Prokaryotic Expression Vector Construction and Expression of Tolerance Relatedgene AtSTK in Arabidopsis

doi:10.3969/j.issn.1000-7091.2013.02.007

Abstract

Abstract: Total RNA was extracted from Arabidopsis leaves. 1212 bp AtSTKgene was amplified by RT-PCR and constructed into the expression vector PET-32a. Then the recombinant plasmid was transformed into E. coli Rosetta. The optimal induction conditions for AtSTKgene expression were 1 mmol/L IPTG and 4 hours induction. Under these conditions,the expression level of the fusion protein was highest. It was found that the form of the fusion protein was inclusion body. So the inclusion body was treated by dissolving and renaturation. Finally,the purified At-STK fusion protein was obtained. This work laid the foundation for the next study on the physical and chemical prop-erties of AtSTK protein.

Key words: Arabidopsis thaliana, Serine/threonine protein kinase, Prokaryotic expression, Salt tolerancegene

CLC Number:

HU Jing-rui, SHEN Jin-bao, LI Jing-lan, LI Xiao-xu, ZHAO Bao-cun,gE Rong-chao. Prokaryotic Expression Vector Construction and Expression of Tolerance Relatedgene AtSTK in Arabidopsis[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2013, 28(2): 38-41. doi: 10.3969/j.issn.1000-7091.2013.02.007.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.3969/j.issn.1000-7091.2013.02.007

http://www.hbnxb.net/EN/Y2013/V28/I2/38

References

[1] Chenk P W,Snaar J B E. Signal perception and transduc-tion:the role of protein kinases[J]. Biochim Biophys Ac-ta,1999,1449(1):1-24.
[2] Ferreira Pg. The Arabidopsis functional homolog of the P34cdc2protein kinase[J]. Plant Cell,1991,3(5):531-540.
[3] Lee J H,Montagu M V,Verbruggen N. A highly conserved kinase is an essential component for sress tolerance in yeast and plant cells[J]. Proc Natl Acad Sci USA,1999,9(6):5873-5877.
[4] Robinson J J,Cobb M H. Mitogen-activated protein kinase pathway[J]. Curr Opin Cell Biol,1997,9:180-186.
[5] Pardo J M,Reddy M P,Yang S. Stress signaling through Ca2 +/calmodulin-dependent protein phosphatase calci-neurin mediates salt adaptation in plants[J]. Proc Natl Acad Sci USA,1998,95:9681-9686.
[6] Zhu J K.genetic Analysis of plant salt tolerance using Ar-abidopsis[J]. Plant Physiol,2000,124:941-948.
[7] Zhu J K. Plant salt tolerance review[J]. Trends Plant Sci-ence,2001,6(2):66-71.
[8] Zhu J K. Regulation of ion homeostasis under salt stress review[J]. Current Opinion Plant Biology,2003,6(5): 441-445.
[9] Rudrabhatla P,Rajasekharan R. Developmentally regula-ted dual-specificity kinase that is induced by abiotic stresses[J]. Plant Physiol,2002,130(1):380-390.
[10] Rudrabhatla P,Reddy M M,Rajasekharan R.genome-wide analysis and experimentation of plant serine/threo-nine/tyrosine-specific protein kinases[J]. Plant Mol Bi-ol,2006,60(2):293-319.
[11] Chen W,Wang Y Y,Zhou M J,et al. Prokaryotic expres-sion,purification,and production of polyclonal antibody against human polypeptide N-acetylgalactosaminyl transferase 14[J]. Protein Expression and Purification,2007,56:1-7.
[12] Han X Q,Lin X M,Chen H J,et al. The prokaryotic ex-pression and bioactivity of the recombinant red fire ant venom allergen soli4[J]. Agricultural Sciences in Chi-na,2009,8(2):182-187.
[13] 邓毛子,石春薇,王芳,等.结核分枝杆菌Ag85A蛋白的原核表达、纯化和免疫反应性[J]. 基础医学与临床,2010,30(2):118-119.
[14] Rao C N,Reeder D J,Stack S M,et al. Prokaryotic ex-pression,purification,and reconstitution of biological ac-tivities(antiprotease,antitumor,and heparin-binding) for tissue factor pathway inhibitor-2[J]. Biochemical and Biophysical Research Communications,2000,3(276): 1286-1293.
[15] 胡晓梅,黄建军,饶贤才,等. 分泌型重组铜绿假单胞菌外毒素A基因工程菌的发酵研究[J]. 医学研究生学报,2004,17(5):388-390.
[16] Rudolph H,Bohmg,Lilie H,et al. Folding Proteins
[M]. 2nd ed. New York:IRL Press,1997.
[17] Clark E D B. Refolding of recombinant proteins[J]. Curr Opin Biotechnol,1998,9(2):157-163.
[18] West S M,Chaudhui J B,Howell J A. Improved protein refolding using hollow-fibre membrane dialysis[J]. Bio-technol Bioeng,1998,57(5):590-599.
[19] Suttnar J E,Dyr E,Hamsikova J,et al. Procedure forre-folding and purification of recombinant proteins fromE-sche-richia coliinclusion bodies using a strong anion ex-changer[J]. Chromatography B,1994,656(1):123-126.
[20] Wermer M H,Cloreg M,Gronenborn A M,et al. Refold-ing proteins bygel filtration chromatography[J]. FEBS Lett,1994,345(2-3):125-130.

Please choose a citation manager

Content to export