梨黑斑病菌遗传操作体系的建立与RFP标记转化子的致病性分析
|
摘要
由链格孢(Alternaria alternata)引起的梨黑斑病是我国梨生产上的主要病害之一,导致梨叶与果实产生黑斑症状及早期落叶,对我国梨产业的健康发展构成严重威胁。本实验在前期获得强致病性梨黑斑病菌菌株HN-5基础上,建立该病菌的遗传转化体系。通过优化原生质体制备过程以及转化体系发现梨黑斑病菌HN-5原生质体制备的最优条件为:菌丝在M R液体培养基中培养36 h;以0.7 M NaCl+0.8 M Mannitol为稳渗剂,配置复合细胞壁裂解酶(1%崩溃酶+1%裂解酶+1%纤维素酶+1%蜗牛酶),酶解菌丝3 h,原生质体的产量可达0.5×10~7个·mL~(-1)。通过PEG介导的原生质体遗传转化体系,将含有RFP基因的质粒p KD7转入链格孢菌HN-5中,转化效率为6个·μg~(-1)DNA。PCR检测和转化子荧光观察均表明RFP基因整合到了HN-5基因组中并成功表达。致病性分析发现RFP (Red Fluorescent Protein)标记转化子致病性未发生变化。本研究成功建立了PEG (Polyethylene glycol)介导的梨黑斑病菌遗传转化体系,构建了RFP标记菌株,为研究该病菌的致病机理奠定基础。
Pear black spot disease is one of the major diseases of pear in China. It causes black spot symptoms and early defoliation,which seriously threatens pear production. Based on a previously obtained strain (HN-5)of Alternaria alternata with strong pathogenicity,we established a genetic transformation system of this fungus.We improved the conditions for increasing protoplast yield of HN-5 strain as below : the mycelia were cultured in MR medium for 36 hours,useing 0.7 M NaCl and 0.8 M Manitol as the osmotic stabilizer; the mycelia were digested with four enzymes mixture (1% lysing enzyme,1% driselase,1% cellulose,and 1% snailase) at 30℃ for 3 hours,and the protoplasts yeild was up to 0.5×10~7·mL~(-1). The plasmid pKD7 with RFP was transferred into HN-5 strain by PEG-mediated protoplast genetic transformation,and six transformants per μg DNA were obtained. Genome PCR validation and fluorescence analysis of the transformants confirmed that the RFP gene was successfully integrated and expressed into the HN-5 genome. There was no change in the pathogenicity of the RFP transformants. In this summary,we successfully established a PEG-mediated genetic transformation ofA. alternata and generated the RFP transgenic strains,which laid the foundation for investigating the pathogenesis of this fungus.
Pear black spot disease is one of the major diseases of pear in China. It causes black spot symptoms and early defoliation,which seriously threatens pear production. Based on a previously obtained strain (HN-5)of Alternaria alternata with strong pathogenicity,we established a genetic transformation system of this fungus.We improved the conditions for increasing protoplast yield of HN-5 strain as below : the mycelia were cultured in MR medium for 36 hours,useing 0.7 M NaCl and 0.8 M Manitol as the osmotic stabilizer; the mycelia were digested with four enzymes mixture (1% lysing enzyme,1% driselase,1% cellulose,and 1% snailase) at 30℃ for 3 hours,and the protoplasts yeild was up to 0.5×10~7·mL~(-1). The plasmid pKD7 with RFP was transferred into HN-5 strain by PEG-mediated protoplast genetic transformation,and six transformants per μg DNA were obtained. Genome PCR validation and fluorescence analysis of the transformants confirmed that the RFP gene was successfully integrated and expressed into the HN-5 genome. There was no change in the pathogenicity of the RFP transformants. In this summary,we successfully established a PEG-mediated genetic transformation ofA. alternata and generated the RFP transgenic strains,which laid the foundation for investigating the pathogenesis of this fungus.
引文
[1]Yang X,Qian G,Fan J,et al.Selection and identification of antagonist Alternaria alternate and initial analysis of its antipathogenic activity(in Chinese)[J].Journal of Nanjing Agricultural University(南京农业大学学报),2014,37(1):68-74.
[2]Yang Q Q,Liu Y L,Wang T,et al.Influence of pathogen infection on early leaf abscission of three Chinese pear cultivars(Pyrus pyrifolia Nakai)(in Chinese)[J].South China Fruits(中国南方果树),2012,41(1):14-16.
[3]Li Y C.Occurrence and infection of Alternaria rot of Pingguoli pear,Pyrus pyrifolia(in Chinese)[J].Acta Phytophylacica Sinica(植物保护学报),2006,33(2):131-135.
[4]Cho Y,Davis J W,Kim K H,et al.A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element(LM E)constructs[J].M olecular Plant-M icrobe Interactions,2006,19(1):7-15.
[5]Xie L,Zhang D,Zhang L P,et al.Isolation and identification of an antagonistic streptomyces strain against Alternaria alternata(in Chinese)[J].Journal of Hebei Normal University(Natural Science Edition)[河北师范大学学报(自然科学版)],2008,32(4):526-528.
[6]Lin R F.Pear blight occurrence and control measures(in Chinese)[J].Fu Jian Agriculture(福建农业),2006,(8):26.
[7]Tanaka S.Studies on black spot disease of the Japanese pear Pirus serotina Rehd[J].M emoirs of the College of Agriculture,Kyoto University,1933,28:1-31.
[8]Zhang Z,Song F,Sun S,et al.The identification of pathogen causing Ya pears black spot(in Chinese)[J].Plant Quarantine(植物检疫),2003,17(4):212-215.
[9]Tanaka A,Shiotani H,Yamamoto M,et al.Insertional mutagenesis and cloning of the genes required for biosynthesis of the host-specific AK-toxin in the Japanese pear pathotype of Alternaria alternata[J].M olecular Plant-M icrobe Interactions:M PM I,1999,12(8):691-702.
[10]Nishimura S,Kohmoto K.Host-specific toxins and chemical structures from Alternaria species[J].Annual Review of Phytopathology,1983,21(1):87-116.
[11]Nagano K.Ringspot disease of pear[J].Journal of the Japanese Horticultural Society,1920,32(3):16-19.
[12]Leung H,Lehtinen U,Karjalainen R,et al.Transformation of the rice blast fungus Magnaporthe grisea to hygromycin B resistance[J].Current Genetics,1990,17(5):409-411.
[13]Proctor R H,Hohn T M,Mccormick S P.Reduced virulence of G ibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene[J].M olecular Plant-M icrobe Interactions,1995,8(4):593-601.
[14]Choquer M,Robin G,Le P P,et al.Ku70 or Ku80deficiencies in the fungus Botrytis cinerea facilitate targeting of genes that are hard to knock out in a w ildtype context[J].Fems M icrobiology Letters,2008,289(2):225-232.
[15]Cho Y,Ohm R A,Grigoriev I V,et al.Fungal-specific transcription factor Ab Pf2 activates pathogenicity in Alternaria brassicicola[J].Plant Journal,2013,75(3):498-514.
[16]Fu J G,Jiang S,Zhu Y Z,et al.Protoplast preparation and mutation for pathogenicity of Alternaria Alternata(FR.)keissler by restriction enzyme mediated integration(REM I)(in Chinese)[J].M ycosystema(菌物学报),2005,24(3):407-413.
[17]Tsuge T,Nishimura S,Kobayashi H.Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive r DNA sequences[J].Gene,1990,90(2):207-214.
[18]Akramiene D,Kondrotas A,Didziapetriene J,et al.Effects of beta-glucans on the immune system[J].M edicina,2007,43(8):597-606.
[19]Bundock P,Den D A,Beijersbergen A,et al.Transkingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae[J].Embo Journal,1995,14(13):3206-3214.
[20]Schiestl R H,Petes T D.Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(17):7585-7589.
[21]Zhao H,Chen F.Genetic transformation of protoplasts from somatic cells of plants(in Chinese)[J].Acta Botanica Boreali-occidentalia Sinica(西北植物学报),2004,24(7):1329-1341.
[22]Zhen L Z,Du Q,Zhou X,et al.A preliminary study on protoplast preparation conditions of Streptomyces gongzhulingensis 769[J].Chinese Agricultural Science Bulletin,2013,29(9):155-158.
[23]Tan W H,Li Y P,Xu Y.Factors affect the formation and regeneration of protoplasts of microorganism(in Chinese)[J].M odern Food Science&Technology(现代食品科技),2006,22(3):263-265.
[24]Zhou F,Zhang Y,Zhou Z,et al.Preparation and regeneration of protoplasts for alligator w eed pathogen Nimbya alternantherae(in Chinese)[J].Acta Phytophylacica Sinica(植物保护学报),2014,41(2):151-156.
[25]Wan Y.Establishment of protoplast transformation system in Alternaria tenuissima using G418 selection marker(in Chinese)[J].Journal of Anhui Agricultural Sciences(安徽农业科学),2008,36(29):12602-12604.
[2]Yang Q Q,Liu Y L,Wang T,et al.Influence of pathogen infection on early leaf abscission of three Chinese pear cultivars(Pyrus pyrifolia Nakai)(in Chinese)[J].South China Fruits(中国南方果树),2012,41(1):14-16.
[3]Li Y C.Occurrence and infection of Alternaria rot of Pingguoli pear,Pyrus pyrifolia(in Chinese)[J].Acta Phytophylacica Sinica(植物保护学报),2006,33(2):131-135.
[4]Cho Y,Davis J W,Kim K H,et al.A high throughput targeted gene disruption method for Alternaria brassicicola functional genomics using linear minimal element(LM E)constructs[J].M olecular Plant-M icrobe Interactions,2006,19(1):7-15.
[5]Xie L,Zhang D,Zhang L P,et al.Isolation and identification of an antagonistic streptomyces strain against Alternaria alternata(in Chinese)[J].Journal of Hebei Normal University(Natural Science Edition)[河北师范大学学报(自然科学版)],2008,32(4):526-528.
[6]Lin R F.Pear blight occurrence and control measures(in Chinese)[J].Fu Jian Agriculture(福建农业),2006,(8):26.
[7]Tanaka S.Studies on black spot disease of the Japanese pear Pirus serotina Rehd[J].M emoirs of the College of Agriculture,Kyoto University,1933,28:1-31.
[8]Zhang Z,Song F,Sun S,et al.The identification of pathogen causing Ya pears black spot(in Chinese)[J].Plant Quarantine(植物检疫),2003,17(4):212-215.
[9]Tanaka A,Shiotani H,Yamamoto M,et al.Insertional mutagenesis and cloning of the genes required for biosynthesis of the host-specific AK-toxin in the Japanese pear pathotype of Alternaria alternata[J].M olecular Plant-M icrobe Interactions:M PM I,1999,12(8):691-702.
[10]Nishimura S,Kohmoto K.Host-specific toxins and chemical structures from Alternaria species[J].Annual Review of Phytopathology,1983,21(1):87-116.
[11]Nagano K.Ringspot disease of pear[J].Journal of the Japanese Horticultural Society,1920,32(3):16-19.
[12]Leung H,Lehtinen U,Karjalainen R,et al.Transformation of the rice blast fungus Magnaporthe grisea to hygromycin B resistance[J].Current Genetics,1990,17(5):409-411.
[13]Proctor R H,Hohn T M,Mccormick S P.Reduced virulence of G ibberella zeae caused by disruption of a trichothecene toxin biosynthetic gene[J].M olecular Plant-M icrobe Interactions,1995,8(4):593-601.
[14]Choquer M,Robin G,Le P P,et al.Ku70 or Ku80deficiencies in the fungus Botrytis cinerea facilitate targeting of genes that are hard to knock out in a w ildtype context[J].Fems M icrobiology Letters,2008,289(2):225-232.
[15]Cho Y,Ohm R A,Grigoriev I V,et al.Fungal-specific transcription factor Ab Pf2 activates pathogenicity in Alternaria brassicicola[J].Plant Journal,2013,75(3):498-514.
[16]Fu J G,Jiang S,Zhu Y Z,et al.Protoplast preparation and mutation for pathogenicity of Alternaria Alternata(FR.)keissler by restriction enzyme mediated integration(REM I)(in Chinese)[J].M ycosystema(菌物学报),2005,24(3):407-413.
[17]Tsuge T,Nishimura S,Kobayashi H.Efficient integrative transformation of the phytopathogenic fungus Alternaria alternata mediated by the repetitive r DNA sequences[J].Gene,1990,90(2):207-214.
[18]Akramiene D,Kondrotas A,Didziapetriene J,et al.Effects of beta-glucans on the immune system[J].M edicina,2007,43(8):597-606.
[19]Bundock P,Den D A,Beijersbergen A,et al.Transkingdom T-DNA transfer from Agrobacterium tumefaciens to Saccharomyces cerevisiae[J].Embo Journal,1995,14(13):3206-3214.
[20]Schiestl R H,Petes T D.Integration of DNA fragments by illegitimate recombination in Saccharomyces cerevisiae[J].Proceedings of the National Academy of Sciences of the United States of America,1991,88(17):7585-7589.
[21]Zhao H,Chen F.Genetic transformation of protoplasts from somatic cells of plants(in Chinese)[J].Acta Botanica Boreali-occidentalia Sinica(西北植物学报),2004,24(7):1329-1341.
[22]Zhen L Z,Du Q,Zhou X,et al.A preliminary study on protoplast preparation conditions of Streptomyces gongzhulingensis 769[J].Chinese Agricultural Science Bulletin,2013,29(9):155-158.
[23]Tan W H,Li Y P,Xu Y.Factors affect the formation and regeneration of protoplasts of microorganism(in Chinese)[J].M odern Food Science&Technology(现代食品科技),2006,22(3):263-265.
[24]Zhou F,Zhang Y,Zhou Z,et al.Preparation and regeneration of protoplasts for alligator w eed pathogen Nimbya alternantherae(in Chinese)[J].Acta Phytophylacica Sinica(植物保护学报),2014,41(2):151-156.
[25]Wan Y.Establishment of protoplast transformation system in Alternaria tenuissima using G418 selection marker(in Chinese)[J].Journal of Anhui Agricultural Sciences(安徽农业科学),2008,36(29):12602-12604.