拟南芥AZI1基因对酿酒酵母生长和蒜薹灰霉菌侵染的抑制作用
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摘要
本工作采用酿酒酵母细胞表达载体pESC和植物细胞表达载体pPZP211分析了拟南芥AZI1基因对真菌的抗性功能。与转空载体pESC的酵母细胞相比,包含pESC-AZI1的酵母细胞在附加2%蔗糖的SC-URA培养基上的生长状况较差。在含有2%半乳糖的诱导培养基上于28℃生长48h后,观察不到转AZI1基因酵母菌落；144h后,转AZI1基因酵母只形成少量菌落。Western免疫印迹分析结果表明,包含pESC-AZI1的酵母细胞经半乳糖诱导后可以有效表达AZI1蛋白。用蒜薹灰霉菌孢子侵染拟南芥AZI1基因过表达和Col-0野生型植株,24h后进行DAB染色,发现Col-0野生型植株叶片的被侵染部位着色较浅,只能产生少量H2O2,病原体可以扩散,而AZI1基因过表达植株叶片在侵染部位着色较深,有大量H2O2产生,表明转化体能够以局部细胞的死亡来阻止病原体侵染周围的细胞。台酚蓝染色结果表明,与AZI1基因过表达植株相比,Col-0野生型植株叶片上有大量细胞死亡。在Col-0野生型植株中,AZI1基因表达受外源水杨酸(?)24h后达到峰值。以上结果显示AZI1基因在拟南芥抵抗生物胁迫过程中具有重要作用。此外,还进行了以下三个方面的工作：一、构建了原核表达载体pET32a-AZI1和pET32a-EARLI1,并成功导入大肠杆菌BL21(DE3)菌株；二、构建了pC AMBI A1302-AZI1-GFP融合表达载体,导入农杆菌LBA4404后对拟南芥和秦烟95进行了遗传转化；三,利用含有pPZP211-AZI1植物表达载体的农杆菌ABI菌株对秦烟95进行了遗传转化,采用PCR及RT-PCR方法对11株转基因植株进行了初步验证。
Resistance of Arabidopsis AZI1 to fungus was analyzed with Saccharomyces cerevisiae expression vector pESC and plant expression vector pPZP211. Compared to yeast cells transformed with empty vector, the growth ability of yeast cells harbouring pESC-AZI1 declined obviously on SC-URA medium containing 2% sucrose. On SC-URA medium supplemented with 2% galactose, yeast cells harbouring pESC-AZI1 could not form colony after 48h incubation at 28℃, while only a few colonies could be found after 144h. Western blot analysis showed that yeast cells harbouring pESC-AZIl could express AZI1 efficiently after induction with galactose. The spores of Botrytis cinerea Pers.ex of garlic sprout were used to infect the leaves of wild-type Col-0 and AZI1 overexpressing transgenic plants. Generation of H2O2 was analyzed by DAB staining 24h after inoculation. In Col-0 wild-type plants, the staining colour of infection sites on leaves were a little lighter, it showed that only small amounts of H2O2 were produced at the infected sites of leaves and the pathogens could spread to surrounding cells. In contrast to this, the infection sites on leaves of AZI1 overexpressing plants were stained intensively by DAB, which would accumulate large amounts of H2O2, indicating the transformants could prevent the spread of pathogen by the death of local cells in infected sites. Trypan blue staining showed that a large number of cells in pathogen infected leaves of wild-type Col-0 plants were died in comparison with the leaves of AZI1 overexpressing plants. Besides, the expression of AZI1 could be induced by salicylic acid and peaked after 24h in Col-0 wild-type plants. All these results suggested that AZI1 play an important role in defense system of Arabidopsis against biotic stresses. In addition, the following three aspects of researches were carried out in this work.1, prokaryotic expression vectors pET32a-AZI1 and pET32a-EARLI1 were constructed and transferred to BL21(DE3) strain of Escherichia coli successfully.2, Plant fusion expression vector pCAMBIA1302-AZI-GFP was constructed and introduced into LBA4404 strain of Agrobacterium tumefaciens which was used in genetic transformation of the wild-type Arabidopsis thaliana ecotype Col-0 and Nicotiana tobacum genotype QinYan95 subsequently. 3, Agrobacterium tumefaciens strain ABI containing pPZP211-AZI1 was adopted in transformation of Nicotiana tobacum genotype QinYan95,11 putative transgenic plants were preliminarily verified by PCR and RT-PCR.
Resistance of Arabidopsis AZI1 to fungus was analyzed with Saccharomyces cerevisiae expression vector pESC and plant expression vector pPZP211. Compared to yeast cells transformed with empty vector, the growth ability of yeast cells harbouring pESC-AZI1 declined obviously on SC-URA medium containing 2% sucrose. On SC-URA medium supplemented with 2% galactose, yeast cells harbouring pESC-AZI1 could not form colony after 48h incubation at 28℃, while only a few colonies could be found after 144h. Western blot analysis showed that yeast cells harbouring pESC-AZIl could express AZI1 efficiently after induction with galactose. The spores of Botrytis cinerea Pers.ex of garlic sprout were used to infect the leaves of wild-type Col-0 and AZI1 overexpressing transgenic plants. Generation of H2O2 was analyzed by DAB staining 24h after inoculation. In Col-0 wild-type plants, the staining colour of infection sites on leaves were a little lighter, it showed that only small amounts of H2O2 were produced at the infected sites of leaves and the pathogens could spread to surrounding cells. In contrast to this, the infection sites on leaves of AZI1 overexpressing plants were stained intensively by DAB, which would accumulate large amounts of H2O2, indicating the transformants could prevent the spread of pathogen by the death of local cells in infected sites. Trypan blue staining showed that a large number of cells in pathogen infected leaves of wild-type Col-0 plants were died in comparison with the leaves of AZI1 overexpressing plants. Besides, the expression of AZI1 could be induced by salicylic acid and peaked after 24h in Col-0 wild-type plants. All these results suggested that AZI1 play an important role in defense system of Arabidopsis against biotic stresses. In addition, the following three aspects of researches were carried out in this work.1, prokaryotic expression vectors pET32a-AZI1 and pET32a-EARLI1 were constructed and transferred to BL21(DE3) strain of Escherichia coli successfully.2, Plant fusion expression vector pCAMBIA1302-AZI-GFP was constructed and introduced into LBA4404 strain of Agrobacterium tumefaciens which was used in genetic transformation of the wild-type Arabidopsis thaliana ecotype Col-0 and Nicotiana tobacum genotype QinYan95 subsequently. 3, Agrobacterium tumefaciens strain ABI containing pPZP211-AZI1 was adopted in transformation of Nicotiana tobacum genotype QinYan95,11 putative transgenic plants were preliminarily verified by PCR and RT-PCR.
引文
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[21]Jose-Estanyol M, Ruiz-Avila L, Puigdomenech P. A maize embryo Specific gene:encode a proline-rich and hydrophobic protein. Plant Cell,1992,4:413-423
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[23]Blanco-Portales R, Lopez-Raez JA, Bellido ML, et al. A strawberry fruit-Specific and ripening-related gene codes for a HyPRP protein involved in polyphenol anchoring. Plant Mol Biol,2004,55:763-780
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