Method for constructing full-length transcription factor yeast two-hybrid library

A yeast two-hybrid and transcription factor technology is applied in the field of patent application for yeast two-hybrid library construction method, which can solve the problems of complex construction process, low library quality and high false positive rate, achieve high screening accuracy, improve library quality, The effect of high screening positive rate

Active Publication Date: 2019-01-25
ZHENGZHOU TOBACCO RES INST OF CNTC
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Problems solved by technology

[0007] The purpose of this application is to provide a method for constructing a full-length transcription factor yeast two-hybrid library, so as to overcome the high false positive rate and low accuracy of existing yeast two-hybrid libraries for transcription factor screening and identification. problems, as well as problems such as complex construction process, high cost, and low quality library in the existing construction process

Method used

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  • Method for constructing full-length transcription factor yeast two-hybrid library
  • Method for constructing full-length transcription factor yeast two-hybrid library
  • Method for constructing full-length transcription factor yeast two-hybrid library

Examples

Experimental program
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specific Embodiment

[0067] The technical solutions of the present application will be further explained below in conjunction with the examples. Before introducing the specific examples, a brief description of some experimental backgrounds in the following examples is as follows.

[0068] biomaterials:

[0069] Red flower big gold dollar ( Nicotiana tabacum ), a commonly used tobacco variety;

[0070] The design and synthesis of relevant primers were provided by Beijing Liuhe Huada Gene Technology Co., Ltd.;

[0071] Yeast two-hybrid vector pGADT7, provided by Shanghai Haike Biotechnology Co., Ltd.;

[0072] Experimental reagents:

[0073] RNA extraction reagent (TRIzol™ Reagent), Invitrogen, USA;

[0074] Reverse transcription reagents (Reverse Transcriptase M-MLV, Recombinant RNase Inhibitor, 10 mMdNTP Mixture, Oligo(dT)18 Primer), Baori Medical Biotechnology (Beijing) Co., Ltd., China;

[0075] Blunt-ended DNA polymerase Pfu (2×EasyPfu PCR SuperMix), a product of Beijing TransGenBiotech C...

Embodiment

[0083] In this example, taking Safflower Dajinyuan tobacco material as an example, the corresponding full-length transcription factor yeast two-hybrid library was constructed. The specific construction process is briefly described as follows.

[0084] (1) Screening of transcription factors and design of primers for full-length open reading frame amplification,

[0085] Based on the genome information of the target crop, screen full-length transcription factors and design PCR amplification primers for their corresponding full-length open reading frames; the specific screening principles and operating steps of full-length transcription factors are briefly introduced as follows.

[0086] First, transcription factors with incomplete ORFs were removed from those predicted in the genome. Specifically, using the transcription factors that have been reported in Arabidopsis, rice and other plants, based on the homologous comparison, the transcription factors in the genome of the common...

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Abstract

The invention belongs to the technical field of genetic engineering, in particular to a patent application matter of a yeast two-hybrid library construction method. A method for constructing a full-length transcription factor library of tobacco genome specifically comprises transcription factor screening and designing full-length amplification primers of an open reading frame, preparing a cDNA template, PCR amplification, phosphorylation treatment, linking a recombinant linker, constructing a library plasmid of full-length transcription factors, plasmid transformation and constructing a library, and that like. A method for construct a yeast two-hybrid library of full-length transcription factors is a further comprehensive improvement of existing construction methods, it has the advantagesof high positive screening rate of transcription factors, high screening accuracy and high quality of constructed libraries. Meanwhile, it can save construction time and construction cost because of the optimization of related operation steps, so it has good practical value and popularization significance.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and specifically relates to a patent application for a yeast two-hybrid library construction method. Background technique [0002] Transcription factor (transcription factor, TF), also known as trans-acting factor ( trans -actingelement), is a class of cis-acting elements that can specifically bind to eukaryotic promoter regions ( cis -actingelement) protein, whose function is to regulate the opening, closing and expression level of genes, and the spatiotemporal expression of transcription factors directly determines the physiological functions of plants such as growth and development, stress response and secondary metabolism. On the other hand, studies have found that there are also interactions between transcription factors, which can combine with each other to form complexes, directly bind to the promoters of regulatory genes, and jointly regulate the transcription levels of rela...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/81C12N15/29C12N15/10C12N1/19C40B50/06C40B40/02
CPCC07K14/415C12N15/1096C12N15/81C40B40/02C40B50/06C12Q2531/113C12Q2525/191
Inventor 罗朝鹏杨军谢小东张剑锋魏攀李锋武明珠王中李泽锋曹培健
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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