Construction method of cDNA library for screening sticktight stress related genes

Abstract

The invention belongs to the technical field of biotechnology, and particularly relates to a construction method of a cDNA library for screening sticktight stress related genes. The method disclosed by the invention is scientific and reasonable in design, and comprises the following steps: firstly, extracting total RNA (Ribonucleic Acid) of a spanishneedles herb plant, and then purifying by using Oligo magnetic beads to obtain mRNA; after the mRNA is reversed into cDNA, amplification and homogenization are performed, and small fragments are removed, so that the size of cDNA fragments in the library is about 1kb; the method comprises the following steps: constructing spanishneedles herb cDNA (complementary deoxyribonucleic acid) onto a pYES2-NTB carrier through homologous recombination, and transforming escherichia coli by plasmids to obtain a spanishneedles herb cDNA library; carrying out quality identification on the library; according to the invention, through SMART library establishment, a cDNA library capable of screening sticktight stress related genes can be obtained, and the storage capacity and quality of the library are identified.

Description

Technical Field

[0001] The invention belongs to the technical field of biotechnology, and particularly relates to a method for constructing a cDNA library for screening Bidens pilosa adversity-related genes. Background Art

[0002] The construction of a cDNA library that can screen for adversity-related genes in Bidens pilosa has far-reaching significance, which is mainly reflected in the following aspects: 1) Understanding the response mechanism of adversity: By constructing a cDNA library and screening for adversity-related genes in Bidens pilosa, researchers can have a deeper understanding of the physiological and molecular response mechanisms of Bidens pilosa under adverse conditions. These adversities may include drought, salinity, high temperature, low temperature, heavy metal pollution, etc. Studying the expression and regulation of these genes will help reveal how Bidens pilosa adapts to and resists these adverse environments. 2) Discovering stress-resistant gene resources: The cDNA library contains a large amount of gene information expressed by Bidens pilosa under adverse conditions. Through screening and identification, gene resources with stress resistance can be discovered. These genes may encode proteins related to stress resistance, such as antioxidant enzymes, osmotic regulating substance synthases, etc. The discovery of these genes not only helps to understand the molecular basis of stress resistance, but also may provide valuable gene resources for the cultivation of stress-resistant crops. 3) Promoting crop genetic improvement: The screened stress-resistant genes can be introduced into other crops through genetic engineering technology to cultivate new varieties with stress resistance. This is of great significance for improving crop yield and quality, as well as responding to challenges such as global climate change and environmental pollution. The stress-resistant genes obtained through cDNA library screening provide new ways and possibilities for crop genetic improvement. 4) Promote biotechnology research: Constructing cDNA libraries and screening stress-related genes is an important technology in the field of biotechnology. The development of this technology will not only help promote research progress in related disciplines such as genetic engineering and molecular biology, but may also provide new ideas and methods for new drug development, bioreactor construction and other fields.

[0003] In the prior art, there is no cDNA library for screening adversity-related genes of Bidens pilosa. Therefore, the inventors hope to provide a method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa. Summary of the invention

[0004] The purpose of the present invention is to overcome the above-mentioned problems existing in traditional technologies and to provide a method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa.

[0005] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0006] The present invention provides a method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa L., comprising the following steps:

[0007] S1, extract total RNA from Bidens pilosa;

[0008] S2, Oligo magnetic beads to purify mRNA;

[0009] S3, synthesis of three-frame cDNA;

[0010] S4, cDNA homogenization and small fragment removal;

[0011] S5, double-stranded cDNA and pYES2-NTB homologous recombination;

[0012] S6, plasmid transformation into E. coli;

[0013] S7, library identification;

[0014] S8. Library plasmid extraction.

[0015] Furthermore, in step S1, the specific steps of extracting Bidens pilosa total RNA are as follows:

[0016] 1) Pre-cool the mortar, pestle and spoon with liquid nitrogen, grind the Bidens pilosa sample into powder in liquid nitrogen, transfer it to a centrifuge tube, add Buffer PR1 to the sample, immediately shake and mix, and centrifuge;

[0017] 2) Place the gDNA filter column in a collection tube, transfer the supernatant to the filter column, centrifuge, discard the gDNA filter column, and pipette the supernatant in the collection tube into a new centrifuge tube;

[0018] 3) Add 0.3-0.5 times the volume of the supernatant anhydrous ethanol to the filtrate and mix by pipetting;

[0019] 4) Place the RNA adsorption column in a collection tube and transfer the mixture to the column for centrifugation twice;

[0020] 5) Discard the waste liquid, put the column back into the collection tube, add Buffer PR2 to the column and centrifuge;

[0021] 6) Discard the waste liquid, put the column back into the collection tube, add Buffer PR3 to the column and centrifuge;

[0022] 7) Discard the waste liquid, put the column back into the collection tube, add anhydrous ethanol to the column and centrifuge;

[0023] 8) Discard the waste liquid, put the column back into the collection tube, centrifuge, discard the waste liquid, place the column at room temperature, and thoroughly dry the remaining rinse liquid;

[0024] 9) Transfer the column to a new centrifuge tube, add RNase-free ddH2O to the center of the adsorption membrane, let it stand at room temperature, and centrifuge;

[0025] 10) Collect the filtrate.

[0026] Furthermore, in step S2, the specific steps of purifying mRNA with Oligo magnetic beads are as follows:

[0027] 1) Dilute total RNA with RNase-free H2O in an RNase-free PCR tube;

[0028] 2) Pipette the magnetic beads and add them to the total RNA sample, pipette and beat to mix; place the sample in a PCR instrument to allow the mRNA to bind to the magnetic beads;

[0029] 3) Place the sample on a magnetic rack and remove the supernatant after the solution becomes clear;

[0030] 4) Remove the sample from the magnetic rack, add wash buffer and mix well, place it on the magnetic rack, and remove the supernatant after the solution becomes clear;

[0031] 5) Remove the sample from the magnetic stand, add Tris buffer to resuspend the magnetic beads, place in a PCR instrument, and elute the mRNA;

[0032] 6) Add binding buffer, pipette and mix well, and leave at room temperature to allow the mRNA to bind to the magnetic beads;

[0033] 7) Place the sample on a magnetic rack and remove the supernatant after the solution becomes clear;

[0034] 8) Remove the sample from the magnetic rack, add wash buffer and mix well, place it on the magnetic rack, and remove the supernatant after the solution becomes clear;

[0035] 9) Add RNase-free H2O, pipette to mix, and immediately place on a magnetic rack. After the solution is clarified, pipette the supernatant into a new RNase-free PCR tube.

[0036] Furthermore, in step S3, the specific steps of synthesizing the three-frame cDNA are as follows:

[0037] 1) Add reagents in the following ratios to a 0.2 ml PCR tube:

[0038]

[0039]

[0040] 2) React at 72°C for 3 min, immediately place on ice, and add reagents according to the following ratio:

[0041]

[0042] 3) react at 42°C for 90 min, then terminate at 70°C for 15 min;

[0043] 4) Cool to room temperature, add 1 μl RNase H, and react at 37°C for 30 min to obtain single-stranded cDNA:

[0044] 5) Primers P1, P2, and P3 are three-frame primers; add reagents in the following ratios in a 0.2 ml PCR tube:

[0045]

[0046] 6) Perform PCR according to the procedure.

[0047] Furthermore, in step S4, the specific steps of cDNA normalization and small fragment removal are as follows:

[0048] 1) Mix the following reagents in a 0.2 ml PCR tube:

[0049]

[0050]

[0051] 2) Divide the above solution into 4 equal parts and place them in 0.2 ml PCR tubes;

[0052] 3) Place the PCR tube in a PCR machine at 98°C for 2 minutes;

[0053] 4) Quickly transfer the PCR tube to the prepared PCR instrument and react at 68°C for 5 hours;

[0054] 5) DSN digestion;

[0055] 6) Prepare the following concentrations of DSN enzyme before hybridization:

[0056] Mix 1 l DSN storage buffer and 1 l DSN enzyme and mark it as 1 / 2DSN;

[0057] Mix 3 l DSN storage buffer and 1 l DSN enzyme and mark it as 1 / 4DSN;

[0058] Directly obtain 1 l DSN storage buffer and mark it as control;

[0059] 7) Preheat DSN master buffer at 68°C;

[0060] 8) Add the reagents in the following ratios and mix well.

[0061]

[0062] 9) React the four PCR tubes at 68°C for 25 min;

[0063] 10) Add 10 l 2×DSN stop buffer to each tube and mix well;

[0064] 11) Place at room temperature for 5 minutes and store at -20°C;

[0065] 12) PCR amplification after DSN digestion;

[0066] 13) Add reagents to four PCR tubes, mix well, and mark them as DSNP1, 1 / 2DSNP1, 1 / 4DSNP1, and controlP1 respectively; add reagents in the following ratios:

[0067]

[0068]

[0069] 14) Gently tap to mix all components, centrifuge briefly, and place in a PCR machine preheated at 95°C;

[0070] 15) Start PCR according to the program;

[0071] 16) Using Clontech CHROMA SPIN TM +TE-1000Columns for small fragment removal.

[0072] Furthermore, in step S5, the specific steps of homologous recombination of double-stranded cDNA and pYES2-NTB are as follows:

[0073] 1) Add the following reagents to a 0.2 ml PCR tube on ice:

[0074]

[0075] 2) Incubate at 37°C for 30 min and place on ice;

[0076] 3) Add the following reagents to a sterile 1.5 ml centrifuge tube:

[0077]

[0078] 4) After mixing, place at -20°C for 30 min, centrifuge at 13000 rpm for 15 min, and discard the supernatant;

[0079] 5) Add 1 ml of 75% ethanol to wash the precipitate, centrifuge at 8000 rpm for 2 min, and discard the supernatant;

[0080] 6) Repeat step 5) once;

[0081] 7) Allow to dry at room temperature for 5-10 min, add 10 μl TE buffer to dissolve the precipitate, and pipette up and down 60 times.

[0082] Furthermore, in step S6, the specific steps of transforming the plasmid into Escherichia coli are as follows:

[0083] 1) Pre-cool the electroporation cuvette on ice, mix the recombinant product and electroporation competent cells, and transfer them into the electroporation cuvette, taking care not to generate bubbles;

[0084] 2) Set the parameters of the electroporator, add liquid SOC medium immediately after electroporation, transfer to a new centrifuge tube, and culture at 37°C with shaking for 1 hour;

[0085] 3) Take 10 μl of bacterial solution and dilute it 10,000 times, then take 100 μl of it and spread it on LB plate containing Amp antibiotics, and invert and culture it at 37°C overnight; add glycerol to the remaining bacterial solution to a final concentration of 20%, which is the library bacterial solution.

[0086] Furthermore, in step S7, the specific sub-steps of library identification are as follows:

[0087] 1) Library titer (cfu / ml) = number of clones / 0.1ml×10000

[0088] Library capacity (cfu) = titer × bacterial volume

[0089] 2) Clone identification: randomly select 24 clones as templates and add reagents according to the following system:

[0090]

[0091] 3) Perform PCR according to the procedure.

[0092] Furthermore, in step S8, the specific steps of library plasmid extraction are as follows:

[0093] 1) Take the overnight cultured bacterial solution and dispense it into centrifuge tubes, centrifuge, discard the supernatant, and collect the bacterial cells;

[0094] 2) Add buffer RS ​​and resuspend the bacterial pellet by pipetting or vortexing;

[0095] 3) Add lysis buffer LB, gently invert upside down 6-8 times to fully lyse the bacteria, and leave at room temperature;

[0096] 4) Add binding solution BD, gently invert up and down 6-8 times to mix thoroughly, centrifuge for min, and collect the supernatant;

[0097] 5) Add 1 / 10 volume of endotoxin-free solution ER, invert to mix, and place on ice until the solution becomes clear or slightly turbid. In the process of ice bathing, invert and mix 2 to 3 times;

[0098] 6) Leave at room temperature, and when the solution becomes turbid again, invert and mix;

[0099] 7) Centrifuge at room temperature and separate the layers. The upper aqueous phase is DNA, and the lower blue oily phase is endotoxin.

[0100] 8) Collect the upper aqueous phase into a new centrifuge tube, add 0.5 times the volume of isopropanol, and mix thoroughly by inversion;

[0101] 9) Insert the DNA adsorption column E1 into the collection tube, add the pre-treated mixed solution obtained in step 8), centrifuge, and discard the waste liquid;

[0102] 10) Add deproteinized solution PL, centrifuge, and discard the waste liquid;

[0103] 11) Place the DNA adsorption column E1 back into the collection tube, add rinse solution W, centrifuge at room temperature, and discard the waste solution;

[0104] 12) Repeat step 11);

[0105] 13) Place the DNA adsorption column E1 back into the collection tube, centrifuge the empty column at room temperature to remove the remaining rinse solution W, and air dry at room temperature;

[0106] 14) Place the DNA adsorption column E1 into a new collection tube, add the elution solution to the center of the DNA adsorption column E1, place at room temperature, centrifuge, collect the filtrate, and store at -20°C, which is the library plasmid.

[0107] The beneficial effects of the present invention are:

[0108] The method of the present invention is scientifically and reasonably designed. First, the total RNA of the Bidens pilosa plant is extracted, and then the mRNA is purified by using Oligo magnetic beads; the mRNA is reversed into cDNA and then amplified and normalized, and small fragments are removed, so that the size of the cDNA fragments in the library is about 1 kb; the Bidens pilosa cDNA is constructed into a pYES2-NTB vector through homologous recombination, and the plasmid is transformed into Escherichia coli to obtain the Bidens pilosa cDNA library; and the quality of the library is identified; the present invention can obtain a cDNA library capable of screening Bidens pilosa adversity-related genes through SMART library construction, and the library capacity and quality are identified.

[0109] Of course, any product implementing the present invention does not necessarily need to achieve all of the above advantages at the same time. BRIEF DESCRIPTION OF THE DRAWINGS

[0110] In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the accompanying drawings required for describing the embodiments will be briefly introduced below. Obviously, the accompanying drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other accompanying drawings can be obtained based on these accompanying drawings without paying creative work.

[0111] Figure 1 This is a schematic diagram of the results of RNA extraction and electrophoresis detection;

[0112] M: Maker, 1-2: Bidens pilosa sample RNA;

[0113] Figure 2 is a schematic diagram of ds cDNA synthesis;

[0114] M: Maker, 1: P1-F / P4-R amplifies ds cDNA; 2: P2-F / P4-R amplifies ds cDNA; 3: P3-F / P4-R amplifies ds cDNA;

[0115] Figure 3 Schematic diagram of agarose gel electrophoresis detection homogenization and removal of small fragments;

[0116] M: Maker, 1: Purification results after mixing three ds cDNAs;

[0117] Figure 4 Schematic diagram of Escherichia coli colony counting;

[0118] Figure 5 Schematic diagram of library quality identification. DETAILED DESCRIPTION

[0119] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0120] This embodiment provides a method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa, comprising the following steps:

[0121] S1, extracting total RNA from Bidens pilosa;

[0122] S2, Oligo magnetic beads to purify mRNA;

[0123] S3, synthesis of three-frame cDNA;

[0124] S4, cDNA homogenization and small fragment removal;

[0125] S5, double-stranded cDNA and pYES2-NTB homologous recombination;

[0126] S6, plasmid transformation into E. coli;

[0127] S7, library identification;

[0128] S8. Library plasmid extraction.

[0129] In step S1, the specific steps of extracting Bidens pilosa total RNA are as follows:

[0130] 1) Pre-cool the mortar, pestle and spoon with liquid nitrogen, grind 100 mg of Bidens pilosa sample into powder in liquid nitrogen, transfer to a centrifuge tube, add 750 ul Buffer PR1 to the sample, and immediately shake to mix. Centrifuge at 12000 rpm for 5 min;

[0131] 2) Place the gDNA filter column in a collection tube, transfer the supernatant to the filter column, centrifuge at 12,000 rpm for 1 min, discard the gDNA filter column, and pipette the supernatant from the collection tube into a new centrifuge tube;

[0132] 3) Add 0.4 times the volume of the supernatant anhydrous ethanol to the filtrate and mix by pipetting;

[0133] 4) Place the RNA adsorption column in a collection tube, transfer the mixture to the column twice and centrifuge at 12000 rpm for 1 min;

[0134] 5) Discard the waste liquid, put the column back into the collection tube, add 650ul Buffer PR2 to the column and centrifuge at 12000rpm for 1min;

[0135] 6) Discard the waste liquid, put the column back into the collection tube, add 650ul Buffer PR3 to the column and centrifuge at 12000rpm for 1min;

[0136] 7) Discard the waste liquid, put the column back into the collection tube, add 650ul of anhydrous ethanol to the column and centrifuge at 12000rpm for 1min;

[0137] 8) Discard the waste liquid, put the column back into the collection tube, centrifuge at 12000 rpm for 2 min, discard the waste liquid, and place the column at room temperature for several minutes to completely dry the remaining rinse liquid;

[0138] 9) Transfer the column to a new centrifuge tube, add 100ul RNase-free ddH2O to the center of the adsorption membrane, let stand at room temperature for 2 minutes, and centrifuge at 12000rpm for 1 minute;

[0139] 10) Collect the filtrate and take 2ul for electrophoresis and concentration determination. The test results are as follows: Figure 1 shown.

[0140] In step S2, the specific steps of purifying mRNA with Oligo magnetic beads are as follows:

[0141] 1) In an RNase-free PCR tube, dilute 10ug of total RNA to 50ul with RNase-free H2O;

[0142] 2) Pipette 50ul of magnetic beads and add them to the total RNA sample, pipette and mix well; place the sample in a PCR instrument, react at 65°C for 5min and at 25°C for 5min to allow the mRNA to bind to the magnetic beads;

[0143] 3) Place the sample on a magnetic rack and remove the supernatant after the solution becomes clear;

[0144] 4) Remove the sample from the magnetic rack, add 200ul wash buffer and mix well, place it on the magnetic rack, and remove the supernatant after the solution is clarified;

[0145] 5) Remove the sample from the magnetic rack, add 50ul Tris buffer to resuspend the magnetic beads, place in a PCR instrument, react at 80℃ for 2min, and elute the mRNA;

[0146] 6) Add 50ul binding buffer, pipette and mix well, and leave at room temperature for 5 minutes to allow the mRNA to bind to the magnetic beads;

[0147] 7) Place the sample on a magnetic rack and remove the supernatant after the solution becomes clear;

[0148] 8) Remove the sample from the magnetic rack, add 200ul wash buffer and mix well, place it on the magnetic rack, and remove the supernatant after the solution is clarified;

[0149] 9) Add 10ul RNase-free H2O, mix by pipetting, react at 80℃ for 2min, and immediately place on a magnetic rack. After the solution is clarified, carefully pipette the supernatant into a new RNase-free PCR tube.

[0150] In step S3, the specific steps of the synthesis of the three-frame cDNA are as follows:

[0151] 1) Add reagents according to Table 1 in a 0.2 ml PCR tube:

[0152] Table 1 Components and dosage of reverse transcription system

[0153] Reaction Component per rxn mRNA (1ng-500ng) Xμl CDS III / 3'PCR Primer 1μl SMART IV Oligonucleotide 1μl <![CDATA[DEPC H2O]]> up to 5μl Total Volume (μl) 5μl

[0154] 2) React at 72°C for 3 min, immediately place on ice, and add reagents according to Table 2 below:

[0155] Table 2 Components and dosage of reverse transcription system

[0156] Reaction Component per rxn 5×First-Strand Buffer 2.0 l DTT(20mM) 1μl dNTP Mix (10mM) 1μl MMLV Reverse Transcriptase 1μl Total Volume (μl) 5μl

[0157] 3) react at 42°C for 90 min, then terminate at 70°C for 15 min;

[0158] 4) Cool to room temperature, add 1 μl RNase H, and react at 37°C for 30 min to obtain single-stranded cDNA:

[0159] 5) Add reagents according to Table 3 in a 0.2 ml PCR tube: (Primers P1, P2, and P3 are triple-frame primers)

[0160] Table 3 Components and dosages of cDNA amplification system

[0161]

[0162]

[0163] 6) Perform PCR according to the procedure in Table 4:

[0164] Table 4 PCR program

[0165]

[0166] After PCR, 2 μl was taken for electrophoresis detection. The test results were as follows: Figure 2 shown.

[0167] In step S4, the specific steps of cDNA normalization and small fragment removal are as follows:

[0168] 1) Mix the following reagents according to Table 5 in a 0.2 ml PCR tube:

[0169] Table 5 Components and dosage of hybridization system

[0170] Reaction Component per rxn ds cDNA (1 g) X l 2×Hybridization buffer 8μl <![CDATA[ddH2O]]> Xμl Total Volume (μl) 16μl

[0171] 2) Divide the above solution into 4 equal parts and place them in 0.2 ml PCR tubes;

[0172] 3) Place the PCR tube in a PCR machine at 98°C for 2 minutes;

[0173] 4) Quickly transfer the PCR tube to the prepared 68°C PCR machine for 5 hours;

[0174] 5) DSN digestion;

[0175] 6) Prepare the following concentrations of DSN enzyme before hybridization:

[0176] Mix 1 l DSN storage buffer and 1 l DSN enzyme and mark it as 1 / 2DSN;

[0177] Mix 3 l DSN storage buffer and 1 l DSN enzyme and mark it as 1 / 4DSN;

[0178] Directly obtain 1 l DSN storage buffer and mark it as control;

[0179] 7) Preheat DSN master buffer at 68°C;

[0180] 8) Add the reagents in order according to Table 6 and mix well.

[0181] Table 6 Components and dosage of digestion system

[0182]

[0183] 9) React the four PCR tubes at 68°C for 25 min;

[0184] 10) Add 10 l 2×DSN stop buffer to each tube and mix well;

[0185] 11) Place at room temperature for 5 minutes and store at -20℃.

[0186] 12) PCR amplification after DSN digestion;

[0187] 13) Add reagents in order according to Table 7 to 4 PCR tubes, mix well, and mark them as DSNP1, 1 / 2DSNP1, 1 / 4DSNP1, and controlP1 respectively;

[0188] Table 7 Components and dosages of the amplification system after digestion

[0189]

[0190] 14) Gently tap to mix all components, centrifuge briefly, and place in a preheated (95°C) PCR instrument;

[0191] 15) Start PCR according to the program in Table 8:

[0192] Table 8 PCR program

[0193]

[0194] 16) Using Clontech CHROMA SPIN TM +TE-1000 Columns to remove small fragments, and then perform electrophoresis. The results are as follows Figure 3 shown.

[0195] In step S5, the specific steps of homologous recombination between double-stranded cDNA and pYES2-NTB are as follows:

[0196] 1) Add the following reagents to a 0.2 ml PCR tube on ice:

[0197] Table 9 Components and dosages of homologous recombination system

[0198] Reaction Component per rxn ds cDNA 2 l pYES2-NTB 50-200ng 5×CE II Buffer 4μl Exnase II 2μl <![CDATA[ddH2O]]> Up to 20μl

[0199] 2) React at 37°C for 30 min and place on ice.

[0200] 3) Add the following reagents to a sterile 1.5 ml centrifuge tube according to Table 10:

[0201] Table 10 Components and dosage of nucleic acid precipitation system

[0202] Reaction Component per rxn The above products 20μl Nucleic acid precipitation aid 2μl 100% ethanol 55μl

[0203] 4) After mixing, place at -20°C for 30 min, centrifuge at 13000 rpm for 15 min, and discard the supernatant;

[0204] 5) Add 1 ml of 75% ethanol to wash the precipitate, centrifuge at 8000 rpm for 2 min, and discard the supernatant;

[0205] 6) Repeat step 5 once;

[0206] 7) Allow to dry at room temperature for 10 min, add 10 μl TE buffer to dissolve the precipitate, and pipette up and down 60 times.

[0207] In step S6, the specific steps of transforming the plasmid into Escherichia coli are as follows:

[0208] 1) Pre-cool a 2 mm electroporation cuvette on ice, mix 10 μl of the recombinant product and 50 μl of the electroporation competent medium, and transfer them into the cuvette, taking care not to generate bubbles;

[0209] 2) Set the parameters of the electroporator to U = 2.5 kV. Immediately after the electroporation, add 2 ml of liquid SOC medium and transfer to a new 15 ml centrifuge tube. Incubate at 37 °C with shaking for 1 h.

[0210] 3) Take 10 μl of bacterial solution and dilute it 10,000 times, then take 100 μl of it and spread it on LB plate containing Amp antibiotics, and invert and culture it at 37°C overnight. Add glycerol to the remaining bacterial solution to a final concentration of 20%, which is the library bacterial solution.

[0211] In step S7, the specific sub-steps of library identification are as follows:

[0212] 1) Library titer (cfu / ml) = number of clones / 0.1ml×10000

[0213] Library capacity (cfu) = titer × bacterial volume

[0214] The library capacity is as follows Figure 4 Shown: Escherichia coli colony plate, photographed after 12 hours, the dilution factor is 10,000, and the number of colonies on the plate is approximately 1,321.

[0215] 2) Clone identification: randomly select 24 clones as templates and add reagents according to Table 11

[0216] Table 11 Components and dosage of colony PCR system

[0217] Reaction Component per rxn <![CDATA[ddH2O]]> 8μl 2×PCR Mix 10μl pYES2-NTB-F 1μl pYES2-NTB-R 1μl Total Volume(μl) 20μl

[0218] 3) Perform PCR according to the procedure in Table 12:

[0219] Table 12 Colony PCR program

[0220]

[0221] After PCR, 10 μl was taken out for electrophoresis detection. The test results were as follows: Figure 5 shown.

[0222] In step S8, the specific steps of library plasmid extraction are as follows:

[0223] 1) Take 180 mL of overnight cultured bacterial solution and dispense it into a 50 mL centrifuge tube. Centrifuge at 10,000 rpm for 1 min, discard the supernatant, and collect the bacterial cells.

[0224] 2) Add 7.5 mL of buffer RS ​​and resuspend the bacterial pellet by pipetting or vortexing;

[0225] 3) Add 7.5 mL of lysis buffer LB, gently invert up and down 6-8 times to fully lyse the bacteria, and leave at room temperature for 4 minutes;

[0226] 4) Add 7.5 mL of binding solution BD, gently invert up and down 6-8 times to mix thoroughly, centrifuge at 10000 rpm for 10 min, and carefully collect the supernatant;

[0227] 5) Add 1 / 10 volume of the supernatant endotoxin-free solution ER (about 2.4 mL), invert to mix, and place on ice for 5 min until the solution becomes clear and transparent (or slightly turbid). During the ice bath, invert and mix 2-3 times;

[0228] 6) Leave at room temperature for 5 minutes, and when the solution becomes turbid again, mix by inversion;

[0229] 7) Centrifuge at 10,000 rpm for 10 min at room temperature to separate the layers. The upper aqueous phase is DNA, and the lower blue oily phase is endotoxin and other substances;

[0230] 8) Carefully collect the upper aqueous phase into a new centrifuge tube, add 0.5 times the volume of isopropanol (about 11 mL, self-prepared), and mix thoroughly by inversion;

[0231] 9) Place the DNA adsorption column E1 into a 50 mL collection tube, add the pretreatment mixture from step 8, centrifuge at 10,000 rpm for 1 min, and discard the waste liquid;

[0232] 10) Add 10 mL of deproteinized solution PL, centrifuge at 10,000 rpm for 1 min, and discard the waste liquid;

[0233] 11) Place the DNA adsorption column E1 back into the collection tube, add 10 mL of rinse solution W, centrifuge at 12000 rpm for 1 min at room temperature, and discard the waste solution;

[0234] 12) Repeat step 11);

[0235] 13) Place the DNA adsorption column E1 back into the collection tube, centrifuge the empty column at 12000 rpm at room temperature for 3 min to remove the residual rinse solution W, and air dry at room temperature for 3 min;

[0236] 14) Place the DNA adsorption column E1 into a new 50 mL collection tube, add 2 mL of elution buffer to the center of the DNA adsorption column E1, place at room temperature for 2 min, centrifuge at 10,000 rpm for 2 min, collect the filtrate, and store at -20°C, which is the library plasmid.

[0237] The preferred embodiments of the present invention disclosed above are only used to help explain the present invention. The preferred embodiments do not describe all the details in detail, nor do they limit the invention to specific implementation methods. Obviously, many modifications and changes can be made according to the content of this specification. This specification selects and specifically describes these embodiments in order to better explain the principles and practical applications of the present invention, so that those skilled in the art can understand and use the present invention well. The present invention is limited only by the claims and their full scope and equivalents.

Claims

1. A method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa, characterized in that: The steps include: S1, extracting total RNA from Bidens pilosa; S2, Oligo magnetic beads to purify mRNA; S3, synthesis of three-frame cDNA; S4, cDNA homogenization and small fragment removal; S5, double-stranded cDNA and pYES2-NTB homologous recombination; S6, plasmid transformation into E. coli; S7, library identification; S8. Library plasmid extraction.

2. The method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa L. according to claim 1, characterized in that: In step S1, the specific steps of extracting Bidens pilosa total RNA are as follows: 1) Pre-cool the mortar, pestle and spoon with liquid nitrogen, grind the Bidens pilosa sample into powder in liquid nitrogen, transfer it to a centrifuge tube, add Buffer PR1 to the sample, immediately shake and mix, and centrifuge; 2) Place the gDNA filter column in a collection tube, transfer the supernatant to the filter column, centrifuge, discard the gDNA filter column, and pipette the supernatant in the collection tube into a new centrifuge tube; 3) Add 0.3-0.5 times the volume of the supernatant anhydrous ethanol to the filtrate and mix by pipetting; 4) Place the RNA adsorption column in a collection tube and transfer the mixture to the column for centrifugation twice; 5) Discard the waste liquid, put the column back into the collection tube, add Buffer PR2 to the column and centrifuge; 6) Discard the waste liquid, put the column back into the collection tube, add Buffer PR3 to the column and centrifuge; 7) Discard the waste liquid, put the column back into the collection tube, add anhydrous ethanol to the column and centrifuge; 8) Discard the waste liquid, put the column back into the collection tube, centrifuge, discard the waste liquid, place the column at room temperature, and thoroughly dry the remaining rinse liquid; 9) Transfer the column to a new centrifuge tube, add RNase-free ddH2O to the center of the adsorption membrane, let it stand at room temperature, and centrifuge; 10) Collect the filtrate.

3. The method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa L. according to claim 1, characterized in that: In step S2, the specific steps of purifying mRNA with Oligo magnetic beads are as follows: 1) Dilute total RNA with RNase-free H2O in an RNase-free PCR tube; 2) Pipette the magnetic beads and add them to the total RNA sample, pipette and beat to mix; place the sample in a PCR instrument to allow the mRNA to bind to the magnetic beads; 3) Place the sample on a magnetic rack and remove the supernatant after the solution becomes clear; 4) Remove the sample from the magnetic rack, add wash buffer and mix well, place it on the magnetic rack, and remove the supernatant after the solution becomes clear; 5) Remove the sample from the magnetic stand, add Tris buffer to resuspend the magnetic beads, place in a PCR instrument, and elute the mRNA; 6) Add binding buffer, pipette and mix well, and leave at room temperature to allow the mRNA to bind to the magnetic beads; 7) Place the sample on a magnetic rack and remove the supernatant after the solution becomes clear; 8) Remove the sample from the magnetic rack, add wash buffer and mix well, place it on the magnetic rack, and remove the supernatant after the solution becomes clear; 9) Add RNase-free H2O, pipette to mix, and immediately place on a magnetic rack. After the solution is clarified, pipette the supernatant into a new RNase-free PCR tube.

4. The method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa according to claim 1, characterized in that: In step S3, the specific steps of the synthesis of the three-frame cDNA are as follows: 1) Add reagents in the following ratios into a 0.2 ml PCR tube: 2) React at 72°C for 3 min, immediately place on ice, and add reagents according to the following ratio: 3) react at 42°C for 90 min, then terminate at 70°C for 15 min; 4) Cool to room temperature, add 1 μl RNase H, and react at 37°C for 30 min to obtain single-stranded cDNA: 5) Primers P1, P2, and P3 are three-frame primers; add reagents in the following ratios in a 0.2 ml PCR tube: 6) Perform PCR according to the procedure.

5. The method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa L. according to claim 1, characterized in that: In step S4, the specific steps of cDNA normalization and small fragment removal are as follows: 1) Mix the following reagents in a 0.2 ml PCR tube: 2) Divide the above solution into 4 equal parts and place them in 0.2 ml PCR tubes; 3) Place the PCR tube in a PCR machine at 98°C for 2 minutes; 4) Quickly transfer the PCR tube to the prepared PCR instrument and react at 68°C for 5 hours; 5) DSN digestion; 6) Prepare the following concentrations of DSN enzyme before hybridization: Mix 1 l DSN storage buffer and 1 l DSN enzyme and mark it as 1 / 2DSN; Mix 3 l DSN storage buffer and 1 l DSN enzyme and mark it as 1 / 4DSN; Directly obtain 1 l DSN storage buffer and mark it as control; 7) Preheat DSN master buffer at 68°C; 8) Add the reagents in the following ratios and mix well. 9) React the four PCR tubes at 68°C for 25 min; 10) Add 10 l 2×DSN stop buffer to each tube and mix well; 11) Place at room temperature for 5 minutes and store at -20°C; 12) PCR amplification after DSN digestion; 13) Add reagents to four PCR tubes, mix well, and mark them as DSNP1, 1 / 2DSNP1, 1 / 4DSNP1, and controlP1 respectively; add reagents in the following ratios: 14) Gently tap to mix all components, centrifuge briefly, and place in a PCR machine preheated at 95°C; 15) Start PCR according to the program; 16) Using Clontech CHROMA SPIN TM +TE-1000Columns for small fragment removal.

6. The method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa according to claim 1, characterized in that: In step S5, the specific steps of homologous recombination between double-stranded cDNA and pYES2-NTB are as follows: 1) Add the following reagents to a 0.2 ml PCR tube on ice: 2) Incubate at 37°C for 30 min and place on ice; 3) Add the following reagents to a sterile 1.5 ml centrifuge tube: 4) After mixing, place at -20°C for 30 min, centrifuge at 13000 rpm for 15 min, and discard the supernatant; 5) Add 1 ml of 75% ethanol to wash the precipitate, centrifuge at 8000 rpm for 2 min, and discard the supernatant; 6) Repeat step 5) once; 7) Allow to dry at room temperature for 5-10 min, add 10 μl TE buffer to dissolve the precipitate, and pipette up and down 60 times.

7. The method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa according to claim 1, characterized in that: In step S6, the specific steps of transforming the plasmid into Escherichia coli are as follows: 1) Pre-cool the electroporation cuvette on ice, mix the recombinant product and electroporation competent cells, and transfer them into the electroporation cuvette, taking care not to generate bubbles; 2) Set the parameters of the electroporator, add liquid SOC medium immediately after electroporation, transfer to a new centrifuge tube, and culture at 37°C with shaking for 1 hour; 3) Take 10 μl of bacterial solution and dilute it 10,000 times, then take 100 μl of it and spread it on LB plate containing Amp antibiotics, and invert and culture it at 37°C overnight; add glycerol to the remaining bacterial solution to a final concentration of 20%, which is the library bacterial solution.

8. The method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa L. according to claim 1, characterized in that: In step S7, the specific sub-steps of library identification are as follows: 1) Library titer (cfu / ml) = number of clones / 0.1ml×10000 Library capacity (cfu) = titer × bacterial volume 2) Clone identification: randomly select 24 clones as templates and add reagents according to the following system: 3) Perform PCR according to the procedure.

9. The method for constructing a cDNA library for screening adversity-related genes of Bidens pilosa according to claim 1, characterized in that: In step S8, the specific steps of library plasmid extraction are as follows: 1) Take the overnight cultured bacterial solution and dispense it into centrifuge tubes, centrifuge, discard the supernatant, and collect the bacterial cells; 2) Add buffer RS ​​and resuspend the bacterial pellet by pipetting or vortexing; 3) Add lysis buffer LB, gently invert upside down 6-8 times to fully lyse the bacteria, and leave at room temperature; 4) Add binding solution BD, gently invert up and down 6-8 times to mix thoroughly, centrifuge for min, and collect the supernatant; 5) Add 1 / 10 volume of the supernatant of endotoxin-free solution ER, invert to mix, and place on ice until the solution becomes clear or slightly turbid. In the process of ice bathing, invert and mix 2 to 3 times; 6) Leave at room temperature, and when the solution becomes turbid again, mix by inversion; 7) Centrifuge at room temperature and separate the layers. The upper aqueous phase is DNA, and the lower blue oily phase is endotoxin. 8) Collect the upper aqueous phase into a new centrifuge tube, add 0.5 times the volume of isopropanol, and mix thoroughly by inversion; 9) Insert the DNA adsorption column E1 into the collection tube, add the pre-treated mixed solution obtained in step 8), centrifuge, and discard the waste liquid; 10) Add deproteinized solution PL, centrifuge, and discard the waste liquid; 11) Place the DNA adsorption column E1 back into the collection tube, add rinse solution W, centrifuge at room temperature, and discard the waste solution; 12) Repeat step 11); 13) Place the DNA adsorption column E1 back into the collection tube, centrifuge the empty column at room temperature to remove the remaining rinse solution W, and air dry at room temperature; 14) Place the DNA adsorption column E1 into a new collection tube, add the elution solution to the center of the DNA adsorption column E1, place at room temperature, centrifuge, collect the filtrate, and store at -20°C, which is the library plasmid.

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