CN109266677B - Construction method of full-length transcription factor yeast two-hybrid library - Google Patents
Construction method of full-length transcription factor yeast two-hybrid library Download PDFInfo
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Abstract
The invention belongs to the technical field of genetic engineering, and particularly relates to a yeast two-hybrid library construction method, which is suitable for patent application. The method is used for constructing the full-length transcription factor library of the tobacco genome, and specifically comprises the following steps: screening transcription factors, designing an open reading frame full-length amplification primer, preparing a cDNA template, amplifying by PCR, carrying out phosphorylation treatment, connecting a recombinant joint, constructing library plasmids of the full-length transcription factors, transforming the plasmids, constructing a library and the like. The construction method of the full-length transcription factor yeast two-hybrid library is further comprehensive improvement of the existing construction method, has the advantages of high positive screening rate of the transcription factor, high screening accuracy, high quality of the constructed library and the like, and can save construction time and construction cost due to optimization of relevant operation steps, so that the construction method has good practical value and popularization and application significance.
Description
Technical Field
The invention belongs to the technical field of genetic engineering, and particularly relates to a yeast two-hybrid library construction method, which is suitable for patent application.
Background
Transcription Factor (TF), also known as trans-acting factor (F: (F))transActing element), a class of elements capable of specifically binding to cis-acting elements in the promoter region of eukaryotescisAction element) and the space-time expression of transcription factor directly determines the physiological functions of plant such as growth and development, adverse reaction and secondary metabolism. On the other hand, it has been found that there are interactions between transcription factors, which can bind to each other to form a complex, directly bind to the promoter of a regulatory gene, and co-regulate the transcription level of the relevant gene, thereby completing a complex physiological process (Chen D, et al. organic basic helix-loop-helix/bZIP transcription factors for transcription modules that are used for expressing the transcription light and reactive oxygen species signaling in the amplification of Plant cell 2013,25(5): 1657-73). However, in general, relatively few studies have been reported on the correlation between transcription factors.
The yeast two-hybrid (Y2H) system is a common technical method for screening unknown proteins (such as transcription factors) interacting with target proteins (such as transcription factor proteins) in yeast. The main construction process is as follows: constructing a bait vector (for expressing a known protein and DNA binding functional domain fusion protein, wherein the bait vector generally contains a reporter gene or is connected with the reporter gene during construction) and cDNA library plasmids (expressing cDNA and transcription activation functional domain fusion protein, wherein the cDNA library contains cDNA sequences of all proteins in a specific tissue and growth and development period), transferring the 2 plasmids into yeast, and if proteins expressed by the cDNA library plasmids contain proteins interacting with target proteins, starting the expression of the reporter gene so as to screen the proteins interacting with the target proteins. By using yeast two-hybrid technology, interactive transcription factors can be screened. However, when the yeast two-hybrid technology is used to screen the cDNA library containing all proteins for interacting transcription factors in the prior art, the main defects are that: the false positive rate of the screening result is too high, and the result accuracy is also very low (Mitsuda N, et al. effective year one-/two-hybrid screening using a lipid complex of transcription factors in Arabidopsis thaliana Plant and cell physiology 2010, 51(12): 2145-.
In the case of screening for transcription factors, most statistics show that transcription factors account for only a small proportion of all genes. For example, in Arabidopsis, the number of transcription factors is only about 5% of all genes (Riechmann JL, et al. Arabidopsis transcription factors: genome-wide compatible analysis of the same eukakyryotes. Science, 2000, 290: 2105. 2110); the proportion of Transcription Factors in Rice is less than that of all genes, only 2.6% (Xiong Y, et al. Transcription Factors in Rice: A Genome-wide Comparative Analysis between Monocots and Eudicots, Plant Mol Biol 2005, 59: 191-); while some internal statistics show that the proportion of transcription factors in tobacco is as low as only about 1% of all genes. In summary, since the number of other genes in cDNA library is much higher than that of transcription factor, this fact causes the false positive rate to be too high when screening and identifying transcription factor using yeast hybridization technique, and the result accuracy is also greatly reduced (Mitsuda N, et al. effective year one-/two-hybrid screening using a library complex of transcription factors. Plant and cell physiology, 2010, 51(12): 2145-.
In order to improve the efficiency of screening the transcription factors by using the yeast hybridization technology, a mode of independently establishing a transcription factor library is adopted in the construction process of the existing yeast hybridization system, namely, the constructed cDNA library only contains the transcription factors of specific species. For example: in Arabidopsis studies, transcription factor single/double hybrid libraries were created using gateway technology and successfully screened to demonstrate that transcription factor screening and identification using yeast transcription factor single/double hybrid libraries is highly Efficient (Mitsuda N, et al. effective year one-/two-hybrid screening using a library complex of transcription factors in Arabidopsis thaliana. Plant and Cell physiology, 2010, 51(12): 2145-.
When the gateway technology is used for constructing the transcription factor yeast single/double hybrid library by using a site-specific recombination mode, the main process is as follows: firstly, amplifying to obtain a cDNA sequence of a transcription factor; constructing transcription factor entry plasmid through first recombination (BP recombination) to form a primary library; and finally, constructing transcription factor single/double hybrid plasmids through second recombination (LR recombination) to form a transcription factor yeast single/double hybrid library. However, in practice, this technique also has some obvious defects, mainly: 1, 2 recombinases are used after 2 recombinations, and the recombinases are expensive, so that the cost of warehouse building is high; 2, the Gateway technology needs to be recombined twice, and is recombined once more, the homogenization effect can be offset, and due to competitive inhibition during bacteria shaking, part of low abundance and long fragment genes can be lost, so that the library quality is reduced. In conclusion, in order to obtain a transcription factor yeast single/double hybrid library with lower cost, higher efficiency and higher quality, further research and improvement are still necessary for the existing construction method.
Disclosure of Invention
The application aims to provide a construction method of a full-length transcription factor yeast two-hybrid library, so that the problems of high false positive rate and low accuracy when the existing constructed yeast two-hybrid library is used for screening and identifying transcription factors can be solved to a certain extent, and the problems of complex construction process, high cost, low library quality and the like in the existing construction process can be solved.
The construction method is mainly characterized in that the yeast two-hybrid library can be successfully constructed by 1-time recombination of the transcription factor cDNA, and the technical scheme of the application is summarized as follows.
A construction method of a full-length transcription factor yeast two-hybrid library specifically comprises the following steps:
(1) screening transcription factor and designing the full-length amplification primer of open reading frame,
screening full-length transcription factors and designing primers for PCR amplification on the corresponding full-length open reading frames according to the target crop genome information;
the specific screening principle and operation steps of the full-length transcription factor are as follows:
firstly, removing transcription factors with incomplete ORF from predicted transcription factors in genome;
second, screening for transcription factors expressed in at least one tissue or organ thereof;
thirdly, clustering the highly similar transcription factors to obtain a non-redundant transcription factor gene set;
fourthly, designing a primer, carrying out ePCR analysis on the obtained transcription factor primer based on the genome data, and screening a transcription factor with a unique product as a transcription factor for library construction;
finally, based on the operation, finally obtaining the full-length transcription factor, obtaining a corresponding full-length open reading frame according to the full-length transcription factor, and further designing a corresponding primer for PCR amplification for the full-length open reading frame;
when designing corresponding primers for PCR amplification for the full-length open reading frame, the following principles and requirements are specifically referred to:
first, primer design is performed on ORF of each transcription factor using software based on full length transcription factor cds (coding sequence) sequence;
secondly, the length of the primers is 18-28 bp, the annealing temperature is 55-65 ℃ during PCR amplification, the maximum annealing temperature difference between primer pairs is not more than 5 ℃, and the length of a final product is within the range of 200-3000 bp;
thirdly, in order to ensure the accurate reading frame, adding 1 base at the 5' end of the upstream primer; the specifically added base is determined according to the content of the primer, and the specific requirements are as follows: for GC contents less than 40%, C was added; an added T having a GC content of greater than 50%; c or T is randomly added in other cases; the downstream primer carries a stop codon;
(2) preparation of cDNA template
Extracting total RNA of a target crop sample, and performing reverse transcription to synthesize first-strand cDNA;
(3) PCR amplification
Carrying out PCR amplification by using blunt-end DNA polymerase to obtain a full-length open reading frame of the full-length transcription factor;
during PCR amplification, 2-round PCR amplification method is adopted, specifically:
a first round, wherein the first strand cDNA prepared in the step (2) is used as a template during the first round of PCR amplification; the specific reaction procedures and reaction conditions are designed by reference as follows:
first strand cDNA template, 1 μ L;
2×EasyPfu PCR SuperMix，12.5μL；
forward Primer (forward Primer, 10. mu.M), 1. mu.L;
reverse Primer (Reverse Primer, 10. mu.M), 1. mu.L;
ddH2O，9.5μL；
PCR procedure: 94 ℃ for 3 min; 94 deg.C, 30s, 56 deg.C, 30s, 72 deg.C, 2min, 25 cycles; 72 deg.C, 10min, 16 deg.C, 10 min;
a second round, wherein the first round amplification product is used as a template during the second round of PCR amplification; the specific reaction procedures and reaction conditions are designed by reference as follows:
first round PCR product, 2 μ L;
2×EasyPfu PCR SuperMix，25μL；
forward Primer (forward Primer, 10 μ M), 2 μ L;
reverse Primer (Reverse Primer, 10. mu.M), 2. mu.L;
ddH2O，19μL；
PCR procedure: 94 ℃ for 3 min; 94 ℃, 30s, 56 ℃, 30s, 72 ℃, 2min, 32 cycles; 72 deg.C, 10min, 16 deg.C, 10 min;
it should be noted that, in order to ensure the library construction effect and quality, the total product amount of each transcription factor should not be less than 1 μ g after 2 rounds of PCR;
(4) phosphorylation treatment
Carrying out 5' end phosphorylation on the PCR amplification product in the step (3); the specific phosphorylation system and reaction conditions can be designed by reference as follows:
PCR product mixture, 43. mu.L;
10×Blunting Kination Buffer，5μL
Blunting Kination Enzyme Mix，2μL
reaction conditions are as follows: 37 ℃ for 15 minutes, 70 ℃ for 5 minutes;
(5) connecting recombination joint
Connecting the PCR amplification product subjected to 5' end phosphorylation in the step (4) with a recombinant joint; the specific reaction system and reaction conditions can be designed by reference as follows:
10× DNA Ligation Buffer，5μL
5' Adapter，1μL
3' Adapter，1μL
phosphorylated PCR product from step (4), 2. mu.L
T4 DNA Ligase（NEB），1μL
Standing at 16 deg.C overnight, 65 deg.C for 10 min;
(6) construction of library plasmids of full-Length transcription factors
Recombining and connecting the 5' end phosphorylation PCR amplification product connected with the recombination joint in the step (5) with a yeast two-hybrid vector to construct a library plasmid of the full-length transcription factor;
the yeast two-hybrid vector is specifically exemplified by a linearized yeast two-hybrid vector pGADT 7;
(7) plasmid transformation and library construction
And (4) transforming the full-length transcription factor library plasmid constructed in the step (6) into an escherichia coli competent cell, and screening to obtain a full-length transcription factor yeast two-hybrid library.
According to the method for constructing the full-length transcription factor yeast two-hybrid library, the cDNA library adopted in the library construction process only comprises the transcription factor, so that the abundance of the transcription factor is high, and when the library is used for carrying out yeast two-hybrid experiment screening and identification on the transcription factor, the screening positive rate is high, and the result accuracy is greatly improved; on the other hand, in the construction process, a method of directly connecting a transcription factor PCR product with a recombinant joint is adopted, and finally only 1 time of recombination is needed to successfully construct the yeast two-hybrid library plasmid.
In general, the construction method of the full-length transcription factor yeast two-hybrid library is further comprehensive improvement of the existing construction method, and has the advantages of high screening rate of transcription factor positive, high screening accuracy, high quality of the constructed library and the like.
Drawings
FIG. 1 is an electrophoretic detection map of total RNA extracted from test tobacco material (Agilent 2100);
FIG. 2 is a diagram showing an example of the electrophoretic detection of the second round PCR amplification result of the transcription factor;
FIG. 3 is an electrophoretic detection of inserts when the library is amplified using vector primers;
FIG. 4 is an electrophoretic detection map of inserts when a library was amplified by randomly selecting 20 transcription factor primers.
Detailed Description
The technical solution of the present application is further explained with reference to the following embodiments, and before describing the specific embodiments, the following embodiments briefly describe the background of some experiments.
Biological material:
hong Hua Da jin Yuan (Chinese safflower)Nicotiana tabacum) One kind alwaysUsing tobacco varieties;
design and synthesis of related primers, which are provided by the synthesis of Beijing Liu-He Hua Dagen science and technology Limited;
a yeast two-hybrid vector pGADT7, supplied by Shanghai Biotechnology, Inc.;
experimental reagent:
RNA extraction Reagent (TRIzol. Reagent), Invitrogen, usa;
reverse transcription reagent (Reverse transcription M-MLV, recombination RNase Inhibitor, 10 mM dNTP mix, oligo (dT)18 Primer), Baozi physician technology (Beijing) Co., Ltd., China;
blunt-ended DNA polymerase Pfu (2 XEasypfu PCR SuperMix), product of Beijing Quanjin Biotechnology (TransGen Biotech) Ltd;
gel recovery Kit (Gel Purification Kit), product of Beijing codon Biotechnology Ltd;
experimental equipment:
PCR synthesizer, The Applied Biosystems Veriti 96, USA;
NanoDrop 2000 (for RNA concentration determination), Thermo Scientific, usa;
agilent 2100 Bioanalyzer (RNA integrity index, for RIN value determination), Agilent, usa;
GeneChip Scanner 30007G System (expression profiling chip analysis), Affymetrix, USA.
Examples
In this example, the safflower Hongda tobacco material is taken as an example, and a corresponding full-length transcription factor yeast two-hybrid library is constructed, and the specific construction process is briefly described as follows.
Screening transcription factor and designing the full-length amplification primer of open reading frame,
screening full-length transcription factors and designing primers for PCR amplification on the corresponding full-length open reading frames according to the target crop genome information; the specific screening principle and the operation steps of the full-length transcription factor are summarized as follows.
First, transcription factors with incomplete ORFs are removed from the predicted transcription factors in the genome. Specifically, transcription factors reported by plants such as arabidopsis thaliana and rice are used for predicting the transcription factors in the genome of the round golden circle of common tobacco safflower based on homologous comparison. Transcription factors with incomplete ORFs were removed according to the start codon and the stop codon. 4661 tobacco transcription factors were obtained.
Second, the transcription factor expressed in at least one tissue or organ thereof is selected. An Affymetrix Tobacco whole growth period expression profile chip (Tobacco Genome Array, Fromat 49-7875) is utilized to analyze expression profile data, and 3244 transcription factors expressed in at least one tissue of roots, stems, leaves and flowers are screened out according to the standard that the expression value is more than or equal to 4.
Thirdly, clustering the highly similar transcription factors to obtain a non-redundant transcription factor gene set. In operation, the ORTHOMCL V1.4 is used to cluster the highly similar transcription factors (-pi _ cutoff90, -pmatch _ cutoff 80), and based on the clustering result, a non-redundant transcription factor gene set is obtained, and 2266 transcription factors are in total.
Fourthly, designing a primer, carrying out ePCR analysis on the obtained transcription factor primer based on the genome data, and screening a transcription factor with a unique product as a transcription factor for library construction.
It should be noted that, when the ePCR analysis is performed on the primarily obtained transcription factor primers, the comparison conditions are that the similarity is greater than or equal to 80%, the coverage is greater than or equal to 80%, and then 1041 transcription factors with unique products are screened according to the ePCR experiment result.
Finally, based on the operation, finally obtaining the full-length transcription factor, obtaining a corresponding full-length open reading frame according to the full-length transcription factor, and further designing a corresponding primer for PCR amplification for the full-length open reading frame; when designing corresponding primers for PCR amplification for the full-length open reading frame, the following principles and requirements are specifically referred to:
first, Primer design was performed on the ORF of each transcription factor using Primer3 software based on the full-length transcription factor cds (coding sequence) sequence;
secondly, the length of the primers is 18-28 bp, the annealing temperature is 55-65 ℃ during PCR amplification, the maximum annealing temperature difference between primer pairs is not more than 5 ℃, and the length of a final product is within the range of 200-3000 bp;
thirdly, in order to ensure the accurate reading frame, adding 1 base at the 5' end of the upstream primer; the specifically added base is determined according to the content of the primer, and the specific requirements are as follows: for GC contents less than 40%, C was added; an added T having a GC content of greater than 50%; c or T is randomly added in other cases; the downstream primer carries a stop codon.
Finally, 1041 tobacco transcription factor genes and primers for PCR amplification thereof are obtained by screening.
The typical transcription factors obtained by partial screening and the corresponding specific primers for PCR amplification are listed as follows:
(1) the transcription factor Ntab _ TF180 is,
the specific primer sequence design during PCR amplification is as follows:
TF 180-forward primer, 5'-CATGTATCAACCAATTTCGACC-3',
TF 180-reverse primer, 5'-TTAACTGACTAATAGCTGCTCGCC-3';
during PCR amplification, the amplification product is 703bp (adding C base before the upstream amplification primer, so the amplification product has one more base than CDS sequence), and the CDS (coding sequence) sequence of the specific transcription factor Ntab _ TF180 comprises 702bp, as shown in SEQ ID NO.1, which is as follows:
ATGTATCAACCAATTTCGACCGAGCTACCTCCGACGAGTTTCAGTAGTCTCATGCCATGTTTGACGGATACATGGGGTGACTTGCCGTTAAAAGTTGATGATTCCGAAGATATGGTAATTTATGGGCTCTTAAGTGACGCTTTAACTGCCGGATGGACGCCGTTTAATTTAACGTCCACCGAAATAAAAGCCGAGCCGAGGGAGGAGATTGAGCCAGCTACGATTCCTGTTCCTTCAGTGGCTCCACCTGCGGAGACTACGACGGCTCAAGCCGTTGTTCCCAAGGGGAGGCATTATAGGGGCGTTAGGCAAAGGCCGTGGGGGAAATTTGCGGCGGAAATAAGGGACCCAGCTAAAAACGGCGCACGGGTTTGGCTAGGGACTTATGAGACGGCTGAAGAAGCCGCGCTCGCTTATGATAAAGCAGCTTACAGGATGCGCGGCTCCAAGGCTCTATTGAATTTTCCGCATAGGATCGGCTTAAATGAGCCTGAACCGGTTAGACTAACCGCTAAGAGACGATCACCTGAACCGGCTAGCTCGTCAATATCATCGGCTTTGGAAAATGGCTCGCCGAAACGGAGGAGAAAAGCTGTAGCGGCTAAGAAGGCTGAATTAGAAGTGCAAAGCCGATCAAATGCTATGCAAGTTGGGTGCCAGATGGAACAATTTCCAGTTGGCGAGCAGCTATTAGTCAGTTAA。
(2) the transcription factor Ntab _ T593,
the specific primer sequence design during PCR amplification is as follows:
TF 593-forward primer, 5'-CATGACGGACTATAGAATACCAAC-3',
TF 593-reverse primer, 5'-TCATCGCGATTCAGCAAT-3';
during PCR amplification, the amplification product is 1978bp (C base is added before the upstream amplification primer, so that the amplification product has one more base than CDS sequence), and the CDS (coding sequence) sequence of a specific transcription factor Ntab _ TF593 comprises 1977bp, as shown in SEQ ID NO.2, and the specific steps are as follows:
ATGACGGACTATAGAATACCAACGATGACTAATATATGGAGCAATACAACATCCGACGATAACATGATGGAAGCTTTTTTATCTTCTGATCCGTCGTCGTTTTGGGCCGGAACAAATACACCAACTCCACGGAGTTCAGTTTCTCCGGCGCCGGCGCCGGTGACGGGGATTGCCGGAGACCCATTAAAGTCGATGCCGTATTTCAACCAAGAGTCGCTGCAACAGCGACTCCAGACGTTAATCGACGGGGCTCGCGAAGCGTGGACTTACGCCATATTCTGGCAATCGTCTGTTGTGGATTTCGTGAGCCCCTCGGTGTTGGGGTGGGGAGATGGATATTATAAAGGAGAAGAAGACAAGAATAAGCGTAAAACGGCGGCGTTTTCGCCTGATTTTATTACGGAGCAAGAACACCGGAAAAAAGTTCTCCGGGAGCTGAATTCTTTAATTTCCGGCACACAAACTGGTGGTGAAAATGATGCTGTAGATGAAGAAGTAACGGATACTGAATGGTTTTTTCTGATTTCAATGACTCAATCGTTTGTTAACGGAAGCGGGCTTCCGGGCCTGGCTATGTACAGCTCAAGCCCGATTTGGGTTACTGGAAGAGAAAGATTAGCTGCTTCTCACTGTGAACGGGCCCGACAGGCCCAAGGTTTCGGGCTTCAGACTATGGTTTGTATTCCTTCAGCTAATGGTGTTGTTGAGCTCGGGTCAACTGAGTTGATATTCCAGAGCGCTGATTTAATGAACAAGGTTAAAATCTTGTTTGATTTTAATATTGATATGGGCGCGACTACGGGCTCAGGTTCGGGCTCATGTGCTATTCAGGCTGAGCCCGATCCTTCAACCCTTTGGCTTACGGATCCACCTTCCTCAGTTGTGGAAGTCAAGGATTCGTCGAATACAGTTCCTTCAAGTAATAGTAGTAAGCAACTTGTGTTTGGAAATGAGAATTCTGAAAATGTTAATCAAAATTCTCAGCAAACACAAGGATTTTTCACTAGGGAGTTGAATTTTTCCGAATATGGATTTGATGGAAGTAATACTAGGAGTGGAAATGGGAATGTGAATTCTTCGCGTTCTTGCAAGCCTGAGTCTGGCGAAATCTTGAATTTTGGTGATAGTACTAAGAGAAATGCTTCAAGTGCAAATGGGAGCTTGTTTTCGGGCCAATCGCAGTTCGGTCCCGGGCCTGCGGAGGAGAACAAGAACAAGAACAAGAAAAGGTCACCTGCATCAAGAGGAAGCAATGAAGAAGGAATGCTTTCATTTGTTTCGGGTGTGATCTTGCCAAGTTCAAACACGGGGAAGTCCGGTGGAGGTGGCGATTCGGATCATTCAGATCTCGAGGCTTCGGTGGTGAAGGAGGCGGATAGTAGTAGAGTTGTAGACCCCGAGAAGAGGCCGAGGAAACGAGGAAGGAAACCGGCTAACGGGAGAGAGGAGCCATTGAATCATGTGGAGGCAGAGAGGCAAAGGAGGGAGAAATTGAATCAAAGATTCTATGCACTTAGAGCTGTTGTACCAAATGTGTCAAAAATGGATAAAGCATCACTTCTTGGTGATGCAATTGCATTTATCAATGAGTTGAAATCAAAGGTTCAGAATTCTGACTCAGATAAAGATGAGTTGAGGAACCAAATTGAATCTTTAAGGAATGAATTAGCCAACAAGGGATCAAACTATACCGGTCCTCCACCGCCAAATCAAGATCTCAAGATTGTAGATATGGATATCGACGTTAAAGTCATCGGATGGGATGCTATGATTCGTATACAATCTAATAAAAAGAACCATCCAGCCGCGAGGTTAATGGCCGCTCTCATGGAATTGGACTTAGATGTGCACCATGCTAGTGTTTCAGTTGTCAACGAGTTGATGATCCAACAAGCGACAGTGAAAATGGGGAGCCGGCTTTACACGCAAGAGCAGCTTCGGATATCATTGACATCCAGAATTGCTGAATCGCGATGA。
(II) preparation of cDNA template
(1) The Trizol method is adopted to extract the total RNA of the tobacco sample, and the specific operation steps can refer to the following steps:
(1) taking 50mg of tobacco material, grinding into powder with liquid nitrogen, adding 1ml of Trizol, mixing uniformly by vortex, standing at room temperature for 5min, and fully cracking; centrifuging at4 deg.C and 12000g for 10min, and collecting supernatant;
(2) adding 200 μ L chloroform, shaking for 15s, standing at room temperature for 3 min; centrifuging at 12000g for 15min at4 deg.C;
(3) absorbing the upper-layer water phase into a new centrifuge tube, adding 0.5mL of isopropanol, uniformly mixing, and standing at room temperature for 10 min; centrifuging at4 ℃ for 10min at 12000g, discarding the supernatant, and precipitating RNA at the bottom of the tube;
(4) adding 1ml of 75% ethanol, gently oscillating the centrifugal tube, and suspending and precipitating; centrifuging at4 deg.C and 8000g for 5min, and discarding supernatant; drying for 5-10 min on a super clean bench;
with 50. mu.L RNase-free H2Dissolving an RNA sample by using O, and incubating for 5-10 min at 55-60 ℃;
(5) RNA concentration and RNA integrity index (RIN value) were determined.
Specifically, 4 tissue samples of Honghuadajinyuan are taken as materials, total RNA is extracted respectively, and RNA integrity indexes are detected, wherein the specific sample sampling conditions and detection results are shown in the following table:
the total RNA is sampled and subjected to electrophoretic detection, and the result is shown in figure 1, so that an electrophoretic band is clear and is not degraded; meanwhile, the RIN values are all over 8, which further indicates that the extracted RNA has good quality and completely meets the requirements of a library construction experiment.
(2) Reverse transcription to synthesize first strand cDNA
Mixing 4 RNA samples extracted in the step (1) in equal amount for reverse transcription to prepare first strand cDNA, and the specific steps are as follows:
first, a 30 μ L synthesis system was designed as follows:
total RNA, 4 μ g;
Oligo(dT)18 primer，5μL
RNase free H2O，up to 30μL；
rapidly placing on ice for 5min after 10min at 70 deg.C; instantaneous centrifugation, and continuing to design a reaction system as follows:
30 mu L of the denaturation solution in the last step;
5×M-MLV Buffer，10μL；
dNTP Mixture (10 mM each)，2.5μL；
RNase Inhibitor (40 U/μl)，1.25μL；
RTase M-MLV (200 U/μl)，5μL；
RNase free H2O，1.25μL；
cooling on ice after 42 ℃, 1h, 70 ℃ and 15min, and directly using for the subsequent PCR amplification.
(III) PCR amplification
Performing PCR amplification by using a blunt-end DNA polymerase by using the first-strand cDNA as a template to obtain a full-length open reading frame of the full-length transcription factor; during PCR amplification, 2-round PCR amplification mode is adopted for amplification, and the specific process is briefly described as follows.
A first round, wherein the first strand cDNA prepared in the step (2) is used as a template during the first round of PCR amplification; the specific reaction procedures and reaction conditions are designed by reference as follows:
first strand cDNA template, 2. mu.L;
2×EasyPfu PCR SuperMix，12.5μL；
forward Primer (forward Primer, 10. mu.M), 1. mu.L;
reverse Primer (Reverse Primer, 10. mu.M), 1. mu.L;
ddH2O，8.5μL；
PCR procedure: 94 ℃ for 3 min; 94 deg.C, 30s, 56 deg.C, 30s, 72 deg.C, 2min, 25 cycles; 72 deg.C, 10min, 16 deg.C, 10 min;
a second round, wherein the first round amplification product is used as a template during the second round of PCR amplification; the specific reaction procedures and reaction conditions were designed as follows:
first round PCR product, 2 μ L;
2×EasyPfu PCR SuperMix，25μL；
forward Primer (forward Primer, 10 μ M), 2 μ L;
reverse Primer (Reverse Primer, 10. mu.M), 2. mu.L;
ddH2O，19μL；
PCR procedure: 94 ℃ for 3 min; 94 ℃, 30s, 56 ℃, 30s, 72 ℃, 2min, 32 cycles; 72 deg.C, 10min, 16 deg.C, 10 min.
And (3) performing gel cutting purification on the PCR amplification products of the second round, and measuring the concentration, wherein the total amount of the PCR products of each transcription factor is not less than 1 mu g in order to ensure the library construction effect and quality.
Finally, the clone was successfully performed from 1041 transcription factors to 780 (the electrophoresis results of PCR amplification products of partial transcription factors are shown in FIG. 2).
(IV) phosphorylation treatment
Equivalently mixing PCR products of all transcription factors obtained by amplification in the step (three), and carrying out 5' end phosphorylation on DNA; the specific phosphorylation system and reaction conditions are designed as follows:
PCR product mixture, 43. mu.L;
10×Blunting Kination Buffer，5μL；
Blunting Kination Enzyme Mix，2μL；
reaction conditions are as follows: 37 ℃ for 15 minutes, 70 ℃ for 5 minutes; then 7.5. mu.L ammonium acetate, 125. mu.L ethanol, 14000rpm centrifugation for 10min, and 1ml 75% ethanol washing twice, dissolved in 42. mu.L water.
(V) connecting recombination joint
Connecting the PCR amplification product subjected to 5' end phosphorylation in the step (4) with a recombinant joint; when connecting, the linker sequence is designed as follows:
5' Adapter sequence：
5' -CAGATTACGCTCATATG-3'，
3' -GTCTAATGCGAGTATACp-5'；
3' Adapter sequence：
5' -TCATCTGCAGCTCGAGC-3'，
3' -AGTAGACGTCGAGCTCGp-5'；
the specific reaction system and reaction conditions are designed as follows:
10× DNA Ligation Buffer，5μL；
5' Adapter，1μL；
3' Adapter，1μL；
phosphorylated PCR product of step (4), 42. mu.L;
T4 DNA Ligase（NEB），1μL；
standing at 16 deg.C overnight, 65 deg.C for 10 min; the product was purified using an adsorption column (QIAGEN, MinElute PCR Purification Kit) to remove excess linker sequence and other impurities, and dissolved in 20. mu.L of water.
(VI) constructing library plasmid of full-length transcription factor
Recombining and connecting the 5' end phosphorylation PCR amplification product connected with the recombinant joint in the step (5) with a linearized yeast two-hybrid vector pGADT7 to construct a library plasmid of the full-length transcription factor;
in the specific operation, the recombinant reaction system is designed as follows:
the 5' end phosphorylation PCR amplification product connected with the recombinant joint in the step (five) is 14 mu L;
linearized yeast two-hybrid vector pGADT7 (300 ng/. mu.L), 2. mu.L;
5× In-Fusion HD Enzyme Premix（TaKaRa），4μL；
mixing, reacting at 50 deg.C for 60min, and reactingAdding 2 μ L of Proreinase K into the system, and keeping the temperature at 37 ℃ for 15 min; inactivating the recombinase at 75 ℃ for 10min, replenishing water to 100 mu L, and then sequentially adding: glycogen (20. mu.g/. mu.l), 1. mu.L, NH4OAc (7.5M), 50. mu.L, absolute ethanol, 375. mu.L;
mixing, centrifuging at-80 deg.C for over 1 hr, 4 deg.C at 16000rpm for 30min, and carefully removing supernatant;
adding 150 μ l 70% ethanol, washing for 1 time, centrifuging at 16000rpm for 3min at4 deg.C, repeating the above steps once to remove supernatant and avoid triggering DNA precipitation;
airing for 5-10 min at room temperature; resuspend DNA pellet with 10. mu.L DEPC water, blow-suck 30-40 times with gun, centrifuge for 2s instantly, collect, immediately put on ice.
(VII) plasmid transformation and library construction
Transforming the full-length transcription factor library plasmid constructed in the step (6) into an escherichia coli competent cell, and screening to obtain a full-length transcription factor yeast two-hybrid library; the specific operation is as follows:
first, a 1mm electric rotor (Bio-Rad) was pre-cooled for 30min at-20 ℃;
secondly, on ice, 5. mu.l of the ligation product (prepared in step (six)) and 100. mu.l of Escherichia coli DH10B competent cells were added to the pre-cooled electric rotor, mixed well and ice-cooled for 45 min;
thirdly, using an electric conversion instrument (Bio-Rad) to carry out electric shock, and quickly adding 2mL of SOC culture medium into the electric rotating cup after the electric shock;
fourthly, transferring the bacterial liquid to a new 15ml centrifuge tube, culturing for 1h at 37 ℃ and 250rpm to obtain the full-length transcription factor yeast two-hybrid library bacterial liquid;
taking out 10 μ L of the bacterial liquid for detection, adding glycerol to the residual liquid to a final concentration of 20%, and storing at-80 deg.C for later use.
(VIII) quality detection of the library constructed
Diluting 10 μ L of the bacterial liquid obtained in the step (VII) by 1000 times, taking 10 μ L of the bacterial liquid to coat an LB plate, culturing at 37 ℃ overnight, counting the number the next day, and calculating the library capacity. The library capacity CFU calculation method is as follows:
CFU/ml = (number of clones on plate/10. mu.l)X 1000 times X1X 103 μl；
Library total CFU = (CFU/ml) × library pool total volume (ml).
The counting and calculation results show that the library capacity of the full-length transcription factor library constructed in the embodiment is 6 multiplied by 106A total of 2ml, and thus a total of 1.2X 107CFU, reservoir capacity is high.
The 24 clones spread on the LB plate were randomly picked, further amplified by shake bacteria, plasmids were extracted, PCR amplification was performed using vector primers, and the electrophoresis detection chart of the amplification products is shown in FIG. 3. As can be seen, clear bands are amplified by 24 clones, which indicates that the positive rate of the library clone is close to 100%.
In order to further examine the quality of the constructed library, 20 of 780 transcription factors successfully cloned (see the following table specifically) were randomly selected and subjected to PCR amplification using the library plasmid as a template, and the electrophoresis results of the amplified products are shown in FIG. 4.
Randomly selected 20 transcription factors:
in FIG. 4, the amplification results of gene1-gene20 are shown from left to right, and it can be seen that 20 genes can amplify target bands in the library, and the size is correct, which indicates that the constructed tobacco transcription factor yeast two-hybrid library has high quality and can be completely used in the transcription factor yeast two-hybrid screening and identification experiments.
SEQUENCE LISTING
<110> Zhengzhou tobacco institute of China tobacco general Co
<120> construction method of full-length transcription factor yeast two-hybrid library
<130> none
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 702
<212> DNA
<213> Nicotiana tabacum
<400> 1
atgtatcaac caatttcgac cgagctacct ccgacgagtt tcagtagtct catgccatgt 60
ttgacggata catggggtga cttgccgtta aaagttgatg attccgaaga tatggtaatt 120
tatgggctct taagtgacgc tttaactgcc ggatggacgc cgtttaattt aacgtccacc 180
gaaataaaag ccgagccgag ggaggagatt gagccagcta cgattcctgt tccttcagtg 240
gctccacctg cggagactac gacggctcaa gccgttgttc ccaaggggag gcattatagg 300
ggcgttaggc aaaggccgtg ggggaaattt gcggcggaaa taagggaccc agctaaaaac 360
ggcgcacggg tttggctagg gacttatgag acggctgaag aagccgcgct cgcttatgat 420
aaagcagctt acaggatgcg cggctccaag gctctattga attttccgca taggatcggc 480
ttaaatgagc ctgaaccggt tagactaacc gctaagagac gatcacctga accggctagc 540
tcgtcaatat catcggcttt ggaaaatggc tcgccgaaac ggaggagaaa agctgtagcg 600
gctaagaagg ctgaattaga agtgcaaagc cgatcaaatg ctatgcaagt tgggtgccag 660
atggaacaat ttccagttgg cgagcagcta ttagtcagtt aa 702
<210> 2
<211> 1977
<212> DNA
<213> Nicotiana tabacum
<400> 2
atgacggact atagaatacc aacgatgact aatatatgga gcaatacaac atccgacgat 60
aacatgatgg aagctttttt atcttctgat ccgtcgtcgt tttgggccgg aacaaataca 120
ccaactccac ggagttcagt ttctccggcg ccggcgccgg tgacggggat tgccggagac 180
ccattaaagt cgatgccgta tttcaaccaa gagtcgctgc aacagcgact ccagacgtta 240
atcgacgggg ctcgcgaagc gtggacttac gccatattct ggcaatcgtc tgttgtggat 300
ttcgtgagcc cctcggtgtt ggggtgggga gatggatatt ataaaggaga agaagacaag 360
aataagcgta aaacggcggc gttttcgcct gattttatta cggagcaaga acaccggaaa 420
aaagttctcc gggagctgaa ttctttaatt tccggcacac aaactggtgg tgaaaatgat 480
gctgtagatg aagaagtaac ggatactgaa tggttttttc tgatttcaat gactcaatcg 540
tttgttaacg gaagcgggct tccgggcctg gctatgtaca gctcaagccc gatttgggtt 600
actggaagag aaagattagc tgcttctcac tgtgaacggg cccgacaggc ccaaggtttc 660
gggcttcaga ctatggtttg tattccttca gctaatggtg ttgttgagct cgggtcaact 720
gagttgatat tccagagcgc tgatttaatg aacaaggtta aaatcttgtt tgattttaat 780
attgatatgg gcgcgactac gggctcaggt tcgggctcat gtgctattca ggctgagccc 840
gatccttcaa ccctttggct tacggatcca ccttcctcag ttgtggaagt caaggattcg 900
tcgaatacag ttccttcaag taatagtagt aagcaacttg tgtttggaaa tgagaattct 960
gaaaatgtta atcaaaattc tcagcaaaca caaggatttt tcactaggga gttgaatttt 1020
tccgaatatg gatttgatgg aagtaatact aggagtggaa atgggaatgt gaattcttcg 1080
cgttcttgca agcctgagtc tggcgaaatc ttgaattttg gtgatagtac taagagaaat 1140
gcttcaagtg caaatgggag cttgttttcg ggccaatcgc agttcggtcc cgggcctgcg 1200
gaggagaaca agaacaagaa caagaaaagg tcacctgcat caagaggaag caatgaagaa 1260
ggaatgcttt catttgtttc gggtgtgatc ttgccaagtt caaacacggg gaagtccggt 1320
ggaggtggcg attcggatca ttcagatctc gaggcttcgg tggtgaagga ggcggatagt 1380
agtagagttg tagaccccga gaagaggccg aggaaacgag gaaggaaacc ggctaacggg 1440
agagaggagc cattgaatca tgtggaggca gagaggcaaa ggagggagaa attgaatcaa 1500
agattctatg cacttagagc tgttgtacca aatgtgtcaa aaatggataa agcatcactt 1560
cttggtgatg caattgcatt tatcaatgag ttgaaatcaa aggttcagaa ttctgactca 1620
gataaagatg agttgaggaa ccaaattgaa tctttaagga atgaattagc caacaaggga 1680
tcaaactata ccggtcctcc accgccaaat caagatctca agattgtaga tatggatatc 1740
gacgttaaag tcatcggatg ggatgctatg attcgtatac aatctaataa aaagaaccat 1800
ccagccgcga ggttaatggc cgctctcatg gaattggact tagatgtgca ccatgctagt 1860
gtttcagttg tcaacgagtt gatgatccaa caagcgacag tgaaaatggg gagccggctt 1920
tacacgcaag agcagcttcg gatatcattg acatccagaa ttgctgaatc gcgatga 1977
Claims (9)
1. A method for constructing a full-length transcription factor yeast two-hybrid library is characterized by being used for constructing a full-length transcription factor library of a tobacco genome, and specifically comprising the following steps of:
(1) transcription factor screening and open reading frame full-length amplification primer design
Screening full-length transcription factors and designing primers for PCR amplification on the corresponding full-length open reading frames according to the tobacco genome information of the target crops;
the specific screening principle and operation steps of the full-length transcription factor are as follows:
firstly, removing transcription factors with incomplete ORF from predicted transcription factors in genome; in the specific operation: predicting transcription factors in a tobacco safflower large-gold genome by using transcription factors reported in arabidopsis thaliana and rice based on homologous comparison; removing transcription factors with incomplete ORF according to the start codon and the stop codon;
second, screening for transcription factors expressed in at least one tissue or organ thereof; in the specific operation: analyzing expression profile data by using Affymetrix tobacco whole growth period expression profile chip, and screening out transcription factors expressed in at least one tissue of roots, stems, leaves and flowers according to the standard that the expression value is more than or equal to 4;
thirdly, clustering the highly similar transcription factors to obtain a non-redundant transcription factor gene set; during specific operation, highly similar transcription factors are clustered by using ORTHOMCL V1.4, and the clustering standard is as follows: -pi _ cutoff90, -pmatch _ cutoff 80; obtaining a non-redundant transcription factor gene set from the clustering result;
fourthly, designing a primer, carrying out ePCR analysis on the obtained transcription factor primer based on the genome data, and screening a transcription factor with a unique product as a transcription factor for library construction; when the obtained transcription factor primer is subjected to ePCR analysis, the comparison condition is that the similarity is more than or equal to 80 percent, and the coverage is more than or equal to 80 percent;
finally, based on the above operation, finally obtaining the full-length transcription factor;
(2) preparation of cDNA template
Extracting total RNA of a target crop sample, and performing reverse transcription to synthesize first-strand cDNA;
(3) PCR amplification
Carrying out PCR amplification by using blunt-end DNA polymerase to obtain a full-length open reading frame of the full-length transcription factor;
(4) phosphorylation treatment
Carrying out 5' end phosphorylation on the PCR amplification product in the step (3);
(5) connecting recombination joint
Connecting the PCR amplification product subjected to 5' end phosphorylation in the step (4) with a recombinant joint;
(6) construction of library plasmids of full-Length transcription factors
Recombining and connecting the 5' end phosphorylation PCR amplification product connected with the recombination joint in the step (5) with a yeast two-hybrid vector to construct a library plasmid of the full-length transcription factor;
(7) plasmid transformation and library construction
And (4) transforming and screening the full-length transcription factor library plasmid constructed in the step (6) to obtain a full-length transcription factor yeast two-hybrid library.
2. The method for constructing a full-length transcription factor yeast two-hybrid library according to claim 1, wherein in the step (6), the yeast two-hybrid vector is a linearized yeast two-hybrid vector pGADT 7.
3. The method for constructing a full-length transcription factor yeast two-hybrid library according to claim 1, wherein in the step (1), when designing corresponding primers for PCR amplification for the full-length open reading frame, the design principle and requirements are as follows:
firstly, primer design is carried out on ORF of each transcription factor based on the full-length transcription factor CDS sequence;
secondly, the length of the primers is 18-28 bp, the annealing temperature is 55-65 ℃ during PCR amplification, the maximum annealing temperature difference between primer pairs is not more than 5 ℃, and the length of a final product is within the range of 200-3000 bp;
thirdly, in order to ensure the accurate reading frame, adding 1 base at the 5' end of the upstream primer; the specifically added base is determined according to the GC content of the primer, and the specific requirements are as follows: for GC contents less than 40%, C was added; for GC contents greater than 50%, T is added; c or T is randomly added in other cases; the downstream primer carries a stop codon.
4. The method for constructing a full-length transcription factor yeast two-hybrid library according to claim 3,
for the transcription factor Ntab _ TF180, the primer sequence is designed as follows:
TF 180-forward primer, 5'-CATGTATCAACCAATTTCGACC-3',
TF 180-reverse primer, 5'-TTAACTGACTAATAGCTGCTCGCC-3';
for the transcription factor Ntab _ TF593, the primer sequence is designed as follows:
TF 593-forward primer, 5'-CATGACGGACTATAGAATACCAAC-3',
TF 593-reverse primer, 5'-TCATCGCGATTCAGCAAT-3'.
5. The method for constructing a full-length transcription factor yeast two-hybrid library according to claim 1, wherein in the step (3), 2-round PCR amplification mode is adopted:
a first round, wherein the first strand cDNA prepared in the step (2) is used as a template during the first round of PCR amplification;
a second round, wherein the first round amplification product is used as a template during the second round of PCR amplification;
after 2 PCR cycles, the total amount of products of each transcription factor should be not less than 1. mu.g.
6. The method for constructing the full-length transcription factor yeast two-hybrid library according to claim 1, wherein in the step (4), the phosphorylation system and the reaction conditions are as follows:
PCR product mixture, 43. mu.L;
10×Blunting Kination Buffer，5μL；
Blunting Kination Enzyme Mix，2μL；
reaction conditions are as follows: 37 ℃ for 15 minutes, 70 ℃ for 5 minutes.
7. The method for constructing a full-length transcription factor yeast two-hybrid library according to claim 1, wherein in the step (5), the specific reaction system and reaction conditions are as follows:
10× DNA Ligation Buffer，5μL；
5' Adapter，1μL；
3' Adapter，1μL；
phosphorylated PCR product of step (4), 2. mu.L;
T4 DNA Ligase（NEB），1μL；
16 ℃ overnight, 65 ℃ for 10 min.
8. The tobacco full-length transcription factor yeast two-hybrid library constructed by the method for constructing the full-length transcription factor yeast two-hybrid library according to any one of claims 1 to 7.
9. The tobacco full-length transcription factor yeast single hybrid library according to claim 8, comprising a transcription factor Ntab _ TF180 and a transcription factor Ntab _ TF593, wherein the base sequences of the transcription factors are respectively shown in SEQ ID No. 1-2.
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