热处理结合化学处理脱除梨病毒研究及热处理植株中病毒siRNAs鉴定
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摘要
病毒危害可严重影响梨生长势和降低果品产量与品质,栽培无病毒种苗是减轻梨病毒病危害的重要途径,热处理和化学处理是获得无病毒果树种质的主要方法。大量的研究表明持续高温或采用一定浓度的病毒抑制剂处理可以脱除植物病毒,但梨对高温相对敏感,长时间的高温处理易造成梨植株的大量死亡,化学处理也可导致梨植株产生药害,同时需要较长的处理周期。本研究以感染有苹果茎沟病毒(Apple stem grooving virus, ASGV)和苹果褪绿叶斑病毒(Apple chlorotic leaf spot virus, ACLSV)的砂梨“金水2号”离体植株为试验材料,采用热处理(35°C)与化学处理(病毒醚)相结合的方法进行梨病毒脱除的研究,以期提高病毒脱除效果。同时对继代培养过程中梨离体植株的ASGV分子群体结构特点进行了系统分析,测定了一个ASGV砂梨分离物的全长基因组序列。并以热处理的砂梨离体植株为材料,采用高通量测序方法分析了该植株中ASGV的siRNA组成特点,定量分析了其在热处理35d过程中的含量变化。结果如下：
1.以砂梨“黄花”离体植株为研究材料,采用RT-PCR技术对ASGV进行检测的结果表明,该病毒在20次继代培养的“黄花”离体植株中可以稳定增殖。对扩增产物进行克隆,每次继代的植株中挑选大于20个克隆的单链构象多态性(SSCP)分析的结果显示,“黄花”离体植株中存在ASGV的一个优势分子变种,继代培养对ASGV的分子群体结构没有明显影响。
2.根据GenBank登录序列设计合成引物,对来源于砂梨“黄花”离体植株的ASGV的全基因组进行了扩增和序列分析,该分离物的全基因组(除去聚合A尾)由6496个核苷酸组成(命名为ASGV-HH),包含两个开放阅读框(ORF),分别编码241kDa聚合蛋白和36kDa运动蛋白。全基因组序列与已报道的来源于日本的苹果分离物ASGV-P-209,日本的百合分离物CTLV-L和CTLV-Li-23,韩国的砂梨分离物ASGV-P,台湾的柑橘分离物CTLV-K和CTLV-LCd-NA-1,以及美国的柑橘分离物CTLV-ML的相似性为79.8-87.1%,其中,ASGV-HH的复制酶编码区序列与日本的苹果分离物ASGV-P-209(?)似性最高,核苷酸和氨基酸现相似性分别为86.2%和89.9%,其CP和MP的核苷酸序列则与来源于日本的百合分离物CTLV-L同源性最高,分别为93.7%和87.6%。根据该病毒的可变区Ⅱ的氨基酸序列构建系统进化树显示,ASGV-HH与CTLV-L亲缘关系最近,而与来源于韩国的梨分离物ASGV-P亲缘关系较远。
3.以感染ASGV和ACLSV的砂梨“金水2号”离体植株为研究材料,比较分析了热处理(35℃)、化学处理(病毒醚)及二者相结合对梨离体植株生长的影响及脱除这两种病毒的效果,结果显示15-25μg/ml病毒醚处理5-30d对梨离体植株的生长和增殖有明显的促进作用,株高由未处理植株的2.10cm增加至2.57-2.61cm,增殖系数由未处理植株的3.27增加至4.09-8.82。分别于处理30d、35d和40d切取0.5mm和1mm的茎尖进行培养,继代1-2次后采用RT-PCR的方法对再生植株的带毒情况进行检测,结果表明化学处理与热处理相结合可明显提高病毒脱除效果,采用15μg/mL和20μg/mL病毒醚结合35℃处理40d所获再生植株的ACLSV和ASGV脱除率分别由病毒醚单独处理的50.0-60.0%和41.7-50.0%提高至61.5-69.2%和53.8-76.9%,采用25μg/mL病毒醚结合35℃处理40d后切取的0.5-1.0mm茎尖所获再生植株的病毒脱除率达100%。采用该方法对“圆黄梨”、“黄花梨”、“雪花梨”和苹果“潮南”离体植株进行了处理,脱病毒率“圆黄梨”和苹果的为100.0%,“黄花梨”和“雪花梨”的分别为84.6%和64.8%。
4.采用高通量测序方法对经热处理的砂梨“黄花”离体植株中小分子RNA进行了鉴定,共测得16,372,275种,其中与ASGV-HH序列匹配的siRNA有3111种(简称vsiRNA),这些vsiRNA的大小在18-25nt,21nt和22nt所占比例最大,占总数的82.7%。各vsiRNA的出现频率为1-103次。vsiRNA在ASGV-HH基因组的正负链上的分布有一定的偏向性,在正链上分布频率较高,为55.8%。根据vsiRNA所对应ASGV-HH基因组序列,选取与ASGV基因组6个保守结构域的对应的6个vsiRNAs,采用定量RT-PCR (qRT-PCR)分析其在热处理梨植株中相对含量的时间动态变化,结果表明各vsiRNA的含量在热处理初期呈明显下降趋势,而至25d时均出现一个峰值。其中,对应Met、Het、P-Pro和MP的vsiRNA在热处理25d植株中的含量较处理前的植株低,而对应CP和Rdrp的vsiRNA在热处理25d植株中的含量明显高于处理前的植株,分别为处理前的1.3和2.5倍。ASGV的含量随热处理时间的延长呈明显的下降趋势,第5d即开始减低,至第25d时病毒RT-PCR产物的扩增条带已经极其微弱,推测高温促进了病毒RNA沉默的发生。
Virus diseases have serious effect on the growth of pear plants and decrease their fruit quality and productivity. Utilizing certified healthy propagation materials is one of the most effective measures for the controlling of virus diseases. Thermotherapy and chemotherapy are the most widely used methods for the production of virus-free plant materials. Many study results have demonstrated that plant viruses could be eliminated by continuous treatment under high temperature or by using antiviral chemicals at a proper concentration. Pear plants are relatively sensitive to high temperature, which can cause a high death rate. Meanwhile, chemotherapy can induce phytotoxicity of host and usually cost long time. In this study, in order to improve the virus elimination efficiency, sand pear Pyrus pyrifolia cv. Jinshui no.2infected with Apple stem grooving virus (ASGV) and Apple chlorolic leaf spot virus (ACLSV) was used as the experimental material, and thermotherapy (35℃) combined with chemotherapy (Ribavirin) was used to eliminate the viruses from in vitro pears. Meanwhile, the population structure of ASGV in in vitro plants of pear was investigated during20sub-culturings and the full genome of an ASGV isolate from sand pear was sequenced. Moreover, the siRNAs derived from ASGV in in vitro sand pears treated by thermotherapy was characterized by high-throughput sequencing, and the titers of six siRNAs were tested during a35d thermotherapy course. Results obtained are as follows:
1. In vitro sand pear P. pyrifolia cv.'Huanghua' was used as material. The RT-PCR results showed that ASGV can replicate stably in in vitro sand pear cultures during20passage sub-cultures. The RT-PCR products were cloned and single-strand conformation polymorphism (SSCP) of PCR products of more than20clones from each sub-culturing showed that ASGV persisted as a dominant molecular variant in in vitro cultures of 'Huanghua', and continues sub-culturings had no significant effect on the constitution of ASGV molecular variants.
2. The complete genome of an ASGV isolate from in vitro 'Huanghua' was sequenced. The primers used in this study were designed basing on the available sequences from GenBank. The ASGV isolate named ASGV-HH contained6,496nucleotides excluding the poly (A) tail, and consisted of two open reading frames (ORFs), a214kDa polyprotein and a36kDa movement protein, respectively. The full genome of ASGV-HH had identities ranging from79.8%to87.1%with previously reported ASGV isolates ASGV-P-209from apple, CTLV-L and CTLV-Li-23from lily in Japan, ASGV-P from pear in South Korea, CTLV-K and CTLV-LCd-NA-1from citrus in Taiwan, and CTLV-ML from citrus in USA. ASGV-HH had highest identities of86.2%nt and89.9%aa in Rep region with isolate ASGV-P-209. ASGV-HH had highest identities of93.7%and87.6%with isolate CTLV-L in CP and MP, respectively. The phylogenetic tree constructed by amino acid sequences of variable region Ⅱ showed that ASGV-HH was closed to CTLV-L, and separated from isolate ASGV-P in the tree.
3. In vitro cultures of P. pyrifolia cv. Jinshui no.2infected by ACLSV and ASGV, were used as materials to evaluate the effects of thermotherapy at35±0.5℃, chemotherapy with ribavirin, and combinations of chemotherapy and thermotherapy on the growth and virus elimination of in vitro pears. Results showed that the15-25μg/mL ribavirin could significantly improve the growth and proliferation of in vitro pears during5-30d treatment periods. The growth of in vitro pears treated by15-25μg/mL ribavirin was increased from2.10cm to2.57-2.61cm, and the proliferation was increased from3.27to4.09-8.82. Shoot tips of0.5-1.0mm long were excised from in vitro plants treated for30d,35d and40d, and viruses in regenerated plants were detected after one or two sub-culturing by RT-PCR. Results showed that the combination of chemotherapy and thermotherapy could enhance the efficiency of virus elimination. The virus elimination efficiency of treatments of15or20μg/mL ribavirin combined with thermotherapy at35±0.5℃for40d were53.8-76.9%for ACLSV and61.5-69.2%for ASGV, which were much higher than50.0-60.0%and41.7-50.0%of virus elimination rates achieved by treatment with15and20μg/mL ribavirin. A high virus eradication efficiency of100%was achieved by chemotherapy of ribavirin at25μg/mL combined with thermotherapy at35℃for40days, followed by culturing of0.5-1.0mm long meristem-tips. Furthermore, the treatment protocol was used for the virus elimination from other three in vitro-cultured pears,'Huanghua','Xuehua','Yuanhuang', and one in vitro-cultured apple 'Chaonan'. The virus elimination rate of100.0%for 'Yuanhuang' and 'Chaonan', and84.6%and64.8%for,'Huanghua' and 'Xuehua' were achieved, respectively.
4. Small RNAs in in vitro 'Huanghua' treated by thermotherapy were detected by high-throughput sequencing, and a total of16,372,275unique reads was identified. Sequence alignment to the genome of ASGV-HH revealed3,111reads of ASGV siRNAs (named vsiRNA). The sizes of vsiRNAs ranged from18nt to25nt, and vsiRNAs of21nt and22nt account82.7%of total vsiRNAs. The detected frequency of vsiRNAs was 1-103times. There was a slight bias of vsiRNAs toward the sense strand of ASGV genome, which was55.8%. The titers of six vsiRNAs from six conserved regions of the ASGV genome during thermotherapy were analyzed by Real-Time qRT-PCR. The results showed that the titer of vsiRNAs decreased during the primary period of thermotherapy, and then had a peak in the in vitro cultures treated for25days. The titers of vsiRNAs derived from Met, Het, P-Pro and MP in in vitro pear treated by high temperature for25days was lower than that in untreated pears, whereas, the titer of vsiRNAs of CP and Rdrp in in vitro pear treated by high temperature for25days was higher than that in untreated pears for1.3and2.5times, respectively. During thermotherapy, the titer of ASGV in in vitro cultures decreased, and the band of RT-PCR products in agarose gel was very weak when the treatment was prolonged for25days. It was speculated that high temperature enhanced the RNA silencing.
1.以砂梨“黄花”离体植株为研究材料,采用RT-PCR技术对ASGV进行检测的结果表明,该病毒在20次继代培养的“黄花”离体植株中可以稳定增殖。对扩增产物进行克隆,每次继代的植株中挑选大于20个克隆的单链构象多态性(SSCP)分析的结果显示,“黄花”离体植株中存在ASGV的一个优势分子变种,继代培养对ASGV的分子群体结构没有明显影响。
2.根据GenBank登录序列设计合成引物,对来源于砂梨“黄花”离体植株的ASGV的全基因组进行了扩增和序列分析,该分离物的全基因组(除去聚合A尾)由6496个核苷酸组成(命名为ASGV-HH),包含两个开放阅读框(ORF),分别编码241kDa聚合蛋白和36kDa运动蛋白。全基因组序列与已报道的来源于日本的苹果分离物ASGV-P-209,日本的百合分离物CTLV-L和CTLV-Li-23,韩国的砂梨分离物ASGV-P,台湾的柑橘分离物CTLV-K和CTLV-LCd-NA-1,以及美国的柑橘分离物CTLV-ML的相似性为79.8-87.1%,其中,ASGV-HH的复制酶编码区序列与日本的苹果分离物ASGV-P-209(?)似性最高,核苷酸和氨基酸现相似性分别为86.2%和89.9%,其CP和MP的核苷酸序列则与来源于日本的百合分离物CTLV-L同源性最高,分别为93.7%和87.6%。根据该病毒的可变区Ⅱ的氨基酸序列构建系统进化树显示,ASGV-HH与CTLV-L亲缘关系最近,而与来源于韩国的梨分离物ASGV-P亲缘关系较远。
3.以感染ASGV和ACLSV的砂梨“金水2号”离体植株为研究材料,比较分析了热处理(35℃)、化学处理(病毒醚)及二者相结合对梨离体植株生长的影响及脱除这两种病毒的效果,结果显示15-25μg/ml病毒醚处理5-30d对梨离体植株的生长和增殖有明显的促进作用,株高由未处理植株的2.10cm增加至2.57-2.61cm,增殖系数由未处理植株的3.27增加至4.09-8.82。分别于处理30d、35d和40d切取0.5mm和1mm的茎尖进行培养,继代1-2次后采用RT-PCR的方法对再生植株的带毒情况进行检测,结果表明化学处理与热处理相结合可明显提高病毒脱除效果,采用15μg/mL和20μg/mL病毒醚结合35℃处理40d所获再生植株的ACLSV和ASGV脱除率分别由病毒醚单独处理的50.0-60.0%和41.7-50.0%提高至61.5-69.2%和53.8-76.9%,采用25μg/mL病毒醚结合35℃处理40d后切取的0.5-1.0mm茎尖所获再生植株的病毒脱除率达100%。采用该方法对“圆黄梨”、“黄花梨”、“雪花梨”和苹果“潮南”离体植株进行了处理,脱病毒率“圆黄梨”和苹果的为100.0%,“黄花梨”和“雪花梨”的分别为84.6%和64.8%。
4.采用高通量测序方法对经热处理的砂梨“黄花”离体植株中小分子RNA进行了鉴定,共测得16,372,275种,其中与ASGV-HH序列匹配的siRNA有3111种(简称vsiRNA),这些vsiRNA的大小在18-25nt,21nt和22nt所占比例最大,占总数的82.7%。各vsiRNA的出现频率为1-103次。vsiRNA在ASGV-HH基因组的正负链上的分布有一定的偏向性,在正链上分布频率较高,为55.8%。根据vsiRNA所对应ASGV-HH基因组序列,选取与ASGV基因组6个保守结构域的对应的6个vsiRNAs,采用定量RT-PCR (qRT-PCR)分析其在热处理梨植株中相对含量的时间动态变化,结果表明各vsiRNA的含量在热处理初期呈明显下降趋势,而至25d时均出现一个峰值。其中,对应Met、Het、P-Pro和MP的vsiRNA在热处理25d植株中的含量较处理前的植株低,而对应CP和Rdrp的vsiRNA在热处理25d植株中的含量明显高于处理前的植株,分别为处理前的1.3和2.5倍。ASGV的含量随热处理时间的延长呈明显的下降趋势,第5d即开始减低,至第25d时病毒RT-PCR产物的扩增条带已经极其微弱,推测高温促进了病毒RNA沉默的发生。
Virus diseases have serious effect on the growth of pear plants and decrease their fruit quality and productivity. Utilizing certified healthy propagation materials is one of the most effective measures for the controlling of virus diseases. Thermotherapy and chemotherapy are the most widely used methods for the production of virus-free plant materials. Many study results have demonstrated that plant viruses could be eliminated by continuous treatment under high temperature or by using antiviral chemicals at a proper concentration. Pear plants are relatively sensitive to high temperature, which can cause a high death rate. Meanwhile, chemotherapy can induce phytotoxicity of host and usually cost long time. In this study, in order to improve the virus elimination efficiency, sand pear Pyrus pyrifolia cv. Jinshui no.2infected with Apple stem grooving virus (ASGV) and Apple chlorolic leaf spot virus (ACLSV) was used as the experimental material, and thermotherapy (35℃) combined with chemotherapy (Ribavirin) was used to eliminate the viruses from in vitro pears. Meanwhile, the population structure of ASGV in in vitro plants of pear was investigated during20sub-culturings and the full genome of an ASGV isolate from sand pear was sequenced. Moreover, the siRNAs derived from ASGV in in vitro sand pears treated by thermotherapy was characterized by high-throughput sequencing, and the titers of six siRNAs were tested during a35d thermotherapy course. Results obtained are as follows:
1. In vitro sand pear P. pyrifolia cv.'Huanghua' was used as material. The RT-PCR results showed that ASGV can replicate stably in in vitro sand pear cultures during20passage sub-cultures. The RT-PCR products were cloned and single-strand conformation polymorphism (SSCP) of PCR products of more than20clones from each sub-culturing showed that ASGV persisted as a dominant molecular variant in in vitro cultures of 'Huanghua', and continues sub-culturings had no significant effect on the constitution of ASGV molecular variants.
2. The complete genome of an ASGV isolate from in vitro 'Huanghua' was sequenced. The primers used in this study were designed basing on the available sequences from GenBank. The ASGV isolate named ASGV-HH contained6,496nucleotides excluding the poly (A) tail, and consisted of two open reading frames (ORFs), a214kDa polyprotein and a36kDa movement protein, respectively. The full genome of ASGV-HH had identities ranging from79.8%to87.1%with previously reported ASGV isolates ASGV-P-209from apple, CTLV-L and CTLV-Li-23from lily in Japan, ASGV-P from pear in South Korea, CTLV-K and CTLV-LCd-NA-1from citrus in Taiwan, and CTLV-ML from citrus in USA. ASGV-HH had highest identities of86.2%nt and89.9%aa in Rep region with isolate ASGV-P-209. ASGV-HH had highest identities of93.7%and87.6%with isolate CTLV-L in CP and MP, respectively. The phylogenetic tree constructed by amino acid sequences of variable region Ⅱ showed that ASGV-HH was closed to CTLV-L, and separated from isolate ASGV-P in the tree.
3. In vitro cultures of P. pyrifolia cv. Jinshui no.2infected by ACLSV and ASGV, were used as materials to evaluate the effects of thermotherapy at35±0.5℃, chemotherapy with ribavirin, and combinations of chemotherapy and thermotherapy on the growth and virus elimination of in vitro pears. Results showed that the15-25μg/mL ribavirin could significantly improve the growth and proliferation of in vitro pears during5-30d treatment periods. The growth of in vitro pears treated by15-25μg/mL ribavirin was increased from2.10cm to2.57-2.61cm, and the proliferation was increased from3.27to4.09-8.82. Shoot tips of0.5-1.0mm long were excised from in vitro plants treated for30d,35d and40d, and viruses in regenerated plants were detected after one or two sub-culturing by RT-PCR. Results showed that the combination of chemotherapy and thermotherapy could enhance the efficiency of virus elimination. The virus elimination efficiency of treatments of15or20μg/mL ribavirin combined with thermotherapy at35±0.5℃for40d were53.8-76.9%for ACLSV and61.5-69.2%for ASGV, which were much higher than50.0-60.0%and41.7-50.0%of virus elimination rates achieved by treatment with15and20μg/mL ribavirin. A high virus eradication efficiency of100%was achieved by chemotherapy of ribavirin at25μg/mL combined with thermotherapy at35℃for40days, followed by culturing of0.5-1.0mm long meristem-tips. Furthermore, the treatment protocol was used for the virus elimination from other three in vitro-cultured pears,'Huanghua','Xuehua','Yuanhuang', and one in vitro-cultured apple 'Chaonan'. The virus elimination rate of100.0%for 'Yuanhuang' and 'Chaonan', and84.6%and64.8%for,'Huanghua' and 'Xuehua' were achieved, respectively.
4. Small RNAs in in vitro 'Huanghua' treated by thermotherapy were detected by high-throughput sequencing, and a total of16,372,275unique reads was identified. Sequence alignment to the genome of ASGV-HH revealed3,111reads of ASGV siRNAs (named vsiRNA). The sizes of vsiRNAs ranged from18nt to25nt, and vsiRNAs of21nt and22nt account82.7%of total vsiRNAs. The detected frequency of vsiRNAs was 1-103times. There was a slight bias of vsiRNAs toward the sense strand of ASGV genome, which was55.8%. The titers of six vsiRNAs from six conserved regions of the ASGV genome during thermotherapy were analyzed by Real-Time qRT-PCR. The results showed that the titer of vsiRNAs decreased during the primary period of thermotherapy, and then had a peak in the in vitro cultures treated for25days. The titers of vsiRNAs derived from Met, Het, P-Pro and MP in in vitro pear treated by high temperature for25days was lower than that in untreated pears, whereas, the titer of vsiRNAs of CP and Rdrp in in vitro pear treated by high temperature for25days was higher than that in untreated pears for1.3and2.5times, respectively. During thermotherapy, the titer of ASGV in in vitro cultures decreased, and the band of RT-PCR products in agarose gel was very weak when the treatment was prolonged for25days. It was speculated that high temperature enhanced the RNA silencing.
引文
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