拟南芥细胞色素P450 94B3-N25的原核表达
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摘要
为拟南芥细胞色素P450 94B3-N25的优化表达和纯化,利用原核表达技术,成功在大肠埃希菌内表达了拟南芥细胞色素P450 94B3-N25。首先,用热激转化方法将载体pMAL-c2x-IPN-CYP94B3-N25转入Rosseta,利用菌落PCR挑选阳性克隆,设置交叉试验,摸索最适表达温度和时间表达目的蛋白,并在450nm处利用分光光度法测定菌液里有目的蛋白的表达。破碎细胞后,提取细胞总蛋白,SDS-PAGE和Western blot鉴定结果显示目的蛋白表达成功,但蛋白质变性。
Arabidopsis thaliana cytochrome P450 94 B3-N25 was successfully expressed in Escherichia coli using prokaryotic expression technology.pMAL c2 x-IPN-94 B3-N25 was transformed into Rosseta by the method of heat shock,and the positive clones were screened by PCR,with the help of crossing experiment and spectrophotometry,the best temperature and time to express purpose protein were optimized,and the protein at 450 nm in bacteria liquid was detected.We gained the total protein with cell disruption,the results of SDS-PAGE and Western blot showed the protein expressed successfully,but denatured.
Arabidopsis thaliana cytochrome P450 94 B3-N25 was successfully expressed in Escherichia coli using prokaryotic expression technology.pMAL c2 x-IPN-94 B3-N25 was transformed into Rosseta by the method of heat shock,and the positive clones were screened by PCR,with the help of crossing experiment and spectrophotometry,the best temperature and time to express purpose protein were optimized,and the protein at 450 nm in bacteria liquid was detected.We gained the total protein with cell disruption,the results of SDS-PAGE and Western blot showed the protein expressed successfully,but denatured.
引文
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[17]高明煜,吴玉星,朱百涛,等.苹果树腐烂病菌细胞色素P450基因Vmcyp5的功能[J].微生物学报,2018(2):274-283.
[2]汪长发.茉莉酸甲酯对肝癌的抑制作用及其机理研究[D].湖南长沙:中南大学,2013.
[3]薛思鸣,成丹,解洪杰,等.利用拟南芥种群间转录组和表皮毛密度差异信息筛选表皮毛相关功能基因[J],农业生物技术学报,2016,24(8):1138-1146.
[4]高定.纳米羟基磷灰石/壳聚糖/魔芋葡甘聚糖骨替代植入支架材料的生物相容性研究[D].云南昆明:昆明医科大学,2015.
[5]李红利,孙振元,赵梁军,等.茉莉酸类物质对植物生长发育及抗性的影响[J],中国农学通报,2009,25(16):167-172.
[6]Zou Y,Chintamanani S,He P,et al.A gain of function mutation in Msl10triggers cell death and wound-induced hyperaccumulation of jasmonic acid in Arabidopsis[J].J Integr Plant Biol,2016,24(6):600-609.
[7]Liu X L,Lin J,Hu H F,et al.Enhanced production of shikimic acid using a multi-gene co-expression system in Escherichia coli[J].Chin J Nat Med,2016,32(4):286-293.
[8]王芳,吴文惠,蒋霞云.生化综合实验项目的设计及应用——以大肠杆菌质粒DNA的提取、酶切及电泳为例[J].现代农业科技,2016,22(10):328-329.
[9]张迹,李智,张宇,等.一步法制备大肠杆菌BL21(DE3)菌株感受态细胞及转化条件优化[J].江苏农业科学,2016,44(12):529-532.
[10]周鹏.构建含镍铁氢酶基因的重组大肠杆菌及产氢研究[D].四川成都:成都理工大学,2015..
[11]Chiangsom A,Lawanprasert S,Oda S,et al.Inhibitory and inductive effects of Phikud Navakot extract on human cytochrome P450[J].DMPK,2016,16(6):210-217.
[12]杨韵霏,李由然,张梁,等.细菌麦芽糖淀粉酶在枯草芽孢杆菌中的诱导型异源表达[J].微生物学通报,2017(2):263-273.
[13]董栩,许燕,李月亭,等.药用植物中细胞色素P450基因的研究进展[J].云南中医中药杂志,2016,37(3):75-78.
[14]韩丽君,金铁锁.HMGR、FPS、SE和P450基因克隆、生物信息学及表达分析[D].云南昆明:云南中医学院,2016.
[15]石力.朱砂叶螨抗甲氰菊酯P450基因鉴定及其转录调控研究[D].重庆:西南大学,2017.
[16]王晨晨.细胞色素P450酶介导的芳香族化合物代谢机理的研究[D].浙江杭州:浙江大学,2016.
[17]高明煜,吴玉星,朱百涛,等.苹果树腐烂病菌细胞色素P450基因Vmcyp5的功能[J].微生物学报,2018(2):274-283.