梨pgip基因cDNA的克隆与表达
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摘要
植物富含多糖的细胞壁是抵抗病原真菌侵染的屏障。植物致病性真菌在侵染植物的过程中必须首先分泌包括多聚半乳糖醛酸酶(polygalacturonase,PGs)(EC3.2.1.15)在内的酶来降解植物细胞壁才能成功地侵染植物。而在植物的防卫反应中,植物的多聚半乳糖醛酸酶抑制蛋白(polygalacturonase-inhibiting protein,PGIPs)能够抑制真菌的内切多聚半乳糖醛酸酶活性。目前,pgip基因已成为植物抗真菌基因工程的研究热点之一,国内外已先后从苹果、树莓等多种植物中克隆到了pgip基因或片断。
根据GenBank中梨属pgip基因序列保守区设计1对特异引物,以梨优良矮化砧木S_2(Pyrus ussuriensis)叶片总RNA为模板,克隆到1条约1,100bp的cDNA片段,将其与pMD18-T vector连接后转化Escherichia coli JM109,对筛选到的阳性克隆进行序列测定并使用生物信息学方法对所得结果进行综合分析表明:克隆片段为梨pgip基因,命名为Pupgip,GenBank登录号为EU107274,该片段编码330个氨基酸,预测分子量为36,388Da,第1～24个氨基酸残基是信号肽。与梨属其它已获得pgip基因核苷酸序列开放阅读框(Open reading frame,ORF)的同源性为97.5～99.6%,氨基酸序列的同源性为97.6～99.1%。半定量RT-PCR分析显示,该基因在植株各部位表达存在较大差异,茎、叶中的基因表达量明显高于花蕾和花朵。
为了进一研究该基因的功能和诱导表达,将该基因的成熟肽编码区克隆到pET-32a(+)表达载体中,成功地构建了该基因的原核表达质粒pET-PuPGIP,并在E.coli BL21(DE3)pLysS中通过IPTG诱导表达,经SDS-PAGE分析证实获得了该基因的重组蛋白,重组蛋白主要以包涵体形式出现。
Plants posses a polysaccharide-rich cell wall that acts as a barrier against pathogenic fungi.The majority of fungi need to breach this barrier to gain access to the plant cells.In order to infect successfully,plant pathogenic fungi have to secrete a battery of pectic enzymes,the most important ones of which are endo-polygalacturonases(PGs) (EC3.2.1.15),to degrading plant cell walls during the early stages of invasion.Plant polygalacturonase inhibiting proteins(PGIPs) are cell wall-associated glycoproteins which could effectively inhibit the fungal PGs so that limiting fungal colonization.Recently, PGIPs have been identified in numerous plants,such as apple,raspberry and so on.
A 1 038 bp sequence of polygalacturonase-inhibiting protein(PGIP) gene(Genbank Accession Number:EU107274),named Pupgip,was obtained from pear dwarf stock S_2 (Pyrus ussuriensis) by RT-PCR using a pair of specific primers.PCR product was cloned into pMD 18-T vector and then transformed into Escherichia coli JM109.Positive clones were picked out and sequenced.The results revealed that Pupgip encodes a 330 amino acid protein with a predicted molecular weight of 36 388 Da.The predicted signal peptide was formed by 24 residues from 1 to 24.Pupgip showed a 98.4%identity of sequence homology to pgip from Pyrus communis Bartlett(L09264) and 99.1%similarity of deduced amino acid sequences from P.pyrifolia Kousui(AY333102) and Bartlett.
Semi-quantitative RT-PCR analysis demonstrated that Pupgip gene expression has tissue specificity.The results suggested that the relative expression of pgip gene in stems is the highest and at low levels in flowers and buds,respectively.
In this research,a prokaryotic expression plasmid pET-Pupgip,which contains the full encoding region of a Pupgip gene in pear dwarf stock S_2,was constructed.The recombinant PuPGIP protein was expressed in E.coli BL21(DE3) pLysS induced by IPTG. The SDS-PAGE shows that the recombination proteins were mainly appeared as inclusion bodies,a small portion of solvable proteins in the supernatant of the post-sonicated thalli also.
The Pupgip gene isolated from pear dwarf stock S_2 will be useful in the research on the molecular mechanism of the resistance to pear fungal diseases such as pear scab and pear rust.Molecular cloning of pear pgip gene has great potentiality in the disease-resistance breeding,genetic engineering and genomic research of pear.
根据GenBank中梨属pgip基因序列保守区设计1对特异引物,以梨优良矮化砧木S_2(Pyrus ussuriensis)叶片总RNA为模板,克隆到1条约1,100bp的cDNA片段,将其与pMD18-T vector连接后转化Escherichia coli JM109,对筛选到的阳性克隆进行序列测定并使用生物信息学方法对所得结果进行综合分析表明:克隆片段为梨pgip基因,命名为Pupgip,GenBank登录号为EU107274,该片段编码330个氨基酸,预测分子量为36,388Da,第1～24个氨基酸残基是信号肽。与梨属其它已获得pgip基因核苷酸序列开放阅读框(Open reading frame,ORF)的同源性为97.5～99.6%,氨基酸序列的同源性为97.6～99.1%。半定量RT-PCR分析显示,该基因在植株各部位表达存在较大差异,茎、叶中的基因表达量明显高于花蕾和花朵。
为了进一研究该基因的功能和诱导表达,将该基因的成熟肽编码区克隆到pET-32a(+)表达载体中,成功地构建了该基因的原核表达质粒pET-PuPGIP,并在E.coli BL21(DE3)pLysS中通过IPTG诱导表达,经SDS-PAGE分析证实获得了该基因的重组蛋白,重组蛋白主要以包涵体形式出现。
Plants posses a polysaccharide-rich cell wall that acts as a barrier against pathogenic fungi.The majority of fungi need to breach this barrier to gain access to the plant cells.In order to infect successfully,plant pathogenic fungi have to secrete a battery of pectic enzymes,the most important ones of which are endo-polygalacturonases(PGs) (EC3.2.1.15),to degrading plant cell walls during the early stages of invasion.Plant polygalacturonase inhibiting proteins(PGIPs) are cell wall-associated glycoproteins which could effectively inhibit the fungal PGs so that limiting fungal colonization.Recently, PGIPs have been identified in numerous plants,such as apple,raspberry and so on.
A 1 038 bp sequence of polygalacturonase-inhibiting protein(PGIP) gene(Genbank Accession Number:EU107274),named Pupgip,was obtained from pear dwarf stock S_2 (Pyrus ussuriensis) by RT-PCR using a pair of specific primers.PCR product was cloned into pMD 18-T vector and then transformed into Escherichia coli JM109.Positive clones were picked out and sequenced.The results revealed that Pupgip encodes a 330 amino acid protein with a predicted molecular weight of 36 388 Da.The predicted signal peptide was formed by 24 residues from 1 to 24.Pupgip showed a 98.4%identity of sequence homology to pgip from Pyrus communis Bartlett(L09264) and 99.1%similarity of deduced amino acid sequences from P.pyrifolia Kousui(AY333102) and Bartlett.
Semi-quantitative RT-PCR analysis demonstrated that Pupgip gene expression has tissue specificity.The results suggested that the relative expression of pgip gene in stems is the highest and at low levels in flowers and buds,respectively.
In this research,a prokaryotic expression plasmid pET-Pupgip,which contains the full encoding region of a Pupgip gene in pear dwarf stock S_2,was constructed.The recombinant PuPGIP protein was expressed in E.coli BL21(DE3) pLysS induced by IPTG. The SDS-PAGE shows that the recombination proteins were mainly appeared as inclusion bodies,a small portion of solvable proteins in the supernatant of the post-sonicated thalli also.
The Pupgip gene isolated from pear dwarf stock S_2 will be useful in the research on the molecular mechanism of the resistance to pear fungal diseases such as pear scab and pear rust.Molecular cloning of pear pgip gene has great potentiality in the disease-resistance breeding,genetic engineering and genomic research of pear.
引文
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[5].古英洪.桃pgip基因cDNA的克隆及其在大肠杆菌中的表达.[硕士].雅安,四川农业大学,2007
[6].Gotoh Y,Nalumpang S,Isshiki A,et al.A cDNA encoding polygalacturonase-inhibiting protein induced in citrus leaves by polygalacturonase of Alternaria citri.Journal of General Plant Pathology,2002,68(1):57-61
[7].Stotz HU,Powell AL,Damon SE,et al.Molecular characterization of a polygalacturonase inhibitor from Pyrus communis L.cv Bartlett.Plant Physiology,1993,102(1):133-138
[8].Faize M,Sugiyama T,Faize L,et al.Polygalacturonase-inhibiting protein(PGIP) from Japanese pear:possible involvement in resistance against scab.Physiological and Molecular Plant Pathology,2003,63(6):319-327
[9].Simpson CG,MacRae E,Gardner RC.Cloning of a Polygalacturonase Inhibiting Protein from Kiwifruit(Actinidia deliciosa).Plant Physiol,1995,108:1748-1748
[10].Haas BJ,Volfovsky N,Town CD,et al.Full-length messenger RNA sequences greatly improve genome annotation.Genome Biology,2002,3(6):Research0029.0001-0029.0012
[11].Ahsan N,Yoon HS,Jo J.Molecular cloning of a BcPGIP cDNA from Brassica campestris and its expression to several stresses.Plant Science,2005,169(6):1081-1089
[12].Nalumpang S,Gotoh Y,Tsuboi H,et al.Functional characterization of citrus polygalacturonase-inhibiting protein.Journal of General Plant Pathology,2002,68(2):118-127
[13].Nalumpang S,Gotoh Y,Yamasaki Y,et al.Comparison and Characterization of Polygalacturonase-inhibiting Protein Genes from the Genus of Citrus and Its Close Related Genera.Thai J Agri Sci,2002,35:147-164
[14].Chimwamurombe PM,Botha AM,Wingfield MJ,et al.Molecular relatedness of the polygalacturonase-inhibiting protein genes in Eucalyptus species.Theoretical and Applied Genetics,2001,102(4):645-650
[15].Favaron F,D'Ovidio R,Porceddu E,et al.Purification and molecular characterization of a soybean polygalacturonase-inhibiting protein.Planta,1994,195(1):80-87
[16].Mahalingam R,Wang G,Knap HT.Polygalacturonase and polygalacturonase inhibitor protein:gene isolation and transcription in Glycine max-Heterodera glycines interactions.Molecular Plant-microbe Interactions,1999,12(6):490-498
[17].D'Ovidio R,Roberti S,Di Giovanni M,et al.The characterization of the soybean polygalacturonase-inhibiting proteins(Pgip) gene family reveals that a single member is responsible for the activity detected in soybean tissues.Planta,2006,224(3):633-645
[18]. Arendse MS.Dubery IA,Berger DK. Isolation by PCR-based methods of a plant antifungal polygalacturonase-inhibiting protein gene. Electronic Journal of Biotechnology, 1999, 2 (3):152-159
[19]. Yao C,Conway WS.Ren R, et al. Gene encoding polygalacturonase inhibitor in apple fruit is developmentally regulated and activated by wounding and fungal infection. Plant Molecular Biology,1999,39(6): 1231-1241
[20]. Janni M,Di Giovanni M,Roberti S, et al. Characterization of expressed PGIP genes in rice and wheat reveals similar extent of sequence variation to dicot PGIPs and identifies an active PGIP lacking an entire LRR repeat. Theoretical and Applied Genetics, 2006, 113(7): 1233-1245
[21]. D'Ovidio R,Raiola A,Capodicasa C, et al. Characterization of the complex locus of bean encoding polygalacturonase-inhibiting proteins reveals subfunctionalization for defense against fungi and insects. Plant Physiology, 2004, 135(4): 2424-2435
[22]. Nakamura N,Marutani M,Sanematsu S, et al. Phylogenetic Classification of Dof-type Transcription Factors in Pea (Pisum sativum). Plant Biotechnology, 2003, 20: 247-253
[23]. Liang F,Zhang K,Zhou C, et al. Cloning, characterization and expression of the gene encoding polygalacturonase-inhibiting proteins. (PGIPs) of peach [prunus persica (L.) Batch]. Plant Science,2005, 168(2): 481-486
[24]. Abu-Goukh AA.Greve LC,Labavitch JM. Purification and partial characterization of'Bartlett" pear fruit polygalacturonase inhibitors. Physiol Plant Pathol, 1983, 23: 111-122
[25]. De Lorenzo G,D'Ovidio R,Cervone F. The role of polygalacturonase-inhibiting proteins (PGIPs) in defense against pathogenic fungi. Annu Rev Phytopathol, 2001, 39: 313-335
[26]. De Lorenzo G,Ferrari S. Polygalacturonase-inhibiting proteins in defense against phytopathogenic fungi. Current Opinion in Plant Biology, 2002, 5(4): 295-299
[27]. Favaron F. Gel detection of Allium porrum polygalacturonase-inhibiting protein reveals a high number of isoforms. Physiological and Molecular Plant Pathology, 2001, 58(6): 239-245
[28]. Robertsen B. Elicitors of the production of lignin-like compounds in cucumber hypocotyls. Physiol Mol Plant Pathol, 1986,28: 137-148
[29]. C B, W S-C.P A. Plant cell walls. wwwccrcugaedu/～mao/plapath/PP text htm:
[30]. Gomathi V,Gnanamanickam SS. Polygalacturonase-inhibiting proteins in plant defence. Current Science, 2004, 87(9): 1211-1217
[31]. Abu-Goukh AA,Labavitch JM. The in vivo role of "Bartlett" pear fruit polygalacturonase inhibitors.Physiol Plant Pathol, 1983,23: 123-135
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