侵染蚕豆ClYVV的鉴定及其衍生的小干扰RNA的深度测序鉴定研究
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摘要
对安徽省合肥市采集到的具有明显典型花叶症状的蚕豆叶片进行深度测序,鉴定到样品中存在三叶草黄脉病毒(Clover yellow vein virus,Cl YVV),并获得了Cl YVV的完整基因组序列,随后通过直接测序扩增基因组区域获得完整的基因组Cl YVV-HF。获得的Cl YVV-HF基因组由9 585个核苷酸组成,不包括poly(A)尾巴。Cl YVV-HF的全部核苷酸序列与之前报道的来自日本的分离株(AB011819、NC_003536)和来自韩国的分离株(KF975894)分别具有95.45%和95.10%的核苷酸同源性。进一步分析了Cl YVV-HF衍生的小干扰RNA的表征。vsiRNAs的大小为21~22 nt,vsiRNAs的5'末端碱基偏向于A/U。Cl YVV-HF来源的小干扰RNA大多来源于其基因组链中的7个热点,其中5个热点以21 nt vsiRNAs为主。生物学实验表明,Cl YVV可通过汁液摩擦接种多种豆科作物。这是有关中国Cl YVV分离株全部核苷酸序列的首次报道。
The broad bean samples causing prominent mosaic on leaves were collected in Hefei City,Anhui Province in China,and viruses in samples were detected by deep sequencing. The results showed that Clover yellow vein virus( Cl YVV) existed in the broad bean samples and the complete genome Cl YVV-HF was obtained by direct sequencing of the amplified genomic region,subsequently. The Cl YVV genome of China consists of 9 585 nucleotides excluding the poly( A) tail,the full nucleotide sequence of Cl YVV-HF shared a nucleotide identity of 95. 45% and 95. 10%with that of isolates from Japan( AB011819,NC_003536) and an isolate from Korea( KF975894) reported previously. Characterization of Cl YVV-derived small interfering RNAs was further analyzed. The prevalent vsiRNAs size was21-22 nt,and the 5'-terminal base of vsiRNAs was biased towards A/U. The Cl YVV-derived small interfering RNAs were most derived from its genomic-strand RNAs,among the 7 hot spots across the genome,5 hot spots were predominated by 21 nt vsiRNAs. Biological experiments showed that Cl YVV could be used to frictionally inoculate many leguminous crops through juice. This is the first report about the full nucleotide sequence of Cl YVV isolate from China.
The broad bean samples causing prominent mosaic on leaves were collected in Hefei City,Anhui Province in China,and viruses in samples were detected by deep sequencing. The results showed that Clover yellow vein virus( Cl YVV) existed in the broad bean samples and the complete genome Cl YVV-HF was obtained by direct sequencing of the amplified genomic region,subsequently. The Cl YVV genome of China consists of 9 585 nucleotides excluding the poly( A) tail,the full nucleotide sequence of Cl YVV-HF shared a nucleotide identity of 95. 45% and 95. 10%with that of isolates from Japan( AB011819,NC_003536) and an isolate from Korea( KF975894) reported previously. Characterization of Cl YVV-derived small interfering RNAs was further analyzed. The prevalent vsiRNAs size was21-22 nt,and the 5'-terminal base of vsiRNAs was biased towards A/U. The Cl YVV-derived small interfering RNAs were most derived from its genomic-strand RNAs,among the 7 hot spots across the genome,5 hot spots were predominated by 21 nt vsiRNAs. Biological experiments showed that Cl YVV could be used to frictionally inoculate many leguminous crops through juice. This is the first report about the full nucleotide sequence of Cl YVV isolate from China.
引文
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[19]CHEN J,ADAMS M.A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family[J].Archives of Virology,2001,146(4):757-766.
[20]LARKIN M A,BLACKSHIELDS G,BROWN N,et al.Clustal W and Clustal X version 2.0[J].Bioinformatics,2007,23(21):2947-2948.
[21]MI S,CAI T,HU Y,et al.Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5'terminal nucleotide[J].Cell,2008,133(1):116-127.
[22]MONTGOMERY T A,HOWELL M D,CUPERUS J T,et al.Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation[J].Cell,2008,133(1):128-141.
[23]HAGEN C,FRIZZI A,KAO J,et al.Using small RNA sequences to diagnose,sequence,and investigate the infectivity characteristics of vegetable-infecting viruses[J].Archives of Virology,2011,156(7):1209-1216.
[24]KREUZE J F,PEREZ A,UNTIVEROS M,et al.Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs:a generic method for diagnosis,discovery and sequencing of viruses[J].Virology,2009,388(1):1-7.
[25]SHIN J C,KIM M K,KWAK H R,et al.First report of Clover yellow vein virus on Glycine max in Korea[J].Plant Disease,2014,98(9):1283.
[26]RAVELO G,KAGAYA U,INUKAI T,et al.Genetic analysis of lethal tip necrosis induced by Clover yellow vein virus infection in pea[J].Journal of General Plant Pathology,2007,73(1):59-65.
[27]UYEDA I,TAKAHASHI T,TAKAHASHI Y.A c DNA clone to clover yellow vein potyvirus genome is highly infectious[J].Virus Genes,1997,14(3):235-243.
[28]MITTER N,KOUNDAL V,WILLIAMS S,et al.Differential expression of tomato spotted wilt virus-derived viral small RNAs in infected commercial and experimental host plants[J].Plo S One,2013,8(10):e76276.
[29]PANTALEO V,SALDARELLI P,MIOZZI L,et al.Deep sequencing analysis of viral short RNAs from an infected Pinot Noir grapevine[J].Virology,2010,408(1):49-56.
[30]YAN F,ZHANG H,ADAMS M J,et al.Characterization of siRNAs derived from rice stripe virus in infected rice plants by deep sequencing[J].Archives of Virology,2010,155(6):935-940.
[2]尹明华,刘燕,郁雪婷,等.怀玉山高山马铃薯茎尖再生苗6种病毒的DAS-ELISA检测与分析[J].浙江农业学报,2017,29(10):1699-1705.YIN M H,LIU Y,YU X T,et al.DAS-ELISA detection and analysis of six kinds of viruses in plantlets regenerated from Hua-iyushan high mountain potato shoot-tips[J].Acta Agriculturae Zhejiangensis,2017,29(10):1699-1705.(in Chinese with English abstract)
[3]CHUNG B Y,MILLER W A,ATKINS J F,et al.An overlapping essential gene in the Potyviridae[J].Proceedings of the National Academy of Sciences of the United States of America,2008,105(15):5897-5902.
[4]SASAYA T,SHIMIZU T,NOZU Y,et al.Biological,serological,and molecular variabilities of clover yellow vein virus[J].Phytopathology,1997,87(10):1014-1019.
[5]HOLLINGS M,NARIANI T.Some properties of clover yellow vein,a virus from Trifolium repens L.[J].Annals of Applied Biology,1965,56(1):99-109.
[6]FOSTER R,MUSGRAVE D.Clover yellow vein virus in white clover(Trifolium repens)and sweet pea(Lathyrus odoratus)in the North Island of New Zealand[J].New Zealand Journal of Agricultural Research,1985,28(4):575-578.
[7]IREY M,ADKINS S,BAKER C.Clover yellow vein virus Identified in Ammi majus in Florida[J].Plant Disease,2006,90(3):380.
[8]KEHOE M A,COUTTS B A,BUIRCHELL B J,et al.Plant virology and next generation sequencing:experiences with a Potyvirus[J].PLo S One,2014,9(8):e104580
[9]LARSEN R C,MIKLAS P N,EASTWELL K C,et al.A strain of Clover yellow vein virus that causes severe pod necrosis disease in snap bean[J].Plant Disease,2008,92(7):1026-1032.
[10]MORAN J,VAN RIJSWIJK B,TRAICEVSKI V,et al.Potyviruses,novel and known,in cultivated and wild species of the family Apiaceae in Australia[J].Archives of Virology,2002,147(10):1855-1867.
[11]ORTIZ V,CASTRO S,ROMERO J.First report of Clover yellow vein virus in grain legumes in Spain[J].Plant Disease,2009,93(1):106.
[12]PASEV G,KOSTOVA D,TURINA M.A New virulent isolate of Clover yellow vein virus on Phaseolus vulgaris in Bulgaria[J].Journal of Phytopathology,2014,162(11/12):703-711.
[13]YAMAMOTO H.Mosaic disease of bupleurum(Bupleurum griffithii)caused by Clover yellow vein virus[J].Annals of the Phytopathological Society of Japan,2003,69(4):420-421.
[14]HART J P,GRIFFITHS P D.A series of e IF4E alleles at the Bc-3 locus are associated with recessive resistance to Clover yellow vein virus in common bean[J].Theoretical and Applied Genetics,2013,126(11):2849-2863.
[15]LARSEN R,MYERS J.A pod necrosis disease(chocolate pod)of snap bean(Phaseolus vulgaris)in Oregon caused by a strain of Clover yellow vein virus[J].Phytopathology,2006,96:S169.
[16]李长松,朱汉城.侵染菜豆的三叶草黄脉病毒的鉴定[J].山东农业大学学报,1989(4):223-226.LI C S,ZHU H C.Identification of Clover yellow vein virus infecting Phaseolus vulgaris[J].Journal of Shandong Agricultural University,1989(4):223-226.(in Chinese with English abstract)
[17]李长松.侵染扁豆的三叶草黄脉病毒的鉴定[J].中国病毒学,1991,6(3):223-226.LI C S.Identification of Clover yellow vein virus infecting Dolichos lablab[J].Virologica Sinica,1991,6(3):223-226.(in Chinese with English abstract)
[18]TZANETAKIS I E,MARTIN R R.A new method for extraction of double-stranded RNA from plants[J].Journal of Virological Methods,2008,149(1):167-170.
[19]CHEN J,ADAMS M.A universal PCR primer to detect members of the Potyviridae and its use to examine the taxonomic status of several members of the family[J].Archives of Virology,2001,146(4):757-766.
[20]LARKIN M A,BLACKSHIELDS G,BROWN N,et al.Clustal W and Clustal X version 2.0[J].Bioinformatics,2007,23(21):2947-2948.
[21]MI S,CAI T,HU Y,et al.Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5'terminal nucleotide[J].Cell,2008,133(1):116-127.
[22]MONTGOMERY T A,HOWELL M D,CUPERUS J T,et al.Specificity of ARGONAUTE7-miR390 interaction and dual functionality in TAS3 trans-acting siRNA formation[J].Cell,2008,133(1):128-141.
[23]HAGEN C,FRIZZI A,KAO J,et al.Using small RNA sequences to diagnose,sequence,and investigate the infectivity characteristics of vegetable-infecting viruses[J].Archives of Virology,2011,156(7):1209-1216.
[24]KREUZE J F,PEREZ A,UNTIVEROS M,et al.Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs:a generic method for diagnosis,discovery and sequencing of viruses[J].Virology,2009,388(1):1-7.
[25]SHIN J C,KIM M K,KWAK H R,et al.First report of Clover yellow vein virus on Glycine max in Korea[J].Plant Disease,2014,98(9):1283.
[26]RAVELO G,KAGAYA U,INUKAI T,et al.Genetic analysis of lethal tip necrosis induced by Clover yellow vein virus infection in pea[J].Journal of General Plant Pathology,2007,73(1):59-65.
[27]UYEDA I,TAKAHASHI T,TAKAHASHI Y.A c DNA clone to clover yellow vein potyvirus genome is highly infectious[J].Virus Genes,1997,14(3):235-243.
[28]MITTER N,KOUNDAL V,WILLIAMS S,et al.Differential expression of tomato spotted wilt virus-derived viral small RNAs in infected commercial and experimental host plants[J].Plo S One,2013,8(10):e76276.
[29]PANTALEO V,SALDARELLI P,MIOZZI L,et al.Deep sequencing analysis of viral short RNAs from an infected Pinot Noir grapevine[J].Virology,2010,408(1):49-56.
[30]YAN F,ZHANG H,ADAMS M J,et al.Characterization of siRNAs derived from rice stripe virus in infected rice plants by deep sequencing[J].Archives of Virology,2010,155(6):935-940.