PGIP基因的真核表达和SA诱导对苹果叶片中PGIP基因表达的影响
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摘要
多聚半乳糖醛酸酶抑制蛋白(polygalacturonase-inhibiting protein, PGIP)是存在于多种植物中的一种富含LRR的细胞壁结合蛋白，能专一性地抑制真菌分泌的endo-PG酶活性，增强植物的抗病性。因此研究PGlP基因的表达对该基因及其表达产物的利用有重要意义。
本研究以PGIP克隆载体为材料，构建了多种真核表达载体，分别电转入毕赤酵母GS115，并诱导阳性重组酵母表达目标产物；以‘秦冠’、‘礼泉短富’两个苹果品种为材料，研究了不同浓度水杨酸诱导处理对苹果叶片PGIP基因表达的影响。主要结果如下：
1以PGIP基因克隆载体为模板，使用不同引物扩增了4种带有起始、终止密码子的PGIP片段，通过酶切连接分别获得相应的真核表达载体，经电击转化、筛选鉴定共获得了42个重组酵母菌株，经1%甲醇诱导表达，仅转入含起始密码子不含终止密码子PGIP片段的重组酵母菌株有较低的表达，SDS-PAGE电泳分析表明该重组酵母表达的PGIP分子量约为46kDa。
2SA诱导对苹果叶片中PGIP基因的表达存在短期效应和长期效应，不同浓度SA诱导对不同抗病性苹果的PGIP基因表达效应存在差异，两种浓度的SA水杨酸均能诱导‘秦冠’苹果叶片PGIP的表达；但仅高浓度SA处理可以促进感病苹果‘礼泉短富’叶片PGIP基因的表达，低浓度SA处理抑制其表达。
Polygalacturonase-inhibiting protein (PGIP) is a leucine-rich repeated cellwall bindingprotein present in many plants, it could specifically inhibit endo-PG activity secreted byfungus, prevent other pathogenic fungi secrete hydrolytic enzymes destroys the cell, andenhance plant disease resistance. Therefore research PGlP eukaryotic expression and plantexpression have significant meaning for the use of the PGIP gene and their encoded protein.
With the materials of the PGIP gene cloning vectors, a series of variety eukaryoticexpression vectors were constructed and transferred into Pichia pastoris GS115byelectroporation and induced the expression of the target protein. With the material of'Qinguan' and 'Liquan Fuji Spur' apple plant, the effect of Salicylic acid (SA) induction onPGIP gene expression of apple leaves were studied. The main conclusions were as follows:
1. With the template of PGIP cloning vector, four kinds of PGIP fragment with or withstart and stop codon were amplified by PCR Then they were cloned into Pichia pastorissecreted expression vector pPICZαB respectively by enzymatically digested connection.Through efficient electroporation, screening and identification, a total of42recombinantPichia pastoris strains were obtained and induced by1%methanol, analyzed by SDS-PAGE,only one strain transferred by PGIP fragments with start codon and without stop codonexpressed target protein. The molecular weight of expressed PGIP was about46kDa.
2. Salicylic acid induction had long-term and short-term effect on the PGIP geneexpression in apple leaves. The PGIP gene expression level of different disease resistanceapple differed from the inducing concentration:0.1and0.01mmol/L SA treatment bothpromoted the PGIP gene expression of 'Qinguan' while in 'Liquan Fuji Spur'0.1mmol/L SAtreatment promoted the PGIP gene expression but the0.01mmol/L SA treatment inhibited theexpression.
本研究以PGIP克隆载体为材料，构建了多种真核表达载体，分别电转入毕赤酵母GS115，并诱导阳性重组酵母表达目标产物；以‘秦冠’、‘礼泉短富’两个苹果品种为材料，研究了不同浓度水杨酸诱导处理对苹果叶片PGIP基因表达的影响。主要结果如下：
1以PGIP基因克隆载体为模板，使用不同引物扩增了4种带有起始、终止密码子的PGIP片段，通过酶切连接分别获得相应的真核表达载体，经电击转化、筛选鉴定共获得了42个重组酵母菌株，经1%甲醇诱导表达，仅转入含起始密码子不含终止密码子PGIP片段的重组酵母菌株有较低的表达，SDS-PAGE电泳分析表明该重组酵母表达的PGIP分子量约为46kDa。
2SA诱导对苹果叶片中PGIP基因的表达存在短期效应和长期效应，不同浓度SA诱导对不同抗病性苹果的PGIP基因表达效应存在差异，两种浓度的SA水杨酸均能诱导‘秦冠’苹果叶片PGIP的表达；但仅高浓度SA处理可以促进感病苹果‘礼泉短富’叶片PGIP基因的表达，低浓度SA处理抑制其表达。
Polygalacturonase-inhibiting protein (PGIP) is a leucine-rich repeated cellwall bindingprotein present in many plants, it could specifically inhibit endo-PG activity secreted byfungus, prevent other pathogenic fungi secrete hydrolytic enzymes destroys the cell, andenhance plant disease resistance. Therefore research PGlP eukaryotic expression and plantexpression have significant meaning for the use of the PGIP gene and their encoded protein.
With the materials of the PGIP gene cloning vectors, a series of variety eukaryoticexpression vectors were constructed and transferred into Pichia pastoris GS115byelectroporation and induced the expression of the target protein. With the material of'Qinguan' and 'Liquan Fuji Spur' apple plant, the effect of Salicylic acid (SA) induction onPGIP gene expression of apple leaves were studied. The main conclusions were as follows:
1. With the template of PGIP cloning vector, four kinds of PGIP fragment with or withstart and stop codon were amplified by PCR Then they were cloned into Pichia pastorissecreted expression vector pPICZαB respectively by enzymatically digested connection.Through efficient electroporation, screening and identification, a total of42recombinantPichia pastoris strains were obtained and induced by1%methanol, analyzed by SDS-PAGE,only one strain transferred by PGIP fragments with start codon and without stop codonexpressed target protein. The molecular weight of expressed PGIP was about46kDa.
2. Salicylic acid induction had long-term and short-term effect on the PGIP geneexpression in apple leaves. The PGIP gene expression level of different disease resistanceapple differed from the inducing concentration:0.1and0.01mmol/L SA treatment bothpromoted the PGIP gene expression of 'Qinguan' while in 'Liquan Fuji Spur'0.1mmol/L SAtreatment promoted the PGIP gene expression but the0.01mmol/L SA treatment inhibited theexpression.
引文
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