西伯利亚蓼PsPGIP基因的克隆及功能分析
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摘要
多聚半乳糖醛酸酶抑制蛋白(polygalacturonase inhibiting protein,PGIP)是一种能够特异性结合和抑制真菌内切多聚半乳糖醛酸酶(endo-PG)活性的细胞壁结合蛋白,在国内外己在多种单双子叶植物中发现了多聚半乳糖醛酸酶抑制蛋白,本文根据西伯利亚蓼茎抑制消减文库(SSH)中获得的多聚半乳糖醛酸酶抑制蛋白(Polygalacturonase inhibiting proteins)EST序列,采用RACE技术在西伯利亚蓼消减库(SSH)成功克隆了多聚半乳糖醛酸酶抑制蛋白基因(PsPGIP)的cDNA序列,利用生物信息学技术对该基因的序列进行分析。采用实时定量PCR技术分析PsPGIP基因在3％NaHC03胁迫下叶、茎、地下茎表达量的差异性。同时构建PsPGIP基因的植物表达载体,并转入到烟草中,进一步分析非转基因烟草和PsPGIP基因的过量表达在盐胁迫下丙二醛(MDA)含量，SOD.POD活性及可溶性蛋白含量的变化情况以及在不同真菌侵害时叶片受伤害的程度分析,为进一步的抗病育种工作提供理论实践经验。主要研究结果如下：
1.PsPGIP基因的克隆及盐胁迫下的表达分析
成功克隆PsPGIP全长cDNA序列(ACD01043),全长1251 bp,生物信息学分析表明,开放读码框为1020 bp,编码339个氨基酸,具有一段24个残基的保守亮氨酸结构域。该基因具有N端信号肽,具有PGIPs基因家族共有的典型保守区域,属PGIPs家族基因。荧光定量PCR分析表明,PsPGIP在西伯利亚蓼叶、茎、地下茎等器官中均有分布。在3％NaHC03诱导表达中,该基因在叶中表达明显受盐胁迫的诱导。
2.PsPGIP基因对烟草的遗传转化及抗性分析
(1)构建PsPGIP基因的植物表达载体,利用农杆菌介导法将PsPGIP基因转入烟草植株中,并利用PCR和RT-PCR等方法验证PsPGIP基因已经在烟草中成功表达。
(2)选取6个转基因株系进行盐胁迫试验,分析逆境胁迫条件下非转基因野生型烟草和转PsPGIP基因烟草的丙二醛(MDA)含量、SOD活性、POD活性以及可溶性蛋白含量的变化。结果表明,在100 mmol/L NaCl胁迫下,多数转基因株系的POD活性、可溶性蛋白、SOD含量比非转基因野生型烟草高,而MDA含量低于非转基因野生型烟草。说明在盐胁迫条件下,转PsPGIP基因烟草在抵御盐伤害上明显强于非转基因烟草。
(3)进一步分析6个转基因株系抵御真菌伤害方面的差异。结果表明在炭疽病和猝倒病的侵害下,植株表现有明显差异性,转PsPGIP基因植株的抑菌能力明显高于非转基因野生型烟草植株。
Polygalacturonase inhibiting protein (PGIP) are primarily localized in the cell wall and endolpasmic system, and they areinhibitors of endo-polygalacturonase enzyme (endo-PG) from fungi, which refrain plant from fungal infection, PGIP have discovered and studied both in monocotyledonous and dicotyledonous plants. The cDNA sequence of the PGIP was cloned, based on PGIP'EST sequence obtained from SSH library of Polygonum sibiricum Laxm.,using RACE technology, and has been carried on the sequence analysis. Real-time PCR show that the expression of PsPGIP gene is differences in leaves, stems and rhizome of Polygonum sibiricum Laxm. under 3% NaHCO3 stress. At the same time PsPGIP expression vectors have been constructed, and the transgenic plants for the PsPGIP gene were generated. untransgenic tobacco and PsPGIP gene over-expression were selected for the test of 3% NaHC03 tolerance. MDA content, SOD activity, POD activity and were measured. As well as in different fungal infection upon by the leaves of the extent of injury analysis. For further breeding for disease resistance and provide a theoretical work experience. The main research as follow:
1.The full-length cDNA sequence of the Polygalacturonase inhibiting proteins (PGIP) gene was cloned successfully, the gene is named as PsPGIP(ACD01043).The cDNA sequence of the PsPGIP is 1251 bp in length, The open reading frame (ORF) is 1020 bp in length, encoding a deduced amino acid sequence of 339 residues, and 24 residence of a conserved leucine-rich fragment. Sequencing analysis revealed that the gene belongs to the PGIPs family gene as it contains an amino-terminal (N-terminal) signal peptide and typical conservative region of PGIPs family. The analysis by Real time-PCR shows that PsPGIP gene was distributed in leaves,stems and rhizomes of Polygonum sibiricum Laxm.. Under inducing expression under 3% NaHCO3,the espression of PsPGIP gene was induced by salt obviously, by which we can conclude that PsPGIP gene palys a very important role in the salt-resistant.
2.Genetic transformation of PsPGIP and stresss assay of transgenic plants
(1).Construct the expression vectors successfully, the transgenic plants for the PsPGIP gene were generated by an Agrobacterium mediated method. The expression analysis by PCR and RT-PCR showed that PsPGIP was expressed in tobacco.
(2).Six transgenic lines were selected for the test of NaCl tolerance. MDA content, SOD activity, POD activity and soluble protein content were measured under this stress. The results showed that SOD activity, POD activity and soluble protein content in most transgenic lines was higher than that in untransgenic tobacco under 100 mmol/L NaCl conditions. Furthmore, MDA contents in most transformed plants were lower than that in untransgenic tobacco under 100 mmol/L NaCl stress. The results demonstrated that transformed plants exhibits salt stress tolerance were higher than that in untransgenic tobacco.
(3)Six transgenic lines were selected for the test of fungal infection tolerance.The results showed that compared with untransgenic tobacco, all transgenic lines were up-regulated under the F.oxysporum and anthracnose infection stresss, transgenic lines enhanced the tolerance of fungal infection stress.
1.PsPGIP基因的克隆及盐胁迫下的表达分析
成功克隆PsPGIP全长cDNA序列(ACD01043),全长1251 bp,生物信息学分析表明,开放读码框为1020 bp,编码339个氨基酸,具有一段24个残基的保守亮氨酸结构域。该基因具有N端信号肽,具有PGIPs基因家族共有的典型保守区域,属PGIPs家族基因。荧光定量PCR分析表明,PsPGIP在西伯利亚蓼叶、茎、地下茎等器官中均有分布。在3％NaHC03诱导表达中,该基因在叶中表达明显受盐胁迫的诱导。
2.PsPGIP基因对烟草的遗传转化及抗性分析
(1)构建PsPGIP基因的植物表达载体,利用农杆菌介导法将PsPGIP基因转入烟草植株中,并利用PCR和RT-PCR等方法验证PsPGIP基因已经在烟草中成功表达。
(2)选取6个转基因株系进行盐胁迫试验,分析逆境胁迫条件下非转基因野生型烟草和转PsPGIP基因烟草的丙二醛(MDA)含量、SOD活性、POD活性以及可溶性蛋白含量的变化。结果表明,在100 mmol/L NaCl胁迫下,多数转基因株系的POD活性、可溶性蛋白、SOD含量比非转基因野生型烟草高,而MDA含量低于非转基因野生型烟草。说明在盐胁迫条件下,转PsPGIP基因烟草在抵御盐伤害上明显强于非转基因烟草。
(3)进一步分析6个转基因株系抵御真菌伤害方面的差异。结果表明在炭疽病和猝倒病的侵害下,植株表现有明显差异性,转PsPGIP基因植株的抑菌能力明显高于非转基因野生型烟草植株。
Polygalacturonase inhibiting protein (PGIP) are primarily localized in the cell wall and endolpasmic system, and they areinhibitors of endo-polygalacturonase enzyme (endo-PG) from fungi, which refrain plant from fungal infection, PGIP have discovered and studied both in monocotyledonous and dicotyledonous plants. The cDNA sequence of the PGIP was cloned, based on PGIP'EST sequence obtained from SSH library of Polygonum sibiricum Laxm.,using RACE technology, and has been carried on the sequence analysis. Real-time PCR show that the expression of PsPGIP gene is differences in leaves, stems and rhizome of Polygonum sibiricum Laxm. under 3% NaHCO3 stress. At the same time PsPGIP expression vectors have been constructed, and the transgenic plants for the PsPGIP gene were generated. untransgenic tobacco and PsPGIP gene over-expression were selected for the test of 3% NaHC03 tolerance. MDA content, SOD activity, POD activity and were measured. As well as in different fungal infection upon by the leaves of the extent of injury analysis. For further breeding for disease resistance and provide a theoretical work experience. The main research as follow:
1.The full-length cDNA sequence of the Polygalacturonase inhibiting proteins (PGIP) gene was cloned successfully, the gene is named as PsPGIP(ACD01043).The cDNA sequence of the PsPGIP is 1251 bp in length, The open reading frame (ORF) is 1020 bp in length, encoding a deduced amino acid sequence of 339 residues, and 24 residence of a conserved leucine-rich fragment. Sequencing analysis revealed that the gene belongs to the PGIPs family gene as it contains an amino-terminal (N-terminal) signal peptide and typical conservative region of PGIPs family. The analysis by Real time-PCR shows that PsPGIP gene was distributed in leaves,stems and rhizomes of Polygonum sibiricum Laxm.. Under inducing expression under 3% NaHCO3,the espression of PsPGIP gene was induced by salt obviously, by which we can conclude that PsPGIP gene palys a very important role in the salt-resistant.
2.Genetic transformation of PsPGIP and stresss assay of transgenic plants
(1).Construct the expression vectors successfully, the transgenic plants for the PsPGIP gene were generated by an Agrobacterium mediated method. The expression analysis by PCR and RT-PCR showed that PsPGIP was expressed in tobacco.
(2).Six transgenic lines were selected for the test of NaCl tolerance. MDA content, SOD activity, POD activity and soluble protein content were measured under this stress. The results showed that SOD activity, POD activity and soluble protein content in most transgenic lines was higher than that in untransgenic tobacco under 100 mmol/L NaCl conditions. Furthmore, MDA contents in most transformed plants were lower than that in untransgenic tobacco under 100 mmol/L NaCl stress. The results demonstrated that transformed plants exhibits salt stress tolerance were higher than that in untransgenic tobacco.
(3)Six transgenic lines were selected for the test of fungal infection tolerance.The results showed that compared with untransgenic tobacco, all transgenic lines were up-regulated under the F.oxysporum and anthracnose infection stresss, transgenic lines enhanced the tolerance of fungal infection stress.
引文
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