CN113174403B - Method for simultaneously over-expressing N miRNAs - Google Patents

Abstract

The invention discloses a method for simultaneously over-expressing N miRNAs, and belongs to the technical field of biology. The method comprises the following steps: s1, designing a PCR primer aiming at a target miRNA, and obtaining a precursor sequence fragment comprising a coding target miRNA stem-loop structure, a stem-loop structure front end 160-220 bp sequence and a stem-loop structure rear end 160-220 bp sequence through PCR amplification by taking genomic DNA as a template; s2, splicing the precursor sequence fragments of the N miRNAs to obtain a spliced sequence; s3, inserting the splicing sequence into a multiple cloning site of the expression plasmid to obtain an over-expression vector; s4, transferring the over-expression vector into a receptor material, and simultaneously over-expressing N miRNAs. The method of the invention enables a plurality of miRNAs to be expressed normally at the same time, can perform functional research on the miRNAs at the same time, and has important application value in realizing research on agronomic traits of miRNAs for regulating and controlling crops.

Description

Method for simultaneously over-expressing N miRNAs

Technical Field

The invention relates to the technical field of biology, in particular to construction and application of N miRNA simultaneous over-expression vectors.

Background

Plant microRNA (miRNA) is a widely existing sRNA with the length of 20-22 nt in plants, and a precursor gene Pri-miRNA is sheared by two steps of Dicer-liker 1 (DCL 1) to form Pre-miRNA and miRNA duplex respectively, wherein miRNA is degraded, and the other mature miRNA enters AGO protein to form RNA silencing complex (RNA-Induced Silencing Complexes, RISCs) to play a silencing target function.

Research shows that the plant conserved miRNA regulates plant growth and development and resists adversity stress through targeting various transcription factors and signal transduction genes. The effect of miRNA-RISCs on mRNA mainly depends on the complementation degree of complex and target gene transcription sequence, usually one miRNA can target a plurality of genes, and several miRNAs can also regulate one target gene at the same time, based on the complex regulation network, the expression of a plurality of genes is regulated by one miRNA, and the expression of a certain gene can also be finely regulated by the combination of several miRNAs.

Overexpression of the rice miRNA gene is one of important strategies for researching biological functions of the rice miRNA gene. The current common method is to clone the precursor sequence of the target miRNA into a binary vector driven by a specific promoter for transformation, so as to study the functions of the miRNA [ Chen Zhiwen, construction and genetic transformation of six miRNA expression vectors of rice, and 2013, 4 months ]. The method can only study a single rice miRNA, and can not study a plurality of miRNAs which can regulate the same biological process at the same time. The method has weaker expression effect in rice because the promoter commonly used in the vector is a 35S promoter, and has larger limitation on miRNA function research. There is therefore a need for improvements and innovative breakthroughs to the above deficiencies.

Disclosure of Invention

The invention aims to provide a method capable of simultaneously expressing a plurality of miRNAs, so that the plurality of miRNAs are simultaneously and efficiently overexpressed, and the method is used for researching functions of the plurality of miRNAs which have common response to a certain physiological process.

In order to achieve the above purpose, the invention adopts the following technical scheme:

a method for simultaneously over-expressing N miRNAs, wherein N is more than or equal to 2 and less than or equal to 4, comprises the following steps:

s1, designing a PCR primer aiming at a target miRNA, and obtaining a precursor sequence fragment comprising a coding target miRNA stem-loop structure, a stem-loop structure front end 160-220 bp sequence and a stem-loop structure rear end 160-220 bp sequence through PCR amplification by taking genomic DNA as a template;

s2, splicing the precursor sequence fragments of the N miRNAs to obtain a spliced sequence;

s3, inserting the splicing sequence into a multiple cloning site of the expression plasmid to obtain an over-expression vector;

s4, transferring the over-expression vector into a receptor material, and simultaneously over-expressing N miRNAs.

In step S1, PCR primers are designed for each target miRNA corresponding to the precursor genes, and the precursor gene fragments are obtained by utilizing a PCR amplification technology.

The research of the invention discovers that when a plurality of miRNA over-expression vectors are constructed, whether the plurality of miRNAs are successfully expressed at the same time can be realized, the selection of the length of a miRNA precursor sequence is crucial, the 160-220 bp sequence before and after the Pre-miRNA, namely the stem-loop structure is reserved, and the plurality of miRNAs are simultaneously expressed.

Preferably, in step S1, the precursor sequence encodes a target miRNA stem-loop structure, a stem-loop structure front end 190-210 bp sequence and a stem-loop structure rear end 190-210 bp sequence.

In step S2, precursor sequence fragments of N miRNAs are spliced by adopting a molecular biological technology means.

Preferably, the precursor sequence fragments of the N mirnas are spliced using overlap PCR techniques.

In the step S3, a spliced sequence is inserted into a multiple cloning site of an expression plasmid by adopting a molecular biology technical means, so as to obtain a binary vector of a promoter-spliced sequence-terminator. Specifically, the recombinant plasmid is constructed by adopting a double enzyme digestion connection technology, and the method is simple and easy to operate.

Preferably, the promoter of the expression plasmid is Ubi, and the plant promoter Ubi of the maize ubiquitin gene has higher expression activity in rice compared to the CaMV35S promoter. The terminator is a Nos terminator.

More preferably, the expression plasmid is a pCubi1390 binary vector, which is beneficial to subsequent transgenic experiments.

Further, the N miRNAs are at least 2 of rice miR-397a, miR-398a, miR-408 and miR-528.

Research shows that the 4 miRNAs are regulated and controlled in the same physiological path, so that expression vectors for simultaneously expressing the 4 miRNAs are constructed and applied to rice function research.

Preferably, the nucleotide sequence of the precursor sequence encoding miR-397a is shown as SEQ ID NO.1, the nucleotide sequence of the precursor sequence encoding miR-398a is shown as SEQ ID NO.2, the nucleotide sequence of the precursor sequence encoding miR-408 is shown as SEQ ID NO.3, and the nucleotide sequence of the precursor sequence encoding miR-528 is shown as SEQ ID NO. 4.

Specifically, the N miRNAs are rice miR-397a, miR-398a, miR-408 and miR-528, and the over-expression vector comprises a binary vector pCubi1390 and a splicing sequence, wherein the splicing sequence is inserted into a pCubi1390 multiple cloning site and has a nucleotide sequence shown in SEQ ID NO. 5.

The invention provides a method for simultaneously expressing rice miR-397a, miR-398a, miR-408 and miR-528, which comprises the steps of transferring the over-expression vector into receptor rice through agrobacterium-mediated over-expression vector, and culturing, wherein the 4 miRNAs are simultaneously over-expressed.

The invention has the beneficial effects that:

the invention splices precursor sequences of a plurality of miRNAs, connects connecting fragments into a binary vector, constructs an over-expression vector to realize transformation expression in plants, wherein the precursor sequence of each miRNA comprises a stem-loop structure of the miRNA, the expressed miRNA can play the function of the miRNA, and the precursor sequence also comprises 160-220 bp sequences before and after the stem-loop structure, so that the miRNAs can be normally expressed at the same time, and the functional study of the miRNAs can be carried out at the same time. The over-expression vector constructed by the method has important application value in the research of achieving miRNA regulation of agronomic traits of crops such as rice and the like.

Drawings

FIG. 1 shows the cloning of MIR397a, MIR398a, MIR408, MIR528 genes.

FIG. 2 is a plasmid map of the overexpression vector pCubi1390.

FIG. 3 is a flowchart showing the connection of MIR397a, MIR398a, MIR408 and MIR528 gene fragments.

FIG. 4 shows that MIR397a and MIR398a gene fragments were joined by overlap PCR, and MIR408 and MIR528 gene fragments were joined by overlap PCR.

FIG. 5 shows the integration of MIR397a, MIR398a, MIR408 and MIR528 gene fragments into one fragment by overlap PCR.

FIG. 6 shows that pCUbi1390-miRs overexpressing plasmids are transiently expressed in tobacco, and the expression of miR397a, miR398a, miR408 and miR528 is verified.

Detailed Description

The invention will be further illustrated with reference to specific examples, but the invention is not limited thereto.

The test methods used in the following examples are conventional methods unless otherwise specified; the materials, reagents and the like used, unless otherwise specified, are those commercially available.

Example 1

(1) Cloning of rice MIR397a (containing Pre-miR397a sequence), MIR398a (containing Pre-miR398a sequence), MIR408 (containing Pre-miR408 sequence), MIR528 (containing Pre-miR528 sequence).

PCR amplification primers are designed according to MIR397a, MIR398a, MIR408 and MIR528 sequences, as shown in table 1, rice Japanese sunny DNA is used as a template, and the MIR397a, MIR398a, MIR408 and MIR528 precursor genes are obtained through PCR amplification, and the nucleotide sequences are respectively shown as SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.

TABLE 1

MIR397a-F	TTCTAATTTTAGCGTGAACTAAACA
		MIR397a-R	GAATTGCTATGTGCAAACTACGGAG
MIR398a-F	ACATGGATGATCTTTTTCCTTCTCC
		MIR398a-R	GAATAAATATTACCCAAATTTATGA
MIR408-F	GGGCAAATTGGCAGGCTATGAGATG
		MIR408-R	GCCGAATGAAATGCATGTTTTGAGC
MIR528-F	GCTGAAAGCCATAGTACACTGATGA
		MIR528-R	GACATACCTGTAGACAGAATTAGTG

The PCR reaction system is as follows:

PCR reaction procedure: (1) 98 ℃ for 5min; (2) 15s at 98 ℃,15 s at 55 ℃,30 s at 72 ℃ and 30-35 cycles; (3) 10min at 72 ℃; (4) 4℃forever.

The cloning results are shown in FIG. 1.

(2) Rice MIR397a, MIR398a, MIR408, MIR528 gene fragments were ligated by overlapping PCR

After the PCR product (FIG. 1) in this example (1) was subjected to gel recovery and purification, the next overlapping PCR ligation was performed.

The specific operation is as follows:

(1) and designing bridging primers, determining the sequence of each target fragment and the complementary sequence thereof, and designing primers with joints to realize bridging of the front pre-miRNA fragment and the rear pre-miRNA fragment. If the base sequence of MIR397a with the end 25bp length is CTCCGTAGTTTGCACATAGCAATTC, MIR a and the front end 25bp length is ACATGGATGATCTTTTTCCTTCTCC, selecting a proper length overlapping fragment, reversely complementing the front 15 base sequences of the forward primer of the next gene, adding the sequence to the reverse primer of the previous gene, reversely complementing the front 15 base sequences of the reverse primer of the previous gene, and adding the sequence to the forward primer of the next gene.

(2) The cleavage site was added, and pCUbi-1390 vector, whose map is shown in FIG. 2, was used, and KpnI and SpeI were used as cleavage sites. The construction of the desired endonucleases was purchased from Thermo Scientific, the desired ligases from Takara, and the related reactions were performed using the cleavage and ligation systems recommended.

(3) MIR397a, MIR398a, MIR408, MIR528 were ligated using the overlap PCR method, and after obtaining the PCR product of this example (1), the PCR clone was subjected to the next overlap PCR experiment to integrate the four fragments into one sequence. First, MIR397a and MIR398a, MIR408 and MIR528 are joined, and MIR397a reverse primer is replaced by MIR397a-Rov, MIR398a forward primer is replaced by MIR398a-Fov; the reverse primer of MIR408 is replaced by MIR408-Rov, and the forward primer of MIR528 is replaced by MIR528-Fov, as shown in step (1) of FIG. 3, so that the sequence end of MIR397a and the sequence front end of MIR398a have the same sequence of 30 bp; the MIR408 sequence end and the MIR528 sequence front end have the same sequence of 30 bp. Performing a first overlap PCR, as shown in step (2) of FIG. 3, using 2.5. Mu.l each of the recovered MIR397a and MIR398a gel products added with the overlap fragment as a reaction substrate, and performing PCR amplification using MIR397 a-F-KpnI as a forward primer and MIR398a-Rov as a reverse primer; 2.5. Mu.l of each of the recovered MIR408 and MIR528 gel products to which the overlapping fragments were added was used as a reaction substrate, and PCR amplification was performed using MIR408-Fov as a forward primer and MIR 528-R-SpeI as a reverse primer. After the obtained overlapping PCR clone product was subjected to gel recovery, a second overlapping PCR was performed, and as shown in step (3) of FIG. 3, 2.5. Mu.l of each of the gel recovery products MIR397a+MIR398a and MIR408+MIR528, to which the overlapping fragments were added, was used as a reaction substrate, MIR397 a-F-KpnI was used as a forward primer, and MIR 528-R-SpeI was used as a reverse primer, and PCR amplification was performed, thereby obtaining MIR397a, MIR398a, MIR408, MIR528 ligation products having a total length of 2148 bp.

TABLE 2

Note that: in the primers shown in the list, bold bases represent the bridge sequence fragments of the front and rear gene fragments. MIR397 a-F-KpnI and MIR 528-R-SpeI primers were used to construct the vector with cleavage sites, GGTACC indicated as KpnI cleavage site and ACTAGT indicated as SpeI cleavage site.

The first overlap PCR reaction system was as follows:

PCR reaction procedure: (1) 98 ℃ for 5min; (2) 15s at 98 ℃,15 s at 55 ℃, 1min at 72 ℃ and 30-35 cycles; (3) 10min at 72 ℃; (4) 4℃forever.

The cloning results are shown in FIG. 4.

The second overlap PCR reaction system was as follows:

PCR reaction procedure: (1) 98 ℃ for 5min; (2) 15s at 98 ℃,15 s at 55 ℃, 2min at 72 ℃ and 30-35 cycles; (3) 10min at 72 ℃; (4) 4℃forever.

The cloning results are shown in FIG. 5.

(3) Construction of rice multi-miRNA simultaneous over-expression vector based on Ubi promoter

The PCR product (FIG. 5) in this example (2) was subjected to gel recovery and purification, and then the next overexpression vector was constructed. The pCUbi1390 vector (FIG. 2) was subjected to KpnI and SpeI double cleavage, respectively;

the enzyme digestion system is as follows:

and (3) enzyme cutting: 37℃for 15min.

The products after enzyme digestion are recovered and purified and then connected;

the connection system is as follows:

and (3) connection procedure: overnight at 4 ℃;

the ligation product is transformed into escherichia coli DH5 alpha, and amplified, and positive clone is obtained through colony PCR and sequencing screening, and the DNA sequence of the ligation product is shown as SEQ ID NO. 5.

(4) Simultaneous multi-miRNA (micro ribonucleic acid) over-expression vector function verification of rice in tobacco

After the pCubi-1390-miRs over-expression plasmid in the step (3) is obtained, agrobacterium (EHA-105) is transformed by using the pCubi-1390-miRs over-expression plasmid and the pCubi-1390 empty plasmid respectively to realize tobacco infection.

The agrobacterium-infected tobacco system is as follows:

the specific operation is as follows:

1) Amplifying and shaking the successfully detected agrobacterium tumefaciens bacteria liquid overnight at 28 ℃ and 200rpm;

2) Taking 5ml of bacterial liquid, and adding the bacterial liquid into a sterilized 10ml centrifuge tube;

3) Setting 5000g in a centrifuge for 5min, precipitating thalli (room temperature), discarding the supernatant, adding 10ml of penetrating fluid, and re-suspending the thalli;

4) Repeating step 3) to further remove a small amount of antibiotics;

5) Determining the titer of the suspension against the permeate, calculating the dilution factor such that the final suspension (for infestation) is 5ml, OD ₆₀₀ 0.4-0.6;

6) Before infection, putting the tobacco under a white fluorescent lamp for 1h, so that the air holes can be opened easily;

7) Selecting three inverted leaves and four inverted leaves for infection (infection between two veins), selecting two leaves for infection, infecting a bacterial liquid, marking a region to be transferred by using a marker pen, and returning the infected tobacco to a culture chamber;

8) After culturing for 48 hours, taking tissues of the infected part, extracting total RNA by using an RNA extraction reagent RNAiso Plus (Code No. 9108Q) of Takara company, detecting the expression condition of four miRNAs by using a stem-loop method, and the result is shown in figure 6.

Sequence listing

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Claims (4)

1. A method for simultaneously over-expressing N mirnas, wherein the N mirnas are at least 2 of rice miR-397a, miR-398a, miR-408, miR-528, and the method comprises the following steps:

s1, designing a PCR primer aiming at a target miRNA, taking rice genome DNA as a template, and performing PCR amplification to obtain a precursor sequence fragment comprising a stem-loop structure of the target miRNA, wherein the nucleotide sequence of a precursor sequence of the encoded miR-397a is shown as SEQ ID NO.1, the nucleotide sequence of a precursor sequence of the encoded miR-398a is shown as SEQ ID NO.2, the nucleotide sequence of a precursor sequence of the encoded miR-408 is shown as SEQ ID NO.3, and the nucleotide sequence of a precursor sequence of the encoded miR-528 is shown as SEQ ID NO. 4;

s2, splicing the precursor sequence fragments of the N miRNAs to obtain a spliced sequence;

s3, inserting the splicing sequence into a multiple cloning site of an expression plasmid, wherein a promoter of the expression plasmid is Ubi, and obtaining an over-expression vector;

s4, transferring the over-expression vector into a receptor material, and simultaneously over-expressing N miRNAs.

2. The method of simultaneously over-expressing N mirnas according to claim 1, wherein in step S2, the precursor sequence fragments of N mirnas are spliced using an overlap PCR technique.

3. The method of simultaneously over-expressing N mirnas according to claim 1, wherein the expression plasmid is pCUbi1390.

4. The method for simultaneously over-expressing N miRNAs according to claim 3, wherein the N miRNAs are rice miR-397a, miR-398a, miR-408 and miR-528, and the over-expression vector comprises a binary vector pCubi1390 and a splicing sequence, wherein the splicing sequence is inserted into a polyclone site of the pCubi1390 and has a nucleotide sequence shown in SEQ ID NO. 5.

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