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利用中心组合设计(CCD)探究p ET-32a(+)-Fa MYB5融合蛋白原核表达的最优条件。以不同的诱导温度、诱导时间、菌液初始浓度和异丙基-β-D-硫代半乳糖苷(IPTG)终浓度为4个考察因素,以可溶性蛋白含量为考察指标,在大肠杆菌中表达p ET-32a(+)-Fa MYB5融合蛋白,采用玻璃珠法破菌,提取蛋白质后通过SDS-PAGE凝胶电泳鉴定表达产物。经响应面优化后,在37℃培养工程菌至菌液OD600=0.74后,加入异丙基-β-D-硫代半乳糖苷(IPTG)至终浓度为0.40 mmol/L,在25.51℃条件下诱导培养8 h,可获得最高产量的可溶性目的蛋白,为128μg/m L。最优表达条件的获得,为后期纯化该可溶性蛋白和鉴定Fa MYB5蛋白与顺势作用元件的作用情况提供了一定的理论依据。
Abstract：The central composite design methodology(CCD) was used to explore the optimal condition of the prokaryotic expression of p ET-32a(+)-Fa MYB5 fusion gene in E. coli. The formulation of response of surface was designed with induction temperatures, induction time, microbial initial concentrations and final concentration of Isopropyl-beta-D-thiogalactopyranoside(IPTG) as factors and the content of soluble protein as index to express p ET-32a(+)-Fa MYB5 fusion protein in E. coli, in which the glass bead method was adopted to break bacteria and SDS-PAGE gel electrophoresis was used to identify expression product after protein extraction. After the response surface optimization, we cultured the engineering bacteria to reach OD600=0.74 under the condition of 37℃ and then added IPTG to the final concentration of 0.40 mmol/L, which was induced and cultured under the condition of25.51℃ for 8 hours to obtain the highest amount of soluble protein of 128 μg/m L. The obtain of optimal expression condition might provide a theoretical basis for the purification of this soluble protein and identification of the role of the Fa MYB5 protein in acting with the cis-acting element in later stages.
Chen Q.,Jiang L.Y.,Wang Y.,Zhu F.L.,Tang H.R.,and Wang X.R2015,Optimization of prokaryotic expression factors fo blackberry UGT78H2 using responsive surface methodology and protein purification,Zhiwu Yanjiu(Bulletin of Botani cal Research),35(5):724-729(陈清,江雷雨,王燕,朱芳莉汤浩茹,王小蓉,2015,黑莓果实UGT78H2重组蛋白诱导的响应面优化与蛋白纯化,植物研究,35(5):724-729)
Deng Z.G.,Jin L.,Li J.,Wang W.B.,Yang L.,and Wang X.J.,2013Pathways of MYB transcription factors regulating anther de velopment and pollen formation,Xibei Zhiwu Xuebao(Ac ta Botanica Boreali-Occidentalia Sinica),33(4):850-856(邓志刚,金樑,李晶,王文斌,杨龙,王晓娟,2013,MYB类转录因子对花药和花粉发育的调控途径,西北植物学报,33(4):850-856)
Dubos C.,Le Gourrierec J.,Baudry A.,Huep G.,Lanet E.,Debeau jon I.,Routaboul J.M.,Alboresi A.,Weisshaar B.,and Lep iniec L.,2008,MYBL2 is a new regulator of flavonoid biosyn thesis in Arabidopsis thaliana,Plant Journal,55(6):940-953
Frampton J.,2004,MYB transcription factors:their role in growth,differentiation and disease,Proteins and Cell Regu lation,2:6-8
Gonda T.J.,1998,The c-Myb oncoprotein,Int.J.Biochem.Cel Biol.,30(5):547-551
Ishida T.,Kurata T.,Okada K.,and Wada T.,2008,A genetic regulatory network in the development of trichomes and roo hairs,Annual Review Plant Biology,59(1):365-386
Liu L.,Du H.,Tang X.F.,Wu Y.M.,Huang Y.B.,and Tang Y.X.2008,The roles of MYB transcription factors on plant de fense responses and its molecular mechanism,Yichuan(Heredites),30(10):1265-1271(刘蕾,杜海,唐晓凤,吴燕民,黄玉碧,唐益雄,2008,MYB转录因子在植物抗逆胁迫中的作用及其分子机理,遗传,30(10):1265-1271)
Nadeau J.A.,2009,Stomatal development:new signals and fate determinants,Current Opinion Plant Biology,12(1):29-35
Pabo C.O.,and Sauer R.T.,1992,Transcription factors:structura families and principles of DNA recognition,Annual Review Biochemistry,61(1):1053-1095
Qiao M.,Yu Y.C.,and Xiang F.N.,2009,The roles of the Arabidopsi R2R3-MYB transcription factors in the stress responses Shengming Kexue(Chinese Bulletin of Life Sciences),21(1)145-150(乔孟,于延冲,向凤宁,2009,拟南芥R2R3-MYB类转录因子在环境胁迫中的作用,生命科学,21(1):145-150)
Schaart J.G.,Dobos C.,Romero De La Fuente I.,Van Houwelin gen A.M.,De Vos R.C.,Jonker H.H.,Xu W.,Routaboul JM.,Lepiniec L.,and Bovy A.G.,2013,Identification and characterization of MYB-b HLH-WD40 regulatory complex es controlling proanthocyanidin biosynthesis in strawberry(Fragaria×ananassa)fruits,New Phytologist,197(2):454-467
Tanaka Y.,Sasaki N.,and Ohmiya A.,2008,Biosynthesis o plant pigments:anthocyanins,betalains and carotenoids Plant Journal,54(4):733-749
Wang X.Q.,Chen B.J.,and Yin L.P.,2003,The plant MYB tran scription factors,Shengwu Jishu Tongbao(Biotechnology Bulletin),(2):22-25(王希庆,陈柏君,印莉萍,2003,植物中的MYB转录因子,生物技术通报,(2):22-25)
Zhang Q.,Liu S.,Xing Y.,Cao Q.Q.,and Qin L.,2015,Comparision of extraction methods of total RNA from strawberry fruits Zhongguo Nongxue Tongbao(Chinese Agricultural Scienc Bulletin),31(31):146-149(张卿,刘帅,邢宇,曹庆芹,秦岭2015,草莓果实总RNA提取方法的比较,中国农学通报,31(31):146-149)
Zhong R.,Richardson E.A.,and Ye Z.H.,2007,The MYB46transcription factor is a direct target of SND 1 and regulate secondary wall biosynthesis in Arabidopsis,Plant Cell,19(9):2776-2791
Zuo R.,Xu M.L.,Chai G.H.,and Zhou G.K.,2012,Function and regulation mechanism of plant MYB transcription factors Shengming Kexue(Chinese Bulletin of Life Sciences),24(10):1133-1140(左然,徐美玲,柴国华,周功克,2012,植物MYB转录因子功能及调控机制研究进展,生命科学,24(10):1133-1140)
基本信息:
DOI：10.13417/j.gab.036.000388
中图分类号:S668.4;Q943.2
引用信息:
[1]何佳鲜,江雷雨,洪敏,等.草莓FaMYB5基因原核表达的响应面优化[J].基因组学与应用生物学,2017,36(01):388-394.DOI:10.13417/j.gab.036.000388.
基金信息:
四川省四川农业大学创新训练计划项目(201410626028)资助
2016-05-31
2016-05-31
2016-05-31