对转基因植株样本中的农杆菌上T-DNA基因的cDNA 5'末端进行快速扩增（5'RACE）

Yi Zhang

doi:10.21769/BioProtoc.1603

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5’ Rapid Amplification of cDNA Ends (5’ RACE) of Agrobacterial T-DNA Genes within Transformed Plant Sample

对转基因植株样本中的农杆菌上T-DNA基因的cDNA 5'末端进行快速扩增（5'RACE）

YZ Yi Zhang email

发布: 2015年09月20日第5卷第18期 DOI: 10.21769/BioProtoc.1603 浏览次数: 12183

评审: Arsalan DaudiKanika GeraAnonymous reviewer(s)

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Cover of PLOS Pathogens, featuring study using the protocol.

Jan 2015

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Abstract

The T-DNA (transferred-DNA) region of virulent Agrobacterium tumefaciens (A. tumefaciens) strain is transferred and integrated into the plant genome, and thereby the T-DNA genes are expressed in transformed plant cells. This protocol was used to analyze the transcription start sites (TSSs) of agrobacterial T-DNA genes within plant crown gall tumor. Firstly, the stems of Arabidopsis thaliana were inoculated by A. tumefaciens strain C58 and developed crown gall tumor. Subsequently, the mRNA was extracted from the crown gall tumor and then used for amplification of 5’ cDNA ends by 5’ Rapid Amplification of cDNA Ends (5’ RACE) assay. The full-length cDNAs were generated in reverse transcription reactions and used to analyze TSSs. Here, TSSs of three oncogenes, IaaH, IaaM and Ipt were analyzed as examples. This protocol also allows for identification of TSSs of the other agrobacterial T-DNA genes that expressed in plant cells.

Keywords: Agrobacterium tumefaciens (农杆菌介导法)

T-DNA (T-DNA)

5' RACE (5的比赛)

Transcription start site (转录起始位点)

Materials and Reagents

A. tumefaciens strain C58 noc^c (nopaline catabolism) (MAX-PLANCK-GESELLSCHAFT, catalog number: 584 )
Arabidopsis thaliana Col-0
Escherichia coli (E. coli) strain MRF’ (Agilent Technologies, catalog number: 200230-41 )
Dynabeads^® Oligo(dT)25 (Thermo Fisher Scientific, Invitrogen^TM, catalog number: 61005 )
SMARTer^® RACE 5’/3’ Kit (Takara Bio Company, Clontech, catalog number: 634859 )
DreamTaq DNA Polymerase (5 U/µl) (Thermo Fisher Scientific, catalog number: EP0701 )
QIAquick PCR Purification Kit (QIAGEN, catalog number: 28106 )
pGEM^®-T Easy Vector Systems (Promega Corporation, catalog number: A1360 )
Hypodermic Syringes without Needle (5 cc) (Terumo Medical Corporation, catalog number: SS-05L )
Hypodermic Needles (Terumo Medical Corporation, catalog number: NN-2138R )
KB medium (see Recipes)
Agromix buffer (see Recipes)
Lysis buffer (see Recipes)
Washing buffer A (see Recipes)
Washing buffer B (see Recipes)
Elution buffer (see Recipes)
2x binding buffer (see Recipes)

Equipment

Culture tubes 13 ml (SARSTEDT, catalog number: 62.515.028 )
NanoDrop 2000c UV-Vis Spectrophotometer (Thermo Fisher Scientific, catalog number: ND-2000c )
High-performance, Modular Stereomicroscope for Application Based Customization MZ6 (Leica Microsystems, model: MZ6)
Ball mill (RETSCH, model: MM200 )
Thermal cycler (Eppendorf, catalog number: 950000031 )
MiniSpin^®/MiniSpin^® plus Centrifuge (Eppendorf, catalog number: 0 22620100 )
MagneSphere^® Technology Magnetic Separation Stands (Promega Corporation, catalog number: Z5342 )

Procedure

English

中文翻译

文章信息

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如何引用

Readers should cite both the Bio-protocol article and the original research article where this protocol was used:

Zhang, Y. (2015). 5’ Rapid Amplification of cDNA Ends (5’ RACE) of Agrobacterial T-DNA Genes within Transformed Plant Sample. Bio-protocol 5(18): e1603. DOI: 10.21769/BioProtoc.1603.
Zhang, Y., Lee, C. W., Wehner, N., Imdahl, F., Svetlana, V., Weiste, C., Droge-Laser, W. and Deeken, R. (2015). Regulation of oncogene expression in T-DNA-transformed host plant cells. PLoS Pathog 11(1): e1004620.