| 棉花去泛素化酶基因GhOTUD5原核表达条件的优化 |
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| 引用本文： | 兰艳平,刘东,赵岩,张丽,陈义,冯加加,渠云芳,黄晋玲.棉花去泛素化酶基因GhOTUD5原核表达条件的优化[J].分子植物育种,2020(10):3223-3231. |
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| 作者姓名： | 兰艳平 刘东 赵岩 张丽 陈义 冯加加 渠云芳 黄晋玲 |
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| 作者单位： | 山西农业大学农学院 |
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| 基金项目： | 国家重点研发计划项目(2018YFD0100301)资助。 |
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| 摘 要： | 棉花组蛋白去泛素化酶GhOTUD5与生殖发育密切相关。为了获得大量可溶性表达的GhOTUD5蛋白,本试验对目的蛋白原核表达条件进行优化。采用分子克隆方法构建重组表达载体pET-22b-GhOTUD5,转化大肠杆菌(Escherichia coli)BL21(DE3)和Transetta(DE3),用异丙基-β-D-硫代半乳糖苷(isopropylβ-D-1-thiogalactopyranoside,IPTG)诱导目的蛋白表达。在对影响原核表达的四个单因素(菌体密度OD600,诱导剂IPTG终浓度,诱导时间和诱导温度)分别分析的基础上,设计三水平的正交试验L9(43),摸索大量可溶性目的蛋白表达的最优条件。结果显示,目的蛋白GhOTUD5在Transetta(DE3)中成功表达,在BL21(DE3)中不表达。在菌体密度OD600为0.8、诱导温度为28℃、IPTG终浓度为0.7 mmol/L、诱导3 h的条件下可溶性目的蛋白表达量最高,达到13%左右。该研究结果为进一步研究GhOTUD5在棉花育性方面的功能提供了科学数据。
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| 关 键 词： | 棉花 GhOTUD5 基因 原核表达 正交试验 条件优化 |
Optimization of Prokaryotic Expression Conditions of Cotton Deubiquitinase Gene GhOTUD5 |
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| Lan Yanping,Liu Dong,Zhao Yan,Zhang Li,Chen Yi,Feng Jiajia,Qu Yunfang,Huang Jinling.Optimization of Prokaryotic Expression Conditions of Cotton Deubiquitinase Gene GhOTUD5[J].Molecular Plant Breeding,2020(10):3223-3231. |
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| Authors: | Lan Yanping Liu Dong Zhao Yan Zhang Li Chen Yi Feng Jiajia Qu Yunfang Huang Jinling |
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| Institution: | (College of Agriculture,Shanxi Agricultural University,Taigu,030801) |
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| Abstract: | Cotton histone deubiquitinase GhOTUD5 is related to reproductive development.In order to obtain a large amount of soluble expressed GhOTUD5 protein,the prokaryotic expression conditions of the target protein were optimized in this test.Recombinant expression vector pET-22b-GhOTUD5 was constructed by molecular cloning method and transformed into Escherichia coli strain BL21(DE3)and Transetta(DE3).Isopropylβ-D-1-thiogalactopyranoside(IPTG)induced the expression of target protein.Based on the analysis of the four single factors(cell density OD600,IPTG final concentration,induction time and induction temperature)that affect prokaryotic expression,a three-level orthogonal test L9(43)was designed to explore optimal conditions of much soluble target protein expression.The results showed that the target protein GhOTUD5 was expressed successfully in Escherichia coli Transetta(DE3)strain and not in BL21(DE3)strain when induced by IPTG.Under the conditions of the OD600 was 0.8,the induction temperature was 28℃,the final IPTG concentration was 0.7 mmol/L and the induction was 3 h,the expression of soluble target protein was the highest,reaching about 13%.The research results provide a scientific data for further research on the function of GhOTUD5 in fertility. |
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| Keywords: | Cotton GhOTUD5 gene Prokaryotic expression Orthogonal experiment Optimal expression condition |
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