草莓pgip基因的克隆及其序列分析
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摘要
多聚半乳糖醛酸酶抑制蛋白(polygalacturonase-inhibiting proteins,PGIPs)是内切多聚半乳糖醛酸酶(endo-PG)的抑制剂,当病原真菌侵染植物时,它能与病原真菌分泌的endo-PG专一性结合,一方面延缓病原真菌对植物细胞壁的降解,另一方面间接地影响病原真菌的生长繁殖速度,在抵御真菌病害方面发挥重要作用,同时引发植物的防卫反应。它还与大多数植物抗性基因一样,属于富亮氨酸重复(Leucine-richrepeat,LRR)蛋白超家族。pgip基因在植物抗病育种中已成为一重要的候选基因,其分离鉴定是进行转pgip基因研究的基础。
根据蔷薇科植物pgip基因序列保守区域设计两对特异引物,以草莓(Fragaria×ananassa Duch)叶片制备的总DNA和总RNA为模板,采用PCR技术,克隆到了约1,300bp和1,000 bp的DNA和cDNA片段。这两条片段与pMD 18-T载体连接后转化E.coli JM109,对筛选到的阳性克隆进行序列测定与分析结果表明,获得的DNA片段长1,389bp,含一个长度为168bp的内含子,cDNA长1,053 bp。BLAST比对结果表明,克隆到的片段是草莓的pgip基因,命名为Fapgip,GenBank登录号为EU117215和EU117213。该片段含一个长度为999bp的完整开放阅读框,编码332个氨基酸残基组成的蛋白质,具有5个潜在的N-糖基化位点,中心LRR结构域由10个串联的LRRs基序组成。预测该蛋白质分子量为37.1 kDa,等电点为7.67,N端24个氨基酸残基构成的疏水区域为信号肽。序列同源比对结果显示,所克隆的Fapgip基因与蔷薇科其它植物pgip基因核苷酸序列的同源性为75.2～86.5%,推导氨基酸序列的同源性为72.9～87.8%;同源树表明其明显区别于其它pgip基因;进一步的系统进化树分析表明,所克隆的Fapgip(EU117213)除与树莓的pgip(AJ620336)关系较近外,与寿星桃(AY903219)、美洲李(AY883417)和梅(AY903223)等的pgip基因都存在较远的进化关系。
Polygalacturonase inhibiting proteins (PGIPs) are extracellular glycoproteins located in plant cell wall. It is believed to play an important role in the defense against plant pathogenic fungi. PGIPs can specially bind and inhibit or reduce the hydrolytic activity of polygalacturonases (PGs) secreted by plant pathogenic fungi, and limit the growth of plant pathogens, as well as eliciting defense responses in plant. Furthermore, PGIPs belong to the super family of Leucine-rich repeat (LRR) proteins which also include the products of several plant resistance genes. They are considered to be an important candidate gene for genetic engineering to obtain transgenic plants with increased tolerance to fungal infection, which decrease the use of insecticide. Therefore, the basic research of transgenic-engineering for that purpose is to isolate and identify the pgip genes.
Through polymerase chain reaction (PCR) with two pairs of specific primers based on the conserved region of the pgip genes of genus Rosaceae, two fragments about 1,300bp and 1,000 bp was obtained from the total DNA and RNA extracted from the leaves of mature strawberry leaves. After cloning it into pMD18-T vector, the linked DNA was transformed into Escherichia coli JM109. The positive clones were picked out and sequenced. The results showed that the cloned DNA was 1,389bp, which shows a single 168bp intron that is efficiently spliced out of the Fapgip pre-mRNA transcript. The cDNA was 1,053 bp in length and a real pgip gene of stawberry designed as Fapgip in this thesis with an accession number EU117215 and EU117213 in GenBank. The cloned cDNA contains an open reading frame of 999bp, which encodes for a polypeptide of 332 amino acid residues with a molecular mass of 37.1 kDa and a pI of 7.67, and a hydrophobic region of 24 amino acid residues in the N-terminal which was considered to be a signal peptide. The deduced amino acid sequences contain 5 potential N-glycosylation sites, and the center LRR structural domain is composed of 10 tandem LRR motifs. Although the cloned cDNA exhibits a homology of the nucleotide and deduced amino acid sequences of 75.2～86.5% and 72.9～87.8% respectively, which was significantly similar to other pgip genes from the genus Rosaceae, the homology tree shows that it obviously distinguished from the others. And the phylogenetic tree shows that it indeed far from its near species, and belongs to a different branch with the Rubus idaeus.
根据蔷薇科植物pgip基因序列保守区域设计两对特异引物,以草莓(Fragaria×ananassa Duch)叶片制备的总DNA和总RNA为模板,采用PCR技术,克隆到了约1,300bp和1,000 bp的DNA和cDNA片段。这两条片段与pMD 18-T载体连接后转化E.coli JM109,对筛选到的阳性克隆进行序列测定与分析结果表明,获得的DNA片段长1,389bp,含一个长度为168bp的内含子,cDNA长1,053 bp。BLAST比对结果表明,克隆到的片段是草莓的pgip基因,命名为Fapgip,GenBank登录号为EU117215和EU117213。该片段含一个长度为999bp的完整开放阅读框,编码332个氨基酸残基组成的蛋白质,具有5个潜在的N-糖基化位点,中心LRR结构域由10个串联的LRRs基序组成。预测该蛋白质分子量为37.1 kDa,等电点为7.67,N端24个氨基酸残基构成的疏水区域为信号肽。序列同源比对结果显示,所克隆的Fapgip基因与蔷薇科其它植物pgip基因核苷酸序列的同源性为75.2～86.5%,推导氨基酸序列的同源性为72.9～87.8%;同源树表明其明显区别于其它pgip基因;进一步的系统进化树分析表明,所克隆的Fapgip(EU117213)除与树莓的pgip(AJ620336)关系较近外,与寿星桃(AY903219)、美洲李(AY883417)和梅(AY903223)等的pgip基因都存在较远的进化关系。
Polygalacturonase inhibiting proteins (PGIPs) are extracellular glycoproteins located in plant cell wall. It is believed to play an important role in the defense against plant pathogenic fungi. PGIPs can specially bind and inhibit or reduce the hydrolytic activity of polygalacturonases (PGs) secreted by plant pathogenic fungi, and limit the growth of plant pathogens, as well as eliciting defense responses in plant. Furthermore, PGIPs belong to the super family of Leucine-rich repeat (LRR) proteins which also include the products of several plant resistance genes. They are considered to be an important candidate gene for genetic engineering to obtain transgenic plants with increased tolerance to fungal infection, which decrease the use of insecticide. Therefore, the basic research of transgenic-engineering for that purpose is to isolate and identify the pgip genes.
Through polymerase chain reaction (PCR) with two pairs of specific primers based on the conserved region of the pgip genes of genus Rosaceae, two fragments about 1,300bp and 1,000 bp was obtained from the total DNA and RNA extracted from the leaves of mature strawberry leaves. After cloning it into pMD18-T vector, the linked DNA was transformed into Escherichia coli JM109. The positive clones were picked out and sequenced. The results showed that the cloned DNA was 1,389bp, which shows a single 168bp intron that is efficiently spliced out of the Fapgip pre-mRNA transcript. The cDNA was 1,053 bp in length and a real pgip gene of stawberry designed as Fapgip in this thesis with an accession number EU117215 and EU117213 in GenBank. The cloned cDNA contains an open reading frame of 999bp, which encodes for a polypeptide of 332 amino acid residues with a molecular mass of 37.1 kDa and a pI of 7.67, and a hydrophobic region of 24 amino acid residues in the N-terminal which was considered to be a signal peptide. The deduced amino acid sequences contain 5 potential N-glycosylation sites, and the center LRR structural domain is composed of 10 tandem LRR motifs. Although the cloned cDNA exhibits a homology of the nucleotide and deduced amino acid sequences of 75.2～86.5% and 72.9～87.8% respectively, which was significantly similar to other pgip genes from the genus Rosaceae, the homology tree shows that it obviously distinguished from the others. And the phylogenetic tree shows that it indeed far from its near species, and belongs to a different branch with the Rubus idaeus.
引文
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