Prokaryotic Expression Analysis of Resistance-related Gene T1N6_22 from Arabidopsis thaliana

doi:10.7668/hbnxb.2015.01.012

Abstract

Abstract: This study was aimed to construct prokaryotic expression vector of resistance-related gene T1N6_22 from Arabidopsis thaliana and obtain the purified protein T1N6_22 with high-efficiency expression.The CDS of T1N6_22 was amplified by RT-PCR technology using the cDNA of the Arabidopsis Col-0 and was fused into a prokaryotic expression vector pGEX4T-1.Restriction enzyme digestion and sequencing showed that the recombinant vector pGEX4T-1- T1N6_22 -GST was successfully constructed and transformed into E.coli BL21 cells.The results indicated that the pGEX4T-1- T1N6_22 -GST with the predicted molecular weight of about 57 kDa was successfully expressed in E.coli BL21 strain.SDS-PAGE indicated that the best expression quantity was induced with 0.1 mmol/L IPTG treatment for 3 h.These results would provide basis for screening interacting proteins of T1N6_22 and the regulation mechanism in Arabidopsis resistance.

Key words: Arabidopsis thaliana, T1N6_22, Prokaryotic expression, Purification

CLC Number:

S432.1
Q78

JIANG Chen-xi, WENG Qiao-yun, FAN Jin-tao, WANG Guan-yu, DONG Li-ping, XING Ji-hong, DONG Jin-gao. Prokaryotic Expression Analysis of Resistance-related Gene T1N6_22 from Arabidopsis thaliana[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2015, 30(1): 73-76. doi: 10.7668/hbnxb.2015.01.012.

/ / Recommend / Download Citations

URL: http://www.hbnxb.net/EN/10.7668/hbnxb.2015.01.012

http://www.hbnxb.net/EN/Y2015/V30/I1/73

References

[1] Belkhadir Y,Nimchuk Z,Hubert D A,et al.Arabidopsis RIN4 negatively regulates disease resistance mediated by RPS2 and RPM1 downstream or independent of the NDR1 signal modulator and is not required for the virulence functions of bacterial type Ⅲeffectors AvrRpt2 or AvrRpm1[J].Plant Cell,2004,16(10):2822-2835.
[2] Gassmann W,Hinsch M E,Staskawicz B J.The Arabidopsis RPS4 bacterial-resistance gene is a member of the TIR-NBS-LRR family of disease-resistance genes[J].Plant Journal,1999,20(3):265-277.
[3] Mohr T J,Mammarella N D,Hoff T,et al.The Arabidopsis downy mildew resistance gene RPP8 is induced by pathogens and salicylic acid and is regulated by W box cis elements[J].Molecular Plant-Microbe Interactions,2010,23(10):1303-1315.
[4] Rose L E,Bittner-Eddy P D,Langley C H,et al.The maintenance of extreme amino acid diversity at the disease resistance gene, RPP13,in Arabidopsis thaliana [J].Genetics,2004,166(3):1517-1527.
[5] Borhan M H,Holub E B,Beynon J L,et al.The Arabidopsis TIR-NB-LRR gene RAC1 confers resistance to albugo candida(white rust)and is dependent on EDS1 but not PAD4[J].Molecular Plant-Microbe Interactions,2004,17(7):711-719.
[6] Jorgensen T H,Emerson B C.RPW8 and resistance to powdery mildew pathogens in natural populations of Arabidopsis lyrata [J].New Phytologist,2009,182(4):984-993.
[7] Mengiste T,Chen X,Salmeron J,et al.The BOTRYTIS SUSCEPTIBLE1 gene encodes an R2R3MYB transcription factor protein that is required for biotic and abiotic stress responses in Arabidopsis[J].Plant Cell,2003,15(11):2551-2565.
[8] Veronese P,Chen X,Bluhm B,et al.The BOS loci of Arabidopsis are required for resistance to Botrytis cinerea infection[J].Plant Journal,2004,40(4):558-574.
[9] Canonne J,Marino D,Jauneau A,et al.The xanthomonas type Ⅲ effector XopD targets the Arabidopsis transcription factor MYB30 to suppress plant defense[J].Plant Cell,2011,23(9):3498-3511.
[10] Li L,Yu X,Thompson A,et al.Arabidopsis MYB30 is a direct target of BES1 and cooperates with BES1 to regulate brassinosteroid-induced gene expression[J].Plant Journal,2009,58(2):275-286.
[11] Canet J V,Dobon A,Roig A,et al.Structure-function analysis of npr1 alleles in Arabidopsis reveals a role for its paralogs in the perception of salicylic acid[J].Plant Cell and Environment,2010,33(11):1911-1922.
[12] 张红志,蔡新忠.病程相关基因非表达子1(NPR1):植物抗病信号网络中的关键节点[J].生物工程学报,2005,21(4):511-515.
[13] Moffat C S,Ingle R A,Wathugala D L,et al.ERF5 and ERF6 play redundant roles as positive regulators of JA/Et-Mediated defense against Botrytis cinerea in Arabidopsis[J].PLOS One,2012,7(4):e35995.
[14] Xing J H,Weng Q Y,Hao C C,et al. T1N6_22 gene is required for biotic and abiotic stress responses in arabidopsis[J].Russian Journal of Genetics,2012,48(12):1191-1198.
[15] Han X Q,Lin X M,Chen H J,et al.The prokaryotic expression and bioactivity of the recombinant red fire ant venom allergen soli 4[J].Agricultural Sciences in China,2009,8(2):182-187.
[16] 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):117-121.
[17] Rao C N,Reeder D J,Stack S M,et al.Prokaryotic expression,purification,and reconstitution of biological activities(antiprotease,antitumor,and heparin-binding)fortissue factor pathway inhibitor-2[J].Biochemical and Biophysical Research Communications,2000,3(276):1286-1293.
[18] 李波,倪志勇,范玲.棉花 GhCOMT3 基因的原核表达、纯化及鉴定[J].华北农学报,2010,25(z1):12-16.
[19] 杜李继,顾菊,陈光朗,等.拟南芥AtMYB50和AtMYB61蛋白的原核表达研究[J].合肥工业大学学报:自然科学版,2014,37(1):105-109.
[20] Wu C,Wang Y Y,Zou M J,et al.Prokaryotic expression,purification,and production of polyclonal antibody against human polypeptide N-acetylgalactosaminyl transferase 14[J].Protein Expression and Purification,2007,56(1):1-7.

Please choose a citation manager

Content to export