利用酵母双杂交系统筛选与草莓镶脉病毒P6蛋白互作的森林草莓寄主因子
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摘要
【目的】草莓镶脉病毒(Strawberry vein banding virus,SVBV)是侵染草莓的主要病毒,但其侵染草莓的机制尚不清楚。论文以SVBV的P6蛋白为诱饵筛选森林草莓(Fragaria vesca)c DNA文库的寄主因子,为解析SVBV侵染草莓的分子机制提供理论依据。【方法】SVBV接种森林草莓,提取出现明显症状叶片的总RNA,Dnase I处理后,用SMART法反转录合成ds c DNA,均一化处理c DNA并酶切纯化,将<400 bp的短片段去除,其余片段连接到p GAD-T7质粒载体上,构建森林草莓初级c DNA文库。同时将SVBV P6构建到酵母双杂交诱饵载体p GBK-T7上,再将p GBK-P6和p GBK-T7分别转化酵母菌株AH109,阳性酵母菌株接种SD/-Trp液体培养基,鉴定诱饵载体对酵母细胞的毒性。将转化p GBK-P6的酵母菌分别涂布SD/-Trp、SD/-Leu-Trp和SD/-His-Trp平板,测定菌落生长情况,分析P6蛋白对酵母报告基因的自激活活性。然后用森林草莓初级c DNA文库质粒转化含有诱饵载体p GBK-P6的AH109酵母菌株,共转化子依次涂布SD/-Leu-Trp、SD/-Leu-Trp-His和SD/-Trp-Leu-His-Ade/X-α-Gal平板,最终筛选蓝色且长势较好的阳性菌落,提取酵母质粒并测序,Gen Bank中初步比对候选基因,利用Uniprot在线网站的gene ontology(GO)通路注释互作蛋白因子,分析互作蛋白的生物学功能。【结果】3种c DNA文库平均库容超过2.0×106 cfu,平均文库重组率为97%,文库插入片段平均扩增长度>1 kb,表明森林草莓c DNA文库符合试验标准。最终利用SD/-Trp-Leu-His-Ade/X-α-Gal培养基筛选得到230个酵母阳性克隆,经过序列相似性比对,除去重复序列、载体序列和移码序列,共筛得15个与SVBV P6互作的寄主因子。GO通路注释结果表明这些寄主因子参与了13种生物过程,包括泛素化、转录因子调节、防御反应、代谢过程、氧化还原和胞内氨基酸代谢等过程;这15个寄主因子的分子功能多样,包括乙酰转移酶活性、萜烯合酶活性、脱氢酶活性、金属离子结合活性、蛋白激酶活性和水解酶活性等。【结论】成功构建了森林草莓酵母c DNA文库,筛选出15个与SVBV P6互作的森林草莓寄主因子,为进一步探明SVBV与森林草莓互作的分子机理提供了理论依据。
【Objective】 Strawberry vein banding virus(SVBV) is a main virus infecting woodland strawberry(Fragaria vesca), but the SVBV infection mechanisms on woodland strawberry remains unknown. The objective of this study is to provide a theoretical basis for studying the SVBV infection mechanisms on woodland strawberry, SVBV P6 was used as a bait protein to screen the host factors from the c DNA library of woodland strawberry. 【Method】 Woodland strawberries were inoculated with SVBV, and total RNA was extracted from the leaves showed obvious disease symptoms. The total RNA was treated with Dnase I and double-stranded c DNA was synthesized using SMART technology. c DNA was treated with homogenization and enzymatic digestion, and the short fragments with length less than 400 bp were removed. Then the other c DNA fragments were ligated to plasmid vector p GAD-T7 to construct the primary c DNA library of woodland strawberry. Simultaneously, SVBV P6 was ligated into the yeast two-hybrid bait vector p GBK-T7, and the plasmids of p GBK-P6 and p GBK-T7 were transformed into AH109, respectively. The positive yeast clones were grown in the SD/-Trp liquid medium for identifying the toxicity of p GBK-P6 on the yeast AH109. The yeast transformed with p GBK-P6 was grown on the plate of SD/-Trp, SD/-Leu-Trp and SD/-His-Trp medium, respectively, and then the growth situation of the yeast was tested and the self-activating effect of p GBK-P6 on the reporter gene of yeast was analyzed. Then the AH109 containing bait vector p GBK-P6 was transformed with the primary c DNA library of woodland strawberry, the co-transformed yeasts were coated on the plate of SD/-Leu-Trp, SD/-Leu-Trp-His and SD/-Trp-Leu-His-Ade/X-α-Gal medium in turn. Finally, the blue and well grown positive clones were selected. The plasmids of positive yeast clones were extracted and sequenced. The candidate genes were preliminarily compared in the Gen Bank, and the interacted protein factors were annotated and the protein's biological functions were analyzed with gene ontology(GO) pathway of Uniprot online websites. 【Result】 Three libraries with the average capacity more than 2.0×106 cfu were constructed, and the average library recombination rate was 97% and the average amplification sizes of inserts fragment of c DNA library were above 1 kb. It demonstrated that the c DNA library of woodland strawberry measured up to the experiment standard. The 230 positive clones were finally selected by using the SD/-Trp-Leu-His-Ade/X-α-Gal medium. After sequence similarity comparison, removing the repetitive sequences, the vector sequences and the frameshift sequences, the 15 host factors interacted with SVBV P6 were screened. GO pathway annotation showed that the 15 host factors were involved in 13 biological processes, including protein ubiquitination, regulation of transcription factor process, defense response, protein catabolic process, oxidation-reduction process and cellular amino acid metabolic process, etc. Moreover, molecular functions of the 15 host factors are mutiple, including acetyltransferase activity, terpene synthase activity, dehydrogenase activity, metalion binding activity, protease activity and hydrolase activity, etc.【Conclusion】The c DNA library of woodland strawberry was constructed successfully, and 15 host factors of woodland strawberry interacted with SVBV P6 were preliminarily screened. This work can provide a theoretical basis for further exploring the molecular interaction mechanism between SVBV and woodland strawberry.
【Objective】 Strawberry vein banding virus(SVBV) is a main virus infecting woodland strawberry(Fragaria vesca), but the SVBV infection mechanisms on woodland strawberry remains unknown. The objective of this study is to provide a theoretical basis for studying the SVBV infection mechanisms on woodland strawberry, SVBV P6 was used as a bait protein to screen the host factors from the c DNA library of woodland strawberry. 【Method】 Woodland strawberries were inoculated with SVBV, and total RNA was extracted from the leaves showed obvious disease symptoms. The total RNA was treated with Dnase I and double-stranded c DNA was synthesized using SMART technology. c DNA was treated with homogenization and enzymatic digestion, and the short fragments with length less than 400 bp were removed. Then the other c DNA fragments were ligated to plasmid vector p GAD-T7 to construct the primary c DNA library of woodland strawberry. Simultaneously, SVBV P6 was ligated into the yeast two-hybrid bait vector p GBK-T7, and the plasmids of p GBK-P6 and p GBK-T7 were transformed into AH109, respectively. The positive yeast clones were grown in the SD/-Trp liquid medium for identifying the toxicity of p GBK-P6 on the yeast AH109. The yeast transformed with p GBK-P6 was grown on the plate of SD/-Trp, SD/-Leu-Trp and SD/-His-Trp medium, respectively, and then the growth situation of the yeast was tested and the self-activating effect of p GBK-P6 on the reporter gene of yeast was analyzed. Then the AH109 containing bait vector p GBK-P6 was transformed with the primary c DNA library of woodland strawberry, the co-transformed yeasts were coated on the plate of SD/-Leu-Trp, SD/-Leu-Trp-His and SD/-Trp-Leu-His-Ade/X-α-Gal medium in turn. Finally, the blue and well grown positive clones were selected. The plasmids of positive yeast clones were extracted and sequenced. The candidate genes were preliminarily compared in the Gen Bank, and the interacted protein factors were annotated and the protein's biological functions were analyzed with gene ontology(GO) pathway of Uniprot online websites. 【Result】 Three libraries with the average capacity more than 2.0×106 cfu were constructed, and the average library recombination rate was 97% and the average amplification sizes of inserts fragment of c DNA library were above 1 kb. It demonstrated that the c DNA library of woodland strawberry measured up to the experiment standard. The 230 positive clones were finally selected by using the SD/-Trp-Leu-His-Ade/X-α-Gal medium. After sequence similarity comparison, removing the repetitive sequences, the vector sequences and the frameshift sequences, the 15 host factors interacted with SVBV P6 were screened. GO pathway annotation showed that the 15 host factors were involved in 13 biological processes, including protein ubiquitination, regulation of transcription factor process, defense response, protein catabolic process, oxidation-reduction process and cellular amino acid metabolic process, etc. Moreover, molecular functions of the 15 host factors are mutiple, including acetyltransferase activity, terpene synthase activity, dehydrogenase activity, metalion binding activity, protease activity and hydrolase activity, etc.【Conclusion】The c DNA library of woodland strawberry was constructed successfully, and 15 host factors of woodland strawberry interacted with SVBV P6 were preliminarily screened. This work can provide a theoretical basis for further exploring the molecular interaction mechanism between SVBV and woodland strawberry.
引文
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[3]肖敏,张志宏.草莓镶脉病毒研究进展.辽宁农业科学,2005(4):36-38.XIAO M,ZHANG Z H.Research advance in Strawberry vein banding virus.Liaoning Agricultural Sciences,2005(4):36-38.(in Chinese)
[4]MORRIS T J,MULLIN R H,SCHLEGEL D E,COLE A,ALOSI M C.Isolation of a Caulimovirus from strawberry tissue infected with Strawberry vein banding virus.Phytopathology,1980,70(2):156-160.
[5]PETRZIK K,MRAZ I,DULIC-MARKOVIC I.Quarantine Strawberry vein banding virus firstly detected in Slovakia and Serbia.Acta Virologica,1998,42(2):87-89.
[6]FRAZIER N W.Detection of graft-transmissible diseases in strawberry by a modified leaf grafting technique.Plant Disease Reporter,1974,58:203-207.
[7]洪健,李德葆,周雪平.植物病毒分类图谱.北京:科学出版社,2001:12.HONG J,LI D B,ZHOU X P.Classification Atlas of Plant Viruses.Beijing:Science Press,2001:12.(in Chinese)
[8]PETRZIK K,BENES V,MRAZ I,HONETSLEGROVA F J,ANSORGE W,SPAK J.Strawberry vein banding virus-definitive member of the genus Caulimovirus.Virus Genes,1998,16(3):303-305.
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[10]KAREL P,VLADIMIR B,IVAN M,HONETSLEGROVA-FRANOVA J,ANSORGE W,SPAK J.Strawberry vein banding virus-definitive member of the genus Caulimovirus.Virus Genes,1998,16(3):303-305.
[11]LEH V,YOT P,KELLER M.The Cauliflower mosaic virus translational transactivator interacts with the 60S ribosomal subunit protein L18 of Arabidopsis thaliana.Virology,2000,266(1):1-7.
[12]ANDREW J L,JANET L,JUSTIN H,HAMILTON A J,SADANANDOM A,MILNER J J.Cauliflower mosaic virus protein P6 is a suppressor of RNA silencing.Journal of General Virology,2007,88(12):3439-3444.
[13]GAIL A B,JERRY D J,STEPHEN H H.Cauliflower mosaic virus gene VI produces a symptomatic phenotype in transgenic tobacco plants.Proceeding of the National Academy Sciences of the United States of America,1988,85(3):733-737.
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[15]吴建国,蔡丽君,胡梅群,谢荔岩,林奇英,吴祖建,谢联辉.水稻瘤矮病毒P3、P7、P8、Pn9、Pn10、Pnl1、Pnl2的酵母双杂交载体的构建及自激活效应检测.热带作物学报,2009,30(9):1364-1368.WU J G,CAI L J,HU M Q,XIE L Y,LIN Q Y,WU Z J,XIE L H.Construction of yeast two-hybrid vectors of P3,P7,P8,Pn9,Pn10,Pn11 and Pn12 of Rice gall dwarf virus and identification of their self-activation.Chinese Journal of Tropical Crops,2009,30(9):1364-1368.(in Chinese)
[16]何乙坤,钟敏,胡同乐,王树桐,段豪,丁丽,王亚南,曹克强.利用酵母双杂交筛选与苹果褪绿叶斑病毒CP互作的寄主因子.中国农业科学,2014,47(24):4821-4829.HE Y K,ZHONG M,HU T L,WANG S T,DUAN H,DING L,WANG Y N,CAO K Q.Screening of the host factors interacting with CP of Apple chlorotic leaf spot virus by yeast two-hybrid system.Scientia Agricultura Sinica,2014,47(24):4821-4829.(in Chinese)
[17]楼望淮,蒋甲福,陈素梅,房伟民,陈发棣,管志勇,廖园.菊花B病毒外壳蛋白互作蛋白的筛选.南京农业大学学报,2013,36(4):43-48.LOU W H,JIANG J F,CHEN S M,FANG W M,CHEN F D,GUAN Z Y,LIAO Y.Screening of proteins interacting with the coat protein of Chrysanthemum virus B.Journal of Nanjing Agricultural University,2013,36(4):43-48.(in Chinese)
[18]赵艺泽,刘艳,王锡锋.利用酵母双杂交系统筛选介体异沙叶蝉中与小麦矮缩病毒外壳蛋白互作的蛋白质.中国农业科学,2015,48(12):2354-2363.ZHAO Y Z,LIU Y,WANG X F.Screening of putative proteins in vector Psammotettix alienus L.that are interacted with coat protein of Wheat dwarf virus by a split-ubiquitin yeast membrane system.Scientia Agricultura Sinica,2015,48(12):2354-2363.(in Chinese)
[19]肖冬来,邓慧颖,谢荔岩,吴祖建,谢联辉.酵母双杂交系统筛选与水稻黑条矮缩病毒P6互作的水稻蛋白.热带作物学报,2010,31(3):435-438.XIAO D L,DENG H Y,XIE L Y,WU Z J,XIE L H.Screening of rice proteins interacting with P6 of Rice black-streaked dwarf virus from rice c DNA library by yeast two hybrid system.Chinese Journal of Tropical Crops,2010,31(3):435-438.(in Chinese)
[20]蒋琳,魏春红,李毅.病毒基因沉默抑制子及其作用机制.中国科学:生命科学,2012,42(1):16-28.JIANG L,WEI C H,LI Y.Viral suppressor of RNA silencing.Scientia Sinica Vitae,2012,42(1):16-28.(in Chinese)
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