CN110144365B - Plant microRNA expression vector, construction method and application - Google Patents
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Abstract
The invention relates to a construction method of a vector, in particular to a plant microRNA expression vector, a construction method and application thereof, wherein an F/R1 primer and an F/R2 primer are respectively used for cloning a target gene promoter sequence and a target gene promoter sequence containing a section of microRNA combined at the downstream, 35S sequences at the upstream of GFP on a pGFPGUSPlus vector are respectively replaced by a method of NcoI/NcoI double enzyme digestion and T4DNA ligase connection to construct two vectors, a 1# vector miRNA promoter drives GFP, a 2# vector microRNA combines the target gene promoter sequence to drive GFP, transgenic strains transformed by the two vectors are selected, GFP signals in a main root tip meristem region are observed, the GFP signals are contrastively analyzed, expression modes of the plant microRNA before and after regulation and control of the target genes can be clearly displayed, the operation steps of a conventional vector construction method are simplified, the cost is reduced, the working efficiency is improved.
Description
Technical Field
The invention relates to a construction method of a vector, in particular to a plant microRNA expression vector, a construction method and application.
Background
microRNAs (miRNAs) are one of the hot problems in epigenetic research and are widely involved in regulating and controlling the growth, development, stress tolerance and other life activities of plants. The function of the miRNAs mainly depends on the post-transcriptional regulation of the miRNAs on the related target genes, so the mechanism of regulating the related life activities by the miRNAs is analyzed, and the primary task is to determine the regulation mode of the miRNAs on the target genes in the related life activities.
At present, the research method aiming at the regulation and control mode of miRNAs on target genes mainly comprises the following steps: qRT-PCR, RNA in situ hybridization and the construction of a target gene/anti-shearing target gene and GFP reporter gene driven by a target gene promoter. Comparing the three methods, the qRT-PCR method is difficult to perform positioning analysis on the expression patterns of the cell level before and after transcription of the target gene; RNA in-situ hybridization can realize in-situ expression analysis of miRNAs and target genes thereof, but the operation is complicated and the workload is large; the conventional vector construction method can show the expression level of a target gene before and after transcription by comparing the signal of a reporter gene, but in terms of vector construction, a promoter of the gene, a gene coding sequence and a mutated coding sequence need to be cloned simultaneously, so that the workload is high, in addition, the influence of the protein coded by the gene on the spatial conformation of the reporter gene protein cannot be avoided, the constructed vector is easy to appear, and the signal of the reporter gene cannot be seen.
In conclusion, the prior art has the problems that the conventional vector construction method has complicated steps and large workload, and the constructed vector cannot see the signal of the reporter gene due to the influence of the protein of the gene on the spatial conformation of the reporter gene protein.
Disclosure of Invention
The invention simplifies the conventional vector construction method, and can display the expression modes of the target genes before and after transcription by observing the signal of the vector reporter gene.
The first purpose of the invention is to provide a construction method of a plant microRNA expression vector, which comprises the following steps:
designing a primer combination F/R1 aiming at the promoter sequence of the target gene, and cloning the promoter sequence of the target gene by using an F/R1 primer, wherein the sequence is shown as SEQ ID NO: 4, 36 th-2098 th site, performing sequencing verification, namely connecting the correctly sequenced amplified fragment to a pGFPGUSPlus vector by a method of NcoI/NcoI double enzyme digestion and T4DNA ligase connection, namely replacing a 35S sequence at the upstream of GFP on the pGFPGUSPlus vector by a cloned gene promoter sequence at a target, verifying the direction of connecting the fragment to the pGFPGUSPlus vector, wherein a plasmid which is positively connected to the pGFPGUSPlus vector is a successfully constructed 1# vector, and a target gene promoter drives GFP;
designing a primer combination F/R2 aiming at the downstream target gene promoter sequence containing the target microRNA, and cloning the downstream target gene promoter sequence containing the target microRNA by using an F/R1 primer, wherein the sequence is shown as SEQ ID NO: and 5, shown in positions 36-2119, connecting the correctly sequenced amplified fragment to a pGFPGUSPlus vector by a NcoI/NcoI double-enzyme digestion and T4DNA ligase connection method, namely replacing a 35S sequence at the upstream of GFP on the pGFPGUSPlus vector by a target gene promoter sequence containing a section of microRNA combination at the downstream, further verifying the direction of connecting the fragment to the pGFPGUSPlus vector, wherein the plasmid which is positively connected to the pGFPGUSPU vector is a successfully constructed 2# vector, and the target gene promoter containing the target gene sequence of microRNA recognition combination at the downstream drives GFP.
The second purpose of the invention is to provide the vectors 1# and 2# constructed by the construction method.
The third purpose of the invention is to provide an application of the vector in analyzing the regulation and control mode of plant microRNA on target genes of the plant microRNA.
Preferably, the plant is arabidopsis thaliana.
Preferably, the sequence of the primer F is shown as SEQ ID NO: 1 is shown in the specification; the sequence of the primer R1 is shown as SEQ ID NO: 2 is shown in the specification; the sequence of the primer R2 is shown as SEQ ID NO: 3, respectively.
Compared with the prior art, the invention has the following technical effects:
the invention simplifies the conventional vector construction method, can display the expression modes before and after transcription of the target gene by observing the vector reporter gene signal, greatly simplifies the operation steps, reduces the cost and improves the working efficiency; the vector constructed by the method can visually display the tissue cell expression level of the target gene before and after the regulation of the microRNAs, and provides a new method for the regulation mode research of the plant microRNAs on the target gene.
Drawings
FIG. 1 is a schematic diagram of a construction method in example 1 of the present invention (A: pGFPGUSPlus vector; B: a vector for analyzing a transcription level expression pattern of a target gene obtained after replacing 35S promoter with a target gene promoter; C: a sequence of a target gene to which a segment of miRNA is ligated after the target gene promoter by cleavage).
FIG. 2 shows the results of electrophoretic detection of promoter clones in the vectors constructed in example 1 of the present invention (in the figure, M is Mark band, 1-2 is fragment 1, and 3-4 is fragment 2).
FIG. 3 shows the results of electrophoresis detection of the ligation direction of the vector constructed in example 1 of the present invention (in the figure, M is Mark band, 1-5 are clone verification results of fragment 1, and 6-11 are clone verification results of fragment 2).
FIG. 4 shows the screening of transgenic seedlings obtained by transforming Arabidopsis thaliana with two vectors successfully constructed in example 2 of the present invention (A is 1# vector transformation, and B is 2# vector transformation).
FIG. 5 is a GFP signal observation chart of two vectors (A: 1# vector transformation, B: 2# vector transformation) in example 2 of the present invention.
Detailed Description
The present invention is described in detail below with reference to specific embodiments and drawings, but it should be understood that the scope of the present invention is not limited by the specific embodiments. The following examples are generally conducted under conventional conditions, and the materials are commercially available as the materials, and the steps thereof will not be described in detail since they do not relate to the invention.
Example 1
Construction method of plant microRNA expression vector
In the embodiment, a target gene MYB33 of an Arabidopsis miR159 is selected for vector construction (the construction method is shown in figure 1), an expression vector is a pGFPGUSPlus vector (purchased to Peptin biotechnology (Beijing) Co., Ltd.), and a MYB33 gene ATG upstream 2051bp sequence is selected for primer design;
the sequence of the primer F is shown as SEQ ID NO: 1, and the following components:
the sequence of the primer R1 is shown as SEQ ID NO: 2 is shown in the specification;
the sequence of the primer R2 is shown as SEQ ID NO: 3 is shown in the specification;
(1) cloning of the MYB33 promoter sequence
The genome DNA of arabidopsis is used as a template, F/R1 primer combination is used for amplification, and a PCR system comprises: mu.L of template DNA, 1. mu.L of each of the two primers, 10. mu. mol/L of each of the two primers, 5 XBuffer of 10. mu.L, and dNTPs of 2.5mmol/L, 4. mu.L of TransTaq HiFi DNA Polymerase and ddH2O is complemented to 50 mu L;
and (3) PCR reaction conditions: 5min at 94 ℃, 30s at 53 ℃ and 2min at 72 ℃, circulating for 35 times, and storing at 8min at 72 ℃ and 4 ℃.
(2) Cloning downstream target gene promoter sequence containing target microRNA
The genome DNA of arabidopsis is used as a template, F/R2 primer combination is used for amplification, and a PCR system comprises: mu.L of template DNA, 1. mu.L of each of the two primers, 10. mu. mol/L of each of the two primers, 5 XBuffer of 10. mu.L, and dNTPs of 2.5mmol/L, 4. mu.L of TransTaq HiFi DNA Polymerase and ddH2O is complemented to 50 mu L;
and (3) PCR reaction conditions: 5min at 94 ℃, 30s at 53 ℃ and 2min at 72 ℃, circulating for 35 times, and storing at 8min at 72 ℃ and 4 ℃.
Carrying out electrophoresis detection and sequencing verification on the amplification product, wherein the electrophoresis result is shown in figure 2, and the MYB33 promoter clone sequencing verification is shown in 36 th-2098 th position of SEQ ID NO.4 and is marked as a fragment 1; the MYB33 promoter is connected with a 21bp sequence clone sequencing verification that miR159 is sheared and MYB33 is shown in 36 th-2119 th positions of SEQ ID NO.5 and is marked as a fragment 2.
The amplified fragment whose sequencing was confirmed to be correct was ligated to pGFPGUSPlus vector by NcoI digestion and T4DNA ligase ligation.
In order to verify the direction of connecting the fragment 1 and the fragment 2 into pGFPGUSPlus vectors and screen out a forward inserted target vector, a pair of primers (a primer F2 is designed near the 3 'end of a MYB33 promoter, the sequence of which is shown in SEQ ID NO: 6, and a primer R3 is designed near the GFP 5', the sequence of which is shown in SEQ ID NO: 7) is designed, pGFPGUSPlus vector DNA connecting the fragment 1 and the fragment 2 is used as a template, and PCR screening verification is carried out, so that the clone capable of successfully amplifying the target-size fragment is the successfully constructed vector.
The PCR system is as follows: 1 μ L of template DNA, 1 μ L of each of the two primers, and 2 XTAQix and ddH each at a concentration of 10 μmol/L and 10 μ L2O is complemented to 20 mu L;
and (3) PCR reaction conditions: 5min at 94 ℃, 30s at 55 ℃, 40s at 72 ℃, circulating for 35 times, storing at 72 ℃ for 5min and 4 ℃, and carrying out electrophoresis detection and verification.
The verification result is shown in fig. 3, lanes 1 to 5 are the clone verification result of the 1# vector, wherein clones 1, 3 and 5 are all promoter positive insertions, and the successful 1# vector is constructed; lanes 6-11 show the clone verification results of the 2# vector, wherein clones 10 and 11 are both inserted with the promoter in the forward direction, i.e. the successful 2# vector is constructed.
Example 2
The 1# vector and the 2# vector are applied to analysis of the target gene regulation mode of the arabidopsis microRNA.
Respectively transforming Arabidopsis thaliana by using vectors successfully constructed in example 1 (successfully constructed vector 1 and vector 2), and screening by hygromycin to obtain transgenic Arabidopsis thaliana positive seedlings, wherein the result is shown in FIG. 4, further observing GFP signals of a meristematic region of a main root tip of a transgenic strain, as shown in FIG. 5, comparing and analyzing the GFP signals, the GFP signal of the vector 1 represents the expression of the transcription level of a target gene, the GFP signal of the vector 2 represents the expression of the target gene after being transcribed by miRNA shearing, and comparing the GFP signal difference of the transgenic lines of the two vectors can clearly show the expression pattern of MYB33 before and after the meristematic region of the root tip of Arabidopsis thaliana is regulated by miR 159.
The above embodiments are preferred embodiments of the present invention, but the present invention is not limited to the above embodiments, and any other changes, modifications, substitutions, combinations, and simplifications which do not depart from the spirit and principle of the present invention should be construed as equivalents thereof, and all such changes, modifications, substitutions, combinations, and simplifications are intended to be included in the scope of the present invention.
Sequence listing
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Claims (5)
1. A method for constructing a plant microRNA expression vector is characterized by comprising the following steps:
designing a primer combination F/R1 aiming at a promoter sequence of a target gene; cloning of the target gene promoter sequence with the F/R1 primer combination as shown in SEQ ID NO: 4, 36-2098, connecting the correctly sequenced amplified fragment to a pGFPGUSPlus vector by a method of NcoI/NcoI double enzyme digestion and T4DNA ligase connection, namely replacing a 35S sequence at the upstream of GFP on the pGFPGUSPlus vector by a cloned target gene promoter sequence, verifying the direction of connecting the fragment to the pGFPGUSPlus vector, and positively connecting the fragment to the pGFPGUSPP vector to obtain a successfully constructed 1# vector, wherein the target gene promoter drives GFP;
designing a primer combination F/R2 aiming at a downstream target gene promoter sequence containing a target microRNA; and cloning a target gene promoter sequence containing a target gene sequence which is identified and combined by microRNA at the downstream by using an F/R2 primer combination, wherein the target gene promoter sequence is shown as SEQ ID NO: and 5, shown in positions 36-2119, connecting the correctly sequenced amplified fragment to a pGFPGUSPlus vector by means of NcoI/NcoI double enzyme digestion and T4DNA ligase connection, namely replacing a 35S sequence at the upstream of GFP on the pGFPGUSPlus vector by using a target gene promoter sequence containing a target gene sequence combined with microRNA recognition at the downstream, further verifying the direction of connecting the fragment to the pGFPGUSPlus vector, wherein the vector connected to the pGFPGUSPU vector at the forward direction is a successfully constructed 2# vector, and the target gene promoter containing the target gene sequence combined with microRNA recognition at the downstream drives GFP.
2. The 1# and 2# vectors constructed according to the construction method of claim 1.
3. Use of the vector according to claim 2 for analyzing the regulation and control pattern of plant microRNAs on target genes thereof.
4. Use according to claim 3, wherein the plant is Arabidopsis thaliana.
5. The use of claim 4, wherein the primer F has the sequence shown in SEQ ID NO: 1 is shown in the specification; the sequence of the primer R1 is shown as SEQ ID NO: 2 is shown in the specification; the sequence of the primer R2 is shown as SEQ ID NO: 3, respectively.
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| WO2007044057A2 (en) * | 2005-02-24 | 2007-04-19 | The Ohio State University Research Foundation | Methods for quantifying microrna precursors |
| CN103173481A (en) * | 2013-03-01 | 2013-06-26 | 上海中医药大学附属普陀医院 | Plasmid vector containing 3'UTR (untranslated regions) sequence of ABCB1 gene and reporter gene as well as construction method and use of plasmid vector |
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