CN111944885B - Cloning method of Pinus massoniana miRNA precursor gene - Google Patents
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Abstract
The invention discloses a cloning method of a masson pine miRNA precursor gene, belonging to the field of plant molecular biology. The cloning method of the masson pine miRNA precursor gene provided by the invention optimizes a PCR amplification system and PCR reaction conditions of the miRNA precursor gene respectively, controls the annealing temperature to 64-68 ℃, controls the cycle number to 34-35, changes the gel concentration, voltage and electrophoresis time of agarose gel electrophoresis, and realizes successful and efficient cloning of the masson pine miR172 and miR947 precursor gene. The method has strong specificity and high yield of target genes, has no base mismatching phenomenon in the key mature sequence region of precursor genes, and obtains the precursor gene sequence with the target gene sequence homology as high as 100 percent. Is favorable for further constructing a plant expression vector for cultivating transgenic plants, and has good application prospect in the aspects of agricultural and forestry crop genetic improvement and molecular breeding.
Description
Technical Field
The invention belongs to the field of plant molecular biology, and particularly relates to a cloning method of a masson pine miRNA precursor gene.
Background
Pinus massoniana lamb is a peculiar tree species in south China, has the characteristics of wide distribution, strong adaptability, quick growth, large application and the like, plays an important role in forestry production, and is a main raw material in the building material industry, the paper making industry, the chemical fiber industry, the rosin and turpentine chemical industry and the like at present. The expanding propagation mode of the masson pine is mainly carried out through sexual propagation, and people obtain genetically improved seeds in a large scale mainly by building a masson pine seed orchard. However, since 1980, many pinus massoniana clonal seed gardens built face the problems of long childbearing period, unbalanced growth amount of female globeflower and male globeflower, low flowering synchronization and the like, and are difficult to provide enough high-quality seeds for large-area afforestation. The study of the cauda equina floribunda law is one of the important contents of fine variety production research, the yield of pollen and seeds is improved, and greater economic benefits are expected to be created.
mirnas (micrornas) are a group of genome-encoded non-coding RNAs of about 20 to 23 nucleotides in length that are well conserved in species evolution, with mirnas found in plants, animals and fungi being expressed mostly in specific tissues and developmental stages. The tissue specificity and the time sequence of miRNA determine the function specificity of tissues and cells, and show that miRNA plays multiple roles in the regulation process of cell growth and development process. The miRNA is a pre-miRNA which is a miRNA precursor gene after the most original pri-miRNA is processed for one time, and the pre-miRNA is a mature miRNA with the length of about 20-23 nt after being subjected to enzyme digestion by Dicer enzyme. The MiR regulates the expression of plant genes mainly at the post-transcriptional level by mediating the cleavage or the inhibition translation of target gene mRNA, and participates in the biological processes of the morphogenesis, the growth and development of plant organs, the responsiveness to external environmental stress, and the like. With further advances in scientific research, mirnas have also been shown to play an irreplaceable role in plant flowering time regulation and floral organ development. According to the sequencing result in the laboratory, a large number of miRNA precursor genes are obtained from masson pine. A large number of previous researches find that miR172 participates in plant flowering development regulation, and overexpression of miR172 can promote plant flowering. Meanwhile, miR947 is identified in sequencing of the male and female cones sRNA of loblolly pine and Chinese pine, and can participate in the flowering and development regulation process of pine.
So far, no relevant report on the cloning of miRNA precursor genes in masson pine is found. The prior art reports a cloning method of a Pinus koraiensis miRNA precursor gene, and the cloning method of the Pinus koraiensis miRNA precursor gene disclosed by the prior art is used for cloning the Pinus koraiensis miRNA precursor gene, so that the cloning effect is unstable, a target gene cannot be stably cloned, and a primer dimer phenomenon exists. When the target gene is cloned, the single clone is selected through connection transformation, bacteria is shaken and then sent to a bacteria liquid to be sequenced by the department of Ongjingki biology company, and the phenomenon of base mismatching is found.
Disclosure of Invention
Aiming at the problems in the prior art, the technical problem to be solved by the invention is to provide a cloning method of a masson pine miRNA precursor gene, which is used for cloning the masson pine miRNA precursor gene.
In order to solve the technical problems, the technical scheme adopted by the invention is as follows:
a cloning method of a masson pine miRNA precursor gene comprises the following steps:
1) Taking masson pine genome DNA or cDNA as a template, and carrying out PCR amplification to obtain miRNA precursor genes;
2) Recovering and purifying miRNA precursor genes obtained in the step 1) by agarose gel electrophoresis;
3) Connecting the miRNA precursor gene obtained in the step 2) with a cloning vector, and then transforming and screening escherichia coli to obtain a positive strain;
4) And (4) culturing the positive strains obtained in the step 3) by shaking bacteria, recovering a carrier containing miRNA precursor genes, and carrying out PCR detection and sequencing analysis.
Further, in the PCR in step 1), the reaction conditions are as follows: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 55-68 ℃, 1min at 72 ℃ and 30-35 cycles; 7min at 72 ℃.
Further, in the PCR in step 1), the reaction conditions are as follows: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 64-68 ℃, 1min at 72 ℃ and 34-35 cycles; 7min at 72 ℃.
Further, the agarose gel electrophoresis described in step 2) has a gel concentration of 0.01 to 0.015g/mL.
Further, the voltage of the agarose gel electrophoresis in the step 2) is 150-180V, and the agarose gel electrophoresis time is 18-20min.
Further, the cloning Vector in step 3) is pMD19-T simple Vector.
The cloning method of the masson pine miRNA precursor gene is applied to cloning the masson pine miR172 precursor gene.
Further, the upstream and downstream primers used in the PCR amplification of the masson pine miR172 precursor gene are as follows:
miR172Prime F：5′-GCAGCATCATCACGATTCACA-3′，
miR172Prime R：5′-ATGCAGCATCATCAGGATTC-3′。
the cloning method of the Pinus massoniana miRNA precursor gene is applied to cloning of the Pinus massoniana miR947 precursor gene.
Further, the upstream and downstream primers used in the PCR amplification of the masson pine miR172 precursor gene are as follows:
miR947Prime F：5′-CGCAGCAGCAGATTCTGATAGA-3′，
miR947Prime R：5′-GAAACAGTAACAGATTCCGATGCA-3′。
compared with the prior art, the invention has the beneficial effects that:
1) The invention provides a cloning method of a masson pine miRNA precursor gene, which solves the problems and phenomena that the prior art has unstable cloning effect, can not stably clone a target gene and has primer dimer when cloning the masson pine miRNA precursor gene;
2) The invention respectively optimizes the PCR amplification system and the PCR reaction conditions of the Pinus massoniana miRNA precursor gene, the annealing temperature is controlled at 64-68 ℃, the cycle time is 34-35 times, the gel concentration, voltage and electrophoresis time of agarose gel electrophoresis are changed, the Pinus massoniana miRNA precursor gene is efficiently cloned, the precursor genes of Pinus massoniana miR172 and Pinus massoniana miR947 are obtained, and the Pinus massoniana miR172 and Pinus massoniana miR947 play an important role in the flowering development regulation process;
3) The method has strong specificity and high yield of the target gene, no base mismatching phenomenon exists in the key mature sequence region of the precursor gene, and the homology of the obtained precursor gene sequence and the target gene sequence is up to 100 percent. Is favorable for further constructing a plant expression vector for cultivating transgenic plants, and has good application prospect in the aspects of agricultural and forestry crop genetic improvement and molecular breeding.
Drawings
FIG. 1 is the agarose gel electrophoresis picture of miR172 precursor gene of Pinus massoniana cloned by using a Pinus alpina system, wherein M is MakerIII, and the total length of a target gene is 225bp;
FIG. 2 is the result of agarose gel electrophoresis of the precursor gene of Masson pine miR172 cloned in comparative experiment 1 in example 1, in which M is MakerIII;
FIG. 3 is the agarose gel electrophoresis results of the precursor gene of Masson pine miR172 cloned in comparative experiment 2 in example 1, wherein M is MakerIII;
FIG. 4 is the agarose gel electrophoresis results of the precursor gene of Masson pine miR172 cloned in comparative experiment 3 in example 1, wherein M is MakerIII;
FIG. 5 agarose gel electrophoresis image of the cloned masson pine miR947 precursor gene of example 2 and its comparative test, wherein M is MakerIII, and the total length of the target gene is 127bp.
Detailed Description
Materials, reagents and the like used in the following examples are commercially available unless otherwise specified. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.
Example 1: cloning of Pinus massoniana miR172 (miRNA 172) precursor gene (225 bp)
1. Template: extracting masson pine genome DNA and taking plant genome DNA as a template.
The sequences of the upstream primer and the downstream primer are respectively as follows:
miR172Prime F：5′-GCAGCATCATCACGATTCACA-3′
miR172Prime R：5′-ATGCAGCATCATCAGGATTC-3′
2. the reaction system (50. Mu.L) was: 5.0 μ L10 × LA PCR Buffer (Mg) 2+ Free)，5.0μL MgCl 2 (25mM)，8.0μL dNTP Mixture(each 2.5mM)，2.0μL 3′miR172Prime F(10μM)，2.0μL miR172Prime R(10μM)，1μL DNA(1μM)，0.5μL TakaRa LA Taq(5U/μL)26.5. Mu.L of nucleic-free Water. The PCR kit manufacturer is TaKaRa, and the model is No.: RR002A.
3. Reaction procedure: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 64 ℃, 1min at 72 ℃ and 35 cycles; 7min at 72 ℃.
4. Agarose gel electrophoresis:
(1) 0.013g/mL of agarose gel was prepared.
(2) Electrophoresis voltage 150V, electrophoresis time 20min.
The result is shown in FIG. 1, only 1 amplification band appears in the amplification effect, the band is clear and regular, the brightness is high, and the size is consistent with the expected size.
5. Ligation of purified fragments to cloning vectors
The objective DNA molecule was cloned using pMD19-T simple Vector from TaKaRa, and the ligation reaction system and procedure were slightly modified according to the instructions.
Reaction (5 μ L): 2.2. Mu.L of purified recovered PCR product, 0.3. Mu.L of pMD-19Simple vector, 2.5. Mu.L of Solution I. Reaction conditions are as follows: 30 minutes at 16 ℃;4 ℃ overnight.
6. Transformation of E.coli
1) Thawing freshly prepared or frozen E.coli TOP10 competent cells at-70 ℃ on ice;
2) Adding 5 μ L of the ligation product of the purified fragment and the cloning vector into 100 μ L of competent cells, gently mixing, and performing ice bath for about 30 min;
3) Heating in 42 deg.C water bath for 90sec, and rapidly placing on ice for 3-5min;
4) Adding 800 μ L LB liquid culture medium, shaking at 37 deg.C and 100rmp for 1h;
5) Centrifuging at 4000rmp for 3min, sucking off 800 μ L of culture medium at the upper layer, and mixing the rest bacteria solution;
6) Smearing the bacterial liquid on an LB screening culture plate containing Amp, and carrying out inverted culture at 37 ℃ for overnight;
7) Screening positive clones and sequencing analysis: selecting a single colony from a screening culture plate, inoculating the single colony in an LB liquid culture medium, and shaking the bacteria at 37 ℃ and 250rmp overnight; and directly carrying out PCR detection on the recombinant transformant by taking the overnight cultured bacterial liquid as a template.
Reaction system (20.0 μ L): 2.0 μ L10 XPCR Buffer (Mg) 2+ free)，1.5μL MgCl 2 (25 mM), 1.3. Mu.L dNTP mix (each 2.5 mM), 1.0. Mu.L miR172Prime F (10. Mu.M), 1.0. Mu.L miR172Prime R (10. Mu.M), 0.1. Mu.L bacterial suspension, 1.0. Mu.L rTaq, 12.1. Mu.L Milli-Q Water.
Reaction procedure: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 60 ℃, 1min at 72 ℃ and 28 cycles; 7min at 72 ℃.
The clone with positive bacteria liquid PCR detection is sent to the engine biotechnology company for sequencing identification, the obtained masson pine miR172 precursor gene sequence is shown as SEQ ID NO.1, the homology with the miR172 gene on the masson pine genome is 100%, and no base mismatch phenomenon exists in the key mature region of the precursor gene. The concentration of the PCR product after gel recovery and purification is higher than the requirement of a sequencing company.
In this example, a comparative experiment 1 was simultaneously conducted, wherein 14 gradients of annealing temperature between 55 ℃ and 68 ℃ were selected, namely 55 ℃, 56 ℃, 57 ℃, 8230, 68 ℃ and other conditions were not changed, and as a result, it was found that the PCR reaction effect was the best, the band was the most specific and bright, and the result is shown in FIG. 2, after recovery and sequencing, the homology with the target gene was 100%, and no base mismatch occurred in the key mature region of the precursor gene.
In this example, a comparative experiment 2 was also set up, and the cycle number was 30-35 times, i.e., 30, 31 \8230 \ 823035, and 6 different cycle numbers were used, and other conditions were not changed, and as a result, it was found that only when the cycle number was 34,35, the PCR reaction had the best effect, the band was most specific and bright, and the highest content of the target gene was shown in FIG. 3. (the 31-cycle results, while seemingly good, were not accurate in location).
This example sets up comparative experiment 3, agarose gel electrophoresis prepares agarose gels of 0.01g/mL, 0.012g/mL, 0.013g/mL, 0.014g/mL, 0.015g/mL, electrophoresis voltage selects 150V, 160V and 180V respectively, and other conditions are not changed, and the results are shown in FIG. 4. As a result, it was found that the effect of separating and purifying the desired gene by electrophoresis was the best only when the gel concentration of the agarose gel electrophoresis was 0.013g/mL and the electrophoresis voltage was 150V.
In this example, a comparative experiment 4 was set up, and an experiment (somanmann. Alpine pine miRNA171a functional study, university of south china) was performed using a alpine pine precursor cloning system, and the reaction system (50 μ L) was: 5.0. Mu.L 10 × Trans Taq HiFi Buffer I, 4.0. Mu.L dNTP mix (reach 2.5 mM), 1.5. Mu.L miR172Prime F (10. Mu.M), 1.5. Mu.L miR172Prime R (10. Mu.M), 1. Mu.L RT reaction product, 0.5. Mu.L Trans Taq DNA polymerase, 36.5. Mu.L Nuclear-free Water. Mixing, centrifuging instantly, setting on PCR instrument, pre-denaturing at 94 deg.C for 4min, denaturing at 94 deg.C for 30s, annealing at 55 deg.C for 30s, extending at 72 deg.C for 30s, and reacting for 35 cycles; extension for 10min at 72 ℃. After the PCR reaction is finished, the amplified PCR product is taken and then detected in 2% agarose gel electrophoresis, and then photographed in a gel imaging system, and the result is shown in FIG. 1. The comparative experiment shows that the cloning effect is unstable, the target gene cannot be stably cloned, and primer dimer phenomenon exists.
Example 2: cloning method of miR947 (miRNA 947) precursor gene (127 bp)
1. Template: extracting masson pine genome DNA and taking plant genome DNA as a template.
The upstream and downstream primer sequences are respectively
miR947Prime F：5′-CGCAGCAGCAGATTCTGATAGA-3′
miR947Prime R：5′-GAAACAGTAACAGATTCCGATGCA-3′
2. The reaction system (50. Mu.L) was: 5.0 μ L10 × LA PCR Buffer (Mg) 2+ Free)，5.0μL MgCl 2 (25 mM), 8.0. Mu.L dNTP mix (reach 2.5 mM), 2.0. Mu.L miR947Prime F (10. Mu.M), 2.0. Mu.L miR947Prime R (10. Mu.M), 1. Mu.L DNA (1. Mu.M), 0.5. Mu.L TakaRa LA Taq (5U/. Mu.L), 26.5. Mu.L Nuclear-free Water. The PCR kit manufacturer is TaKaRa, and the model is No.: RR002A.
3. Reaction procedure: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 64 ℃, 1min at 72 ℃ and 35 cycles; 7min at 72 ℃.
4. Agarose gel electrophoresis:
(1) 0.013g/mL of agarose gel was prepared.
(2) Electrophoresis voltage is 150V, and electrophoresis time is 20min.
The result is shown in FIG. 5, only 1 amplification band appears in the amplification effect, the band is clear and regular, the brightness is high, and the size is consistent with the expected size.
5. Ligation of purified fragments to cloning vectors
The objective DNA molecule was cloned using pMD19-T simple Vector from TaKaRa, and the ligation reaction system and procedure were slightly modified according to the instructions.
Reaction (5 μ L): mu.L of purified recovered PCR product, 0.3. Mu.L of pMD-19Simple vector, 2.5. Mu.L of Solution I. The reaction conditions are as follows: 30 minutes at 16 ℃;4 ℃ overnight.
6. Transformation of E.coli
1) Thawing freshly prepared or frozen-stored escherichia coli TOP10 competent cells at-70 ℃ on ice;
2) Adding 5 mu L of the connection product of the purified fragment and the cloning vector into 100 mu L of competent cells, gently mixing uniformly, and carrying out ice bath for about 30 min;
3) Thermally shocking in 42 deg.C water bath for 90sec, and rapidly placing on ice for 3-5min;
4) Adding 800 μ L LB liquid culture medium, shaking at 37 deg.C and 100rmp for 1h;
5) Centrifuging at 4000rmp for 3min, sucking off 800 μ L of culture medium at the upper layer, and mixing the rest bacteria solution;
6) Smearing the bacterial liquid on an LB screening culture plate containing Amp, and carrying out inverted culture at 37 ℃ for overnight;
7) Screening positive clones and sequencing analysis: selecting a single colony from a screening culture plate, inoculating the single colony in an LB liquid culture medium, and shaking the culture overnight at 37 ℃ and 250 rmp; and directly carrying out PCR detection on the recombinant transformant by taking the overnight cultured bacterial liquid as a template.
Reaction system (20.0 μ L): 2.0 μ L10 XPCR Buffer (Mg) 2+ free)，1.5μL MgCl 2 (25 mM), 1.3. Mu.L dNTP mix (each 2.5 mM), 1.0. Mu.L miR947Prime F (10. Mu.M), 1.0. Mu.L miR947Prime R (10. Mu.M), 0.1. Mu.L bacterial fluid, 1.0. Mu.L rTaq, 12.1. Mu.L Milli-Q Water.
Reaction procedure: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 60 ℃, 1min at 72 ℃ and 28 cycles; 7min at 72 ℃.
The clone with positive bacteria liquid PCR detection is sent to the Scirpus biotechnology company for sequencing identification, the obtained miR947 precursor gene sequence is shown as SEQ ID NO.2, the homology with the miR947 gene on the masson pine genome is 100%, and the phenomenon of base mismatch does not exist in the key mature region of the precursor gene. The concentration of the PCR product after gel recovery and purification is higher than the requirement of a sequencing company.
In this example, a comparative experiment was also set up, i.e., the agarose gel concentration was 0.01g/mL, the electrophoresis voltage was 180V, and the electrophoresis time was 15min, while the other PCR reaction parameters were unchanged. As a result, it was found that the brightness and the clarity of the electrophoretically amplified band of the comparative experiment were inferior to those of the agarose gel having a gel concentration of 0.013g/mL, an electrophoresis voltage of 150V and an electrophoresis time of 20min, and primer dimer appeared, and the results are shown in FIG. 5.
The miR172 is closely related to the flowering development of pinus massoniana, can play an important role in the regulation and control of pinus massoniana flowers and the flowering development and participates in the flowering development regulation and control network. The over-expression of miR172 can possibly cause the release of masson pine to be advanced, and plays an important role in improving the yield of masson pine. miR947 identified in sequencing of the strobilus sRNA of the loblolly pine and the pinus tabulaeformis shows that the miR947 can be involved in the control of the flowering and development of pine plants. Therefore, miR172 and miR947 have important theoretical and practical significance for further researching development of the red-tail pine flowers.
The foregoing description is intended to be illustrative rather than limiting, and it will be appreciated by those skilled in the art that many modifications, variations or equivalents may be made without departing from the spirit and scope of the invention as defined in the appended claims.
Sequence listing
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<120> method for cloning Pinus massoniana miRNA precursor gene
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Claims (6)
1. A cloning method of a masson pine miRNA precursor gene is characterized by comprising the following steps:
1) Taking masson pine genome DNA or cDNA as a template, and carrying out PCR amplification to obtain miRNA precursor genes;
2) Recovering and purifying miRNA precursor genes obtained in the step 1) by agarose gel electrophoresis;
3) Connecting the miRNA precursor gene obtained in the step 2) with a cloning vector, and then transforming and screening escherichia coli to obtain a positive strain;
4) Shake bacteria culture of the positive strain obtained in step 3), recovering the carrier containing miRNA precursor gene, carrying out PCR detection and sequencing analysis
The Pinus massoniana miRNAs are specifically Pinus massoniana miR172 and Pinus massoniana miR947;
the upstream and downstream primers used in PCR amplification of the masson pine miR172 precursor gene are as follows:
miR172 Prime F: 5'-GCAGCATCATCACGATTCACA-3'，
miR172 Prime R: 5'-ATGCAGCATCATCAGGATTC-3'；
the upstream and downstream primers used in PCR amplification of the masson pine miR947 precursor gene are as follows:
miR947 Prime F: 5'-CGCAGCAGCAGATTCTGATAGA-3' ，
miR947 Prime R: 5'-GAAACAGTAACAGATTCCGATGCA-3'。
2. the method for cloning a masson pine miRNA precursor gene according to claim 1, wherein the PCR in step 1) is performed under the following reaction conditions: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 55-68 ℃, 1min at 72 ℃ and 30-35 cycles; 7min at 72 ℃.
3. The method for cloning a masson pine miRNA precursor gene according to claim 2, wherein the PCR in step 1) is performed under the following reaction conditions: 3min at 94 ℃; 30sec at 94 ℃, 30sec at 64-68 ℃, 1min at 72 ℃ and 34-35 cycles; 7min at 72 ℃.
4. The method for cloning a masson pine miRNA precursor gene according to claim 1, wherein the gel concentration of the agar gel electrophoresis in step 2) is 0.01-0.015g/mL.
5. The method for cloning a masson pine miRNA precursor gene according to claim 1, wherein the voltage of the agar gel electrophoresis in the step 2) is 150-180V, and the time of the agar gel electrophoresis is 18-20min.
6. The method for cloning a masson pine miRNA precursor gene according to claim 1, wherein the cloning Vector in step 3) is pMD19-T simple Vector.
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