMYEV的特异片段。建立了稳定的SMYEV的RTPCR检测体系,并对19个草莓品种和2种野生草莓进行检测,其中12个品种和五叶草莓带有SMYEV。从SMYEV的沈阳分离物SY01上扩增出了932bp的基因组3末端区域,其核酸序列与国外分离物的序列同源性为86%～96%。系统进化树显示SMYEV分为3个组群,沈阳分离物SY01与欧美分离物位于同一组群内,但独立形成1个小的分支。RNA二级结构分析显示SMYEV基因组3末端形成3个茎环结构,不同分离物的序列变化并未引起茎环结构的明显变化。

草莓病毒脱除方法的比较与评价

以感染病毒的草莓品种宝交早生为试材,利用多重RT—PCR同时检测SMoV和SMYEV技术,对茎尖培养、热处理、抗病毒药剂处理3种脱毒方法进行了比较和评价,以期为草莓脱毒苗培育提供重要参考。结果表明,0．2 mm茎尖和0．5 mm茎尖都能够有效地脱除SMoV和SMYEV;热处理能够脱除草莓试管苗中的SMoV,但不能脱除SMYEV,39℃恒温处理30 d,SMoV脱除率达到63．0％;利巴韦林不能清除草莓植株体内的SMoV和SMYEV。综合脱毒率和茎尖分化成苗率2个指标,认为0．5 mm茎尖培养是适宜的草莓病毒脱除方法。

利用PCR技术检测草莓镶脉病毒

采用特异引物的PCR扩增方法 ,对 30个草莓品种进行了草莓镶脉病毒的检测 ,同时用指示植物小叶嫁接法对被检植株进行了病毒检测 ,两种方法结果一致。回收、克隆PCR扩增出的特异DNA片段 ,获得了携有草莓镶脉病毒CP基因片段的载体。测序结果表明 ,得到的特异DNA片段同美国草莓镶脉病毒的株系ATCC 4 5 0 5 8CP基因片段相比 ,同源性为 90 .94 %。

应用多重RT-PCR检测草莓斑驳病毒和草莓轻型黄边病毒

建立了多重RT-PCR同时检测草莓斑驳病毒(SMoV)和草莓轻型黄边病毒(SMYEV)的技术体系,对草莓田间植株和试管苗都可以有效进行检测。多重PCR引物根据引物之间的互补性及引物的Tm值进行筛选。适宜的PCR缓冲液的浓度为2×,退火温度为57℃,延伸温度为66℃。分别利用单一PCR和多重PCR对微茎尖培养获得的草莓品种宝交早生的11个株系进行了脱毒效果鉴定。

四种草莓病毒SMoV、SVBV、SCV、SMYEV多重RT-PCR检测

我国草莓病毒主要有草莓斑驳病毒(SMoV)、草莓镶脉病毒(SVBV)、草毒皱缩病毒(SCV)和草莓轻型黄边病毒(SMYEV),通常发生混合侵染。本研究针对我国4种草莓病毒的外壳蛋白基因部分序列设计引物,通过优化多重RT-PCR反应条件,建立起能同时检测4种草莓病毒的多重RT-PCR检测体系。应用该体系成功对4种病毒复合侵染的草莓材料进行多重RT-PCR扩增,得到278、394、731和861bp共4条特异性条带。测序结果表明,4种病毒序列与相应的参考序列相似性均达98%以上。对草莓茎尖脱毒和叶片离体再生的试管苗样品进行检测,结果显示,该方法可以同步、快速、特异地检测出受侵染寄主植物中的4种病毒,并且灵敏度高。

利用内标为基础的RT-PCR技术检测草莓斑驳病毒

利用改进的CTAB法提取出优质的草莓总RNA,可以稳定地进行RT-PCR.针对草莓斑驳病毒(SMoV)基因组序列设计筛选引物,对病毒基因组的不同区域进行扩增,扩增产物经克隆、测序证明为SMoV的特异片段,与国外序列的同源性为91%~97%.为了监测RNA的质量和RT-PCR反应的正常进行,在检测体系中引入线粒体NADH脱氢酶基因ND2亚基作为内标,内标引物跨越内含子区域,只对剪接后的mRNA进行特异扩增,可以较好地监测整个检测过程.在国内首次建立了SMoV的RT-PCR检测体系,以优质的草莓总RNA为模板,结合扩增内标的检测体系,对草莓病毒指示植物和草莓栽培品种都可以进行快速稳定的检测.

草莓轻型黄边病毒RT-LAMP检测方法的建立

【目的】草莓轻型黄边病毒(Strawberry mild yellow edge virus,SMYEV)是侵染草莓的重要病毒,严重影响草莓果实的产量和品质。研究旨在建立利用反转录等温扩增技术(reverse transcription loop-mediated isothermal amplification,RT-LAMP)检测SMYEV的方法。【方法】根据SMYEV外壳蛋白基因3′端保守序列设计4个特异性引物SMYEV-FIP(5′-CAGATCAGCGACAATTTGGACTCCTGAGGAACTTGCTGCT-3′)、SMYEV-BIP(5′-GCTTTGTC GGGGATCCTGGGAAGGCTAAGTCGAAGAGACC-3′)、SMYEV-F3(5′-TCAAGTTGGTGACCCTTTCC-3′)和SMYEV-B3(5′-CGAGGAAC CAATGTCGTAGC-3′)。对RT-LAMP检测体系中的条件进行优化,设置反应温度为60、61、62、63、64、65℃,反应时间为30、45、60、75 min,分别设置引物SMYEV-FIP/BIP浓度为1.0、1.2、1.4、1.6、1.8μmol·L-1,引物SMYEV-F3/B3浓度0.1、0.15、0.2、0.25、0.3μmol·L-1,设置Mg2+浓度为2、4、6、8、10 mmol·L-1,d NTPs浓度为0、0.4、0.8、1.2、1.6、2.0 mmol·L-1,betaine浓度为0、0.4、0.8、1.0、1.2、1.4 mol·L-1,DTT浓度为2.0、2.4、2.8、3.2、3.6、4.0μmol·L-1等,并对以上不同反应条件进行检测,确定优化后的RT-LAMP检测体系。选用其他常见的草莓病毒以及健康草莓植株叶片中的RNA作为模板,对RT-LAMP体系的特异性进行测定;将SMYEV的RNA进行10倍梯度逐级稀释,分别获得RNA原液、10-1、10-2、10-3、10-4、10-5、10-6和10-7不同稀释液作为模板,比较RT-LAMP和RT-PCR的检测灵敏度。RT-LAMP反应产物可以进行电泳和紫外成像检测,阳性样品出现瀑布状条带,阴性样品无条带,或者在反应产物中加入SYBR green I核酸染料,直接观察检测,阳性样品为绿色,阴性样品为橙色。【结果】建立了一种特异性检测SMYEV的RT-LAMP方法,经过各反应条件优化后的检测体系为1.0μmol·L-1SMYEV-FIP/BIP、0.1μmol·L-1 SMYEV-F3/B3、4 mmol·L-1 Mg2+、1.6 mmol·L-1 d NTPs、0.4 mol·L-1 betaine、2.0μmol·L-1DTT,60℃反应45 min。选用SMYEV、草莓镶脉病毒(Strawberry vein banding virus,SVBV)、草莓斑驳病毒(Strawberry mottle virus,SMo V)和草莓皱缩病毒(Strawberry crinkle virus,SCV)的RNA以及健康草莓植株叶片中的RNA作为模板进行RT-LAMP的特异性检测,结果显示仅有SMYEV的RNA能够扩增出瀑布型条带,建立的RT-LAMP检测体系有很好的特异性。将SMYEV的RNA原液、10-1、10-2、10-3稀释液作为模板进行RT-PCR反应后,检测结果为阳性,随着模板稀释倍数的增加,电泳检测不能观察到RT-PCR的扩增产物。而进行SMYEV的RT-LAMP检测时,RNA原液、10-1、10-2、10-3、10-4和10-5稀释液作为模板时,均可以观察到阳性结果。RT-LAMP检测SMYEV比RT-PCR方法检测的灵敏度高100倍,并且比RT-PCR节省时间,检测结果容易判定。【结论】RT-LAMP方法能快速、简便、特异地检测SMYEV,供科研部门和基层生产单位使用,在种苗繁育、田间调查和海关检疫的过程中进行快速检测。

浙江省红颜草莓镶脉病毒(SVBV)的调查与茎尖脱毒技术研究

为了解浙江省草莓主要产区红颜草莓镶脉病毒的感染情况,建立适宜的草莓脱毒快繁技术体系,调查了浙江省主要草莓产区草莓镶脉病毒的分布情况。PCR检测结果表明,大部分红颜品种的植株感染了SVBV。序列分析表明,所感染的病毒与NCBI中报道的病毒相似性达98%以上。以0.2 mm匍匐茎茎尖为外植体,用MS培养基+0.3 mg·L-16-BA+0.05 mg·L-1GA+0.01 mg·L-1IBA+3%蔗糖(p H值5.8)进行脱毒培养。PCR检测结果显示,茎尖培养植株的脱毒率达到93%。采用以上方法可以培养根系发达、抗性强的植株。试验结果将为建立完善的草莓病毒脱毒技术体系提供一定的技术指导。

草莓镶脉病毒的PCR检测及特异片段的序列分析

用CTAB法从感病的草莓叶片中提取总DNA,以其为模板经PCR扩增获得与预期片段大小一致长约600bp的扩增产物,同时优化PCR反应程序,获得单一特异条带;通过总DNA浓度梯度稀释,进行PCR扩增,结果表明能检测到2.5μg叶组织中病毒的存在。回收PCR特异扩增产物,与pMD18-T载体连接,并进行转化、重组克隆的筛选、重组质粒的酶切鉴定和序列测定。扩增片段序列与已报道SVBVCP基因序列(序列号:Nc_001725)的核苷酸同源性为89.2%,氨基酸同源性为96.3%。该特异片段序列在GenBank中的登记号为AY862389。