选取碳末端富含酸性氨基酸的拟南芥SnRK2.6(sucrose non-fermenting1-related protein kinase 2.6)和人源PDI(protein disulfide isomerase),以及近球形蛋白拟南芥PYL10 (PYR like protein 10),分别将重复酸性氨基酸序列添加到SnRK2.6(1...选取碳末端富含酸性氨基酸的拟南芥SnRK2.6(sucrose non-fermenting1-related protein kinase 2.6)和人源PDI(protein disulfide isomerase),以及近球形蛋白拟南芥PYL10 (PYR like protein 10),分别将重复酸性氨基酸序列添加到SnRK2.6(1-332)、PDI(1-440)、PYL10碳末端,利用大肠杆菌BL21重组表达,经过亲和层析,离子交换层析和分子排阻层析进行纯化,综合利用分析超速离心技术,分子排阻层析技术以及多角度静态光散射技术,研究人为设计的多聚氨基酸末端对蛋白质分子排阻行为,聚合状态和其他水力学性质的影响。结果发现,多聚酸性氨基酸末端虽不影响蛋白质分子的聚合状态,但会明显减少分子排阻色谱中蛋白质的洗脱体积,影响蛋白质分子的斯托克斯半径和轴长比等水力学性质。展开更多
The activities of enzymes responsible for lignification in pepper, pre-inoculation with arbuscular mycorrhizal (AM) fungus of Glomus intraradices and/or infection with pathogenic strain of Phytophthora capsici, and th...The activities of enzymes responsible for lignification in pepper, pre-inoculation with arbuscular mycorrhizal (AM) fungus of Glomus intraradices and/or infection with pathogenic strain of Phytophthora capsici, and the biological control effect of G. intraradices on Phytophthora blight in pepper were investigated. The experiment was carried out with four treatments: (1) plants pre-inoculated with G. intraradices (Gi), (2) plants pre-inoculated with G. intraradices and then infected with P. capsici (Gi+Pc), (3) plants infected with P. capsici (Pc), and (4) plants without any of the two microorganisms (C). Mycorrhizal coloni-zation rate was reduced by about 10% in pathogen challenged plants. Root mortality caused by infection of P. capsici was com-pletely eliminated by pre-inoculation with antagonistic G. intraradices. On the ninth day after pathogen infection, Peroxidase (POD) activity increased by 116.9% in Pc-treated roots but by only 21.2% in Gi+Pc-treated roots, compared with the control, respectively. Polyphenol oxidase (PPO) and Phenylalanine ammonia-lyase (PAL) activities gradually increased during the first 3 d and dramatically decreased in Pc-treated roots but slightly decreased in Gi+Pc-treated roots, respectively. On the ninth day after pathogen infection, PPO and PAL decreased by 62.8% and 73.9% in Pc-treated roots but by only 19.8% and 19.5% in Gi+Pc-treated roots, compared with the control, respectively. Three major POD isozymes (45 000, 53 000 and 114 000) were present in Pc-treated roots, while two major bands (53 000 and 114 000) and one minor band (45 000) were present in spectra of Gi+Pc-treated roots, the 45 000 POD isozyme was significantly suppressed by G. intraradices, suggesting that the 45 000 POD isozyme was induced by the pathogen infection but not induced by the antagonistic G. intraradices. A 60 000 PPO isozyme was induced in Pc-treated roots but not induced in Gi+Pc-treated roots. All these results showed the inoculation of antagonistic G. intraradices alleviates root mortality, activates changes of lignification-related enzymes and induces some of the isozymes in pepper plants infected by P. capsici. The results suggested that G. intraradices is a potentially effective protection agent against P. capsici.展开更多
文摘选取碳末端富含酸性氨基酸的拟南芥SnRK2.6(sucrose non-fermenting1-related protein kinase 2.6)和人源PDI(protein disulfide isomerase),以及近球形蛋白拟南芥PYL10 (PYR like protein 10),分别将重复酸性氨基酸序列添加到SnRK2.6(1-332)、PDI(1-440)、PYL10碳末端,利用大肠杆菌BL21重组表达,经过亲和层析,离子交换层析和分子排阻层析进行纯化,综合利用分析超速离心技术,分子排阻层析技术以及多角度静态光散射技术,研究人为设计的多聚氨基酸末端对蛋白质分子排阻行为,聚合状态和其他水力学性质的影响。结果发现,多聚酸性氨基酸末端虽不影响蛋白质分子的聚合状态,但会明显减少分子排阻色谱中蛋白质的洗脱体积,影响蛋白质分子的斯托克斯半径和轴长比等水力学性质。
文摘脂肪酶广泛应用于食品加工、生物柴油制备等领域。为了有效提高微生物脂肪酶的可利用度,将来源于南极嗜冷杆菌属(antarctic psychrotrophic bacterium,Psychrobacter sp.7195)的嗜冷脂肪酶(Lip7195)在大肠杆菌系统中进行高效可溶性表达优化,并进行酶学性能表征。首先对Lip7195基因进行大肠杆菌偏好密码子优化,并通过添加多聚阳离子氨基酸标签等手段,优化重组Lip7195的可溶性表达。结果显示,添加多聚阳离子氨基酸标签的方法有效增加了目的蛋白的可溶性。对纯化后的重组酶进行酶学定性,结果显示,在40℃、p H 9.0时,重组Lip7195酶活性最高,比酶活最高达10.9 U/mg;在30~40℃、p H 8.0~10.0范围,重组Lip7195具有较好的稳定性;Co^(2+)对酶活力有激活作用。研究结果表明,在保持嗜冷脂肪酶特有的酶学性质基础上,添加多聚阳离子氨基酸标签有效改善了其在原核表达中易形成包涵体的问题。
基金Project supported by Korea Science and Engineering Foundation(KOSEF) through the Agricultural Plants Stress Research Center(APSRC) at Chonnam National University, Korea
文摘The activities of enzymes responsible for lignification in pepper, pre-inoculation with arbuscular mycorrhizal (AM) fungus of Glomus intraradices and/or infection with pathogenic strain of Phytophthora capsici, and the biological control effect of G. intraradices on Phytophthora blight in pepper were investigated. The experiment was carried out with four treatments: (1) plants pre-inoculated with G. intraradices (Gi), (2) plants pre-inoculated with G. intraradices and then infected with P. capsici (Gi+Pc), (3) plants infected with P. capsici (Pc), and (4) plants without any of the two microorganisms (C). Mycorrhizal coloni-zation rate was reduced by about 10% in pathogen challenged plants. Root mortality caused by infection of P. capsici was com-pletely eliminated by pre-inoculation with antagonistic G. intraradices. On the ninth day after pathogen infection, Peroxidase (POD) activity increased by 116.9% in Pc-treated roots but by only 21.2% in Gi+Pc-treated roots, compared with the control, respectively. Polyphenol oxidase (PPO) and Phenylalanine ammonia-lyase (PAL) activities gradually increased during the first 3 d and dramatically decreased in Pc-treated roots but slightly decreased in Gi+Pc-treated roots, respectively. On the ninth day after pathogen infection, PPO and PAL decreased by 62.8% and 73.9% in Pc-treated roots but by only 19.8% and 19.5% in Gi+Pc-treated roots, compared with the control, respectively. Three major POD isozymes (45 000, 53 000 and 114 000) were present in Pc-treated roots, while two major bands (53 000 and 114 000) and one minor band (45 000) were present in spectra of Gi+Pc-treated roots, the 45 000 POD isozyme was significantly suppressed by G. intraradices, suggesting that the 45 000 POD isozyme was induced by the pathogen infection but not induced by the antagonistic G. intraradices. A 60 000 PPO isozyme was induced in Pc-treated roots but not induced in Gi+Pc-treated roots. All these results showed the inoculation of antagonistic G. intraradices alleviates root mortality, activates changes of lignification-related enzymes and induces some of the isozymes in pepper plants infected by P. capsici. The results suggested that G. intraradices is a potentially effective protection agent against P. capsici.
