CN109486855B - Construction method and application of poplar secondary xylem protoplast circular RNA overexpression system - Google Patents

Abstract

The invention discloses a construction method and application of a poplar secondary xylem protoplast circular RNA overexpression system, and belongs to the technical field of genetic engineering. The construction method comprises the following steps: molecular biological verification of circular RNA; constructing a circular RNA overexpression recombinant plasmid; transforming poplar protoplast with PEG mediated recombinant plasmid; molecular biological detection of circular RNA overexpression in transformed recombinant plasmid protoplasts. The overexpression system can be applied to regulation and control of the expression condition of the host gene after transcription.

Description

Construction method and application of poplar secondary xylem protoplast circular RNA overexpression system

Technical Field

The invention belongs to the technical field of genetic engineering, and particularly relates to a construction method and application of a poplar secondary xylem protoplast circular RNA overexpression system.

Background

Circular RNA is a non-coding RNA which is widely noticed by scientists in recent years, and is also a latest research hotspot in the field of RNA. According to the difference of splicing sources of the circular RNA, the circular RNA is divided into circular RNA formed by circularizing an exon, circular RNA formed by circularizing an intron and exon-intron circular RNA obtained by reserving the intron in the middle of the circularized exon, and different from the traditional linear RNA (containing 5 'end and 3' end), the circular RNA is in a closed circular structure, is not influenced by RNA exonuclease, is more stable in expression, is not easy to degrade and has various varieties, and some circular RNA is 10 times higher than the corresponding linear RNA. Because the circular RNA has the characteristics of stability, miRNA cavernous body, universality, space-time specificity, conservation, tissue specificity and the like, the circular RNA can regulate and control important biological processes such as neurodevelopment, aging, tumor and the like, and is also predicted to be a novel biomarker in the future.

All life phenomena of organisms such as growth, senescence, diseases, aging, death and the like are related to genes, gene research is always a hotspot and key point of scientific research, and circular RNA is a special existence mode of genes. Circular RNA is ubiquitous in plants, and is frequently studied in Arabidopsis and rice, but is almost blank in forest trees. Based on the special properties of the circular RNA, in order to research the influence of the circular RNA on gene expression and post-transcriptional regulation, a genetic transformation method is needed to express the circular RNA in a large amount in cells, however, when the genetic transformation work of forests still stays on the bottleneck of low transformation efficiency and long period, the research on the regulation mechanism of the circular RNA by using the traditional genetic transformation system is very time-consuming and labor-consuming. In addition, the research of the circular RNA in animals is mature, the research of the circular RNA in plants is only on arabidopsis thaliana and rice for a long time, and the research on trees is very little, so that another shortcut is needed for researching the gene expression and the regulation and control after transcription of the circular RNA in the trees.

In order to explore the gene expression and post-transcriptional regulation of circular RNA in forest trees, a method capable of efficiently genetically transforming the circular RNA needs to be explored, so that a researcher needs to select a proper expression system and construct a proper expression framework according to the success rate of the expression system and the deep knowledge of the gene expression regulation rule. To date, no reports have been found on the overexpression of the circular RNA system in forest trees, and the methods used in this study are more advanced:

verifying the authenticity of the circular RNA by adopting a two-step method, digesting linear transcripts by RNase R, enriching circular transcripts as a first step, and verifying the head-to-tail connection of circular RNA sequences at a Back-splicing junction by adopting a first-generation sequencing method as a second step;

the DNA of the natural linear host gene is used as a template to construct a vector, so that the manual connection work of multiple genes and complex enzyme cutting sites is reduced;

the 35S promoter is adopted to drive a final vector of the target gene and GFP, and the expression of the target gene and GFP is strongly started; the direct, rapid and stable protoplast method is used for transformation, and the success of the over-expression system of the circular RNA is preliminarily judged by observing the fluorescence efficiency of GFP;

in order to solve the problems of low genetic transformation efficiency and long period, the method for transforming the protoplast can achieve stable expression only by culturing for 12 hours, and then researches the influence of over-expressed circular RNA on gene expression and post-transcriptional regulation. The method realizes the establishment of a circular RNA overexpression system in the protoplast of the forest tree species (poplar secondary xylem) for the first time, has the efficiency far higher than that of a common genetic transformation system, and can specifically research the expression regulation and control of the circular RNA on the host gene at the transcription level and the post-transcription level.

Disclosure of Invention

The invention aims to provide a construction method and application of a poplar secondary xylem protoplast circular RNA overexpression system.

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

a method for constructing a poplar secondary xylem protoplast circular RNA overexpression system comprises the following steps:

(1) molecular biological verification of circular RNA: designing back-to-back primers of the corresponding circular RNA genes according to the circular RNA gene sequence obtained by transcriptome sequencing, carrying out PCR amplification and sequencing, and verifying the authenticity of the circular RNA genes through experiments;

(2) construction of circular RNA overexpression recombinant plasmid: cloning a poplar circular RNA host gene by PCR, taking the circular RNA host gene obtained by PCR cloning as a target gene, and constructing a circular RNA over-expression recombinant plasmid by using a Gateway method;

(3) transforming poplar secondary xylem protoplast by PEG mediated circular RNA overexpression recombinant plasmid:

(4) molecular biological detection of circular RNA overexpression in transformed recombinant plasmid protoplasts.

Furthermore, the sequence of the circular RNA gene obtained by the transcriptome sequencing is shown as SEQ ID NO.1 to SEQ ID NO. 13.

Further, the back-to-back primers are: based on the corresponding circular RNA gene sequence obtained by transcriptome sequencing, back-to-back primers are designed, and the specific sequences of the primers are shown as SEQ ID NO. 14-SEQ ID NO. 39.

Further, the PCR cloning of poplar circular RNA host genes is as follows: the poplar Genomic DNA was used as template, the "back-to-back" primers of the corresponding circular RNA genes were used as primers, and the poplar circular RNA host genes were PCR cloned using high fidelity Taq enzyme (Novogene, No. NHP007L, China).

Further, the construction of the circular RNA overexpression recombinant plasmid by the Gateway method comprises the following steps: construction of a band comprising endogenous flanking sequences upstream and downstream of the host GeneattB-A circular RNA overexpression framework of a linker sequence; pDONR207 was used as the entry vector for Gateway cloning; with pUC19-35s-sGFP (Chen)et al, 2011; Lin et al, 2013; Lin et al2014) is a final vector;

furthermore, the circular RNA gene of Potri.004G162600 is used as the host gene and comprises the band of the endogenous flanking sequence at the upstream and the downstream of the host geneattB-The primer sequences of the joint are shown as SEQ ID NO 40 and SEQ ID NO 41.

Further, the PEG mediated recombinant plasmid for transforming the poplar secondary xylem protoplast comprises the following steps:

(1) separating protoplasts: adding 40 mL of cell wall enzymolysis liquid into 2 g of the secondary xylem stem segments of the populus trichocarpa, and performing enzymolysis for 3 h at room temperature in a dark place; centrifuging for 3 min at 500 g, discarding the upper layer enzymolysis solution, adding 30 mL MMG solution into the precipitate, and slightly shaking back and forth for 1.5 min to release protoplast; then filtering the protoplast in the MMG by using a three-layer 200-mesh nylon net, centrifuging for 3 min at 500 g in a new 50 mL tube, quickly pouring out the supernatant, and adding 1mL of MMG to resuspend the protoplast; counting the number of protoplasts with a hemocytometer, and adjusting the number of protoplasts to 2 × 10⁵~8×10⁵Each per milliliter;

(2) PEG-mediated recombinant plasmid transformation of protoplasts: protoplasts, plasmid ligated to the target gene and PEG solution were gently mixed in EP tube at a ratio of 10:1:11, ddH₂O and no load are used as controls, and the incubation is carried out for 10 min at room temperature; adding 2 times ofMixing the WI solution with the solution, inverting the mixture for seven times, and centrifuging at 500 g for 3 min; removing the supernatant by suction, and adding 10 mL of WI solution to resuspend the protoplasts; spread on a petri dish (Jetbiofil, No.170512-073, China) soaked with 1% BSA in percentage by mass/volume for 12 h in the dark.

Further, cell wall enzymatic hydrolysate: 1.5% (wt/vol) Cellulase R-10, 0.4% (wt/vol) Macerozyme R-10, 20 mM MES (pH 5.7), 0.5M mannitol, 20 mM KCl, 10 mM CaCl₂and 0.1% (wt/vol) BSA; MMG solution: 0.25M mannitol, 4 mM MES (pH 5.7) and 15 mM MgCl₂；PEG solution：40% (wt/vol) PEG 4000, 0.2 M mannitol and 100 mM CaCl₂(ii) a WI solution: 0.25M mannitol, 4 mM MES (pH 5.7), 20 mM KCl.

Further, the expression condition of the circular RNA in the protoplast of the transformed recombinant plasmid is detected, Total RNA of the protoplast of the transformed circular RNA is extracted, cDNA is obtained by RT-PCR reverse transcription, and the expression condition of the circular RNA is detected by semi-quantitative PCR.

The application of the poplar protoplast circular RNA overexpression system comprises the following steps: an over-expression circular RNA system is constructed, so that the over-expression circular RNA regulates the expression of host genes.

Further, constructing an over-expression circular RNA system by taking Potri.004G162600 as a host gene, and detecting the expression regulation and control effect of the circular RNA on the host gene; designing a linear primer of the host gene, wherein the sequence of the primer is as follows: 46 and 47, and the semi-quantitative PCR detection shows that the over-expression of the circular RNA enhances the expression of the host gene.

The invention has the advantages that: the invention discloses a poplar secondary xylem protoplast circular RNA overexpression system, and provides a method for efficiently and stably transforming circular RNA protoplasts so as to overexpress circular RNA. The method is to establish the cyclic RNA overexpression system in the forest tree species, namely poplar secondary xylem protoplast for the first time, the construction method of the overexpression system is simple, convenient and quick, the efficiency is far higher than that of a common genetic transformation system, and the method can be used for researching the transcriptional expression and the post-transcriptional level regulation of specific cyclic RNA in forest tree species host genes.

Drawings

FIG. 1 schematic diagram of "back-to-back" primer design for circular RNA.

FIG. 2 circular RNA enrichment after RNase R (R +) digestion. Taking 18S rRNA and EF1 alpha as reference genes; the top left "-" is: the bands of the linear internal reference genes 18S rRNA and EF1 alpha are not digested by RNase R (R-), and the band of the left lower part "+" is the band weakening condition of the linear internal reference genes 18S rRNA and EF1 alpha after RNase R (R +). The right is a comparison of the bands of 13 circular RNAs digested with RNase R (-) and RNase R (+). Only Potri.003-G001000 was not enriched, but was confirmed to be the authenticity of the circular RNA by conventional sequencing.

FIG. 3 bands containing endogenous flanking sequences upstream and downstream of the geneattB-Schematic representation of the circular RNA overexpression framework of linker sequences.

FIG. 4 contains a schematic representation of the final vector structure of the circular RNA of the endogenous flanking sequences upstream and downstream of the gene. 35S: a 35S promoter; nos T terminator.

FIG. 5 is a protoplast transformation map showing 15% transformation efficiency.

FIG. 6 transformation observations of individual protoplasts under a Laica microscope.

FIG. 7 is a map of the position of the Potri.004G162600 gene (left) and a gel electrophoresis image of the circular RNA overexpression (right). In the left picture, the top is published poplar gene structure annotation (grey is UTR, black is CDS, thin line is intron), the lower part is third generation single molecule sequencing result and second generation assembly structure schematic diagram, and the position of the marked arrow is the position of the designed primer; EF1 α in the right panel is the leveled reference gene, M: DNA Marker; p: protoplasts (Protoplast); EV: empty vector (Empty vector); OX: over-expressing circular RNA (overexpression of circRNA); *: emphasizing the location of the correct strip.

FIG. 8 is a position diagram (left) of the host gene of Potri.004G162600 and a gel electrophoresis diagram (right) showing the increase in the expression level of the host gene in which the selective splicing event has occurred. In the left picture, the top is published poplar gene structure annotation (grey is UTR, black is CDS, thin line is intron), the lower part is third generation single molecule sequencing result and second generation assembly structure schematic diagram, and the position of the marked arrow is the position of the designed primer; EF1 α in the right panel is the leveled reference gene, M: DNA Marker; p: protoplasts (Protoplast); EV: empty vector (Empty vector); OX: over-expressing circular RNA (overexpression of circRNA); *: emphasizing the location of the correct strip.

Detailed Description

Example 1 extraction of Populus nucleic acid

(1) Material treatment

Selecting 6 healthy Chinese white poplar plants growing for 3-9 months, taking xylem below the seventh leaf at the upper end and above the soil surface at the lower end of the morphology as a material, scraping secondary xylem by using an operating knife after peeling, putting the peeled xylem into tinfoil marked in advance, and immediately putting the peeled Chinese white poplar into liquid nitrogen for quick freezing. The wood part material was ground into a powdery sample using a high throughput tissue disruptor (QIAGEN tissue lyser II), mixed well, placed in an RNase-free 50 mL tube (harvesting), and placed in a freezer at-80 ℃ for a long period of time.

(2) Extraction of xylem Genomic DNA and Total RNA

A sample of the powder obtained by treating the above-mentioned material was dispensed in 120 mg portions in a 1.5 mL RNase-free tube, and Genomic DNA of xylem was extracted using a Plant Genomic DNA Kit (TIANGEN, No. DP305, China), and Total RNA thereof was extracted using an RNAprep Pure Plant Kit (Polysaccharides & Polyphenonics-rich) (TIANGEN, No. DP441, China). The specific procedure was performed according to the instructions, and the RNA extraction was performed aseptically. And (3) detecting the quality of DNA and RNA by using a NanoDrop 2000 spectrophotometer, and selecting the DNA and RNA with good quality to perform downstream vector construction and genome sequencing work.

（3）RT-PCR

Mu.g of Total RNA was subjected to reverse transcription using Random Primer using PrimeScript. RTM. II 1st Strand cDNA Synthesis Kit (TaKaKa, No. 6210A). First, RNA template denaturation reaction: cooling for 5 min at 65 deg.C on ice for 2 min; then, reverse transcription reaction: 10 min at 30 ℃ and 60 min at 42 ℃; finally, enzyme inactivation reaction: the reaction system is 20 mu L at 70 ℃ for 15 min. All PCR reactions were performed in a PCR instrument (Thermofisiher, No. 4483636). The obtained reverse transcription product, i.e., cDNA, was diluted 5-fold in a lump, and downstream gene verification experiments were performed using the diluted cDNA as a template.

Example 2 molecular biological validation of circular RNA

13 circular RNA genes are obtained through transcriptome sequencing, the sequences of the genes are shown as SEQ ID NO:1 to SEQ ID NO:13, in order to verify the authenticity of the circular RNA, PRAPI software is used for designing back-to-back primers (figure 1), and PCR amplification and sequencing experiments are used for verification.

Mu.g of Total RNA (54. mu.L) was taken and divided into two 1.5 mL RNase-free centrifuge tubes, one for RNase R digestion and the other for control. Next, 3.1. mu.L of 10 XRNase R Reaction Buffer and 1. mu.L of RNase R (20U/. mu.L) were added to each of the treatment tube and the control tube in this order (1. mu.L of RNase-free water was used in place of RNase R in the control tube), and they were mixed well, microcentrifuged and incubated in a water bath at 37 ℃ for 10 min. Then, 30. mu.L of phenol-chloroform-isoamyl alcohol =25:24:1 was added to terminate the digestion reaction, and the mixture was mixed well and centrifuged at 13,000g for 5 min at 4 ℃. Transferring the upper aqueous phase into a new 1.5 ml RNase-free centrifuge tube, sequentially adding 6 muL of 4M LiCl, 1 muL of glycogen and 90 muL of precooled absolute ethyl alcohol (-20 ℃), gently turning the mixture up and down for several times to uniformly mix the liquid, and placing the mixed liquid at-80 ℃ to precipitate RNA for more than or equal to 1 h. 9. mu.L of each of the treatment tube and the control tube was subjected to RT-PCR using a Random Primer to obtain cDNA. The reaction system of each of the 13 circular RNA genes is: reverse transcription of RNA in the treatment tube or the control tube to obtain 2 μ L cDNA, 15 μ L Premix Taq (TaKaRa), 0.5 μ L of back-to-back forward and reverse primers of corresponding circular RNA, and adding ddH₂O to 30 μ L; 45 cycles of PCR amplification were performed. The PCR amplification products were observed by 2% agarose gel electrophoresis, and the cyclic RNA was verified using EF 1. alpha. and 18S rRNA as reference genes (FIG. 2). The sequences of the circular RNA back-to-back primers are shown as SEQ ID NO. 14-SEQ ID NO. 39; the sequences of the primers of the internal reference genes are shown in SEQ ID NO. 40-SEQ ID NO. 43.

As can be seen from fig. 2: by RNase R (R)⁺) After digestion, 12 circular RNA genes in 13 circular RNA genes are enriched, while 18S rRNA and EF1 alpha of common linear reference genes are subjected toRNase R(R⁺) The band after digestion is more RNase R (R)^-) Is obviously weakened. Only Potri.003G001000 is in a non-enrichment phenomenon, but the authenticity of the circular RNA is verified through a sequencing mode. RNase R can digest linear transcripts and can not digest circular RNA without 3 'end and 5' end, and the authenticity of the circular RNA is preliminarily judged according to the characteristics.

The cDNA obtained after RNase R digestion was used as a template, and 5. mu.L of 10 XPCR Buffer, 5. mu.L of 2 mM dNTPs, and 3. mu.L of 25 mM MgSO 3 were mixed₄The "back-to-back" forward and reverse primers (Table 1) were 1.5. mu.L each, 1. mu.L of KOD-Plus-Neo (TOYOBO, No. KOD-401), and ddH was added₂The target band was amplified in 35 cycles in an O to 50. mu.L system at 98 ℃ for 10 s, 55 ℃ for 30 s, and 68 ℃ for 10 s. The "back-to-back" primers were used as the sequencing primers, and the purified expected bands were sequenced to confirm whether they were authentic circular RNAs.

The specific method of the sequencing and comparison link is as follows: obtaining the gene sequence of the circular RNA through transcriptome sequencing; the genome sequence of the target strip takes two starting ends of back-to-back positive and negative primers as the starting end and the tail end of the target strip, and is subjected to sequence comparison with the gene sequence of the circular RNA obtained by sequencing of a transcriptome; the authenticity of the circular RNA is verified if the sequenced sequence is ligated at the back-splicing junction and matches the sequence of the circular RNA obtained from transcriptome sequencing.

EXAMPLE 3 construction of circular RNA overexpression vectors

(1) Bands comprising endogenous flanking sequences upstream and downstream of the host geneattB-Framework acquisition of linker sequences

A partial sequence of the host gene of Potri.004G162600(Chr04:18369599-18370055) was selected as the target gene. Design of 5' end bandattB-Site-specific linkers and primers including endogenous flanking sequences of the gene of interest (FIG. 3). The primer sequences are as follows:

the forward primer is shown in SEQ ID NO: 44:

Ptr-trans-130-F:5’-GGGGACAAGTTTGTACAAAAAAGCAGGCTTCGTATATGCAGGCCTGTCTTTGCT-3’

the reverse primer is shown in SEQ ID NO: 45:

Ptr-trans-130-R:5’-GGGGACCACTTTGTACAAGAAAGCTGGGTCCTAAACAGAAGACGGACACAATTTTGT-3’

the underlined part is the recognition site for Gateway, i.e.attB-A recognition site.

200 ng Genomic DNA as template, 2.5. mu.L of each of the above adapter-carrying forward and reverse primers, 2 XUnique HiQ^TMPfu Master Mix (No Dye) (Novogene, No. NHP007L, China) 25. mu.L, plus ddH₂The O to 50. mu.L system was used to amplify the band of interest for 30 cycles of PCR at 98 ℃ for 10 s, 50 ℃ for 20 s, and 72 ℃ for 1 min. Add 10. mu.L of 6 XDNA loading buffer (TIANGEN, No. RT201-02, China) to the PCR product, mix well, add all samples to 2% agarose gel, and perform electrophoresis at 140V for 30 min. The band of interest was excised, and the band of interest was purified using the Universal DNA Purification Kit (TIANGEN, No. DP214, China) to obtain a band containing the endogenous flanking sequences upstream and downstream of the gene of interestattB-A circular RNA overexpression framework of the linker sequence to obtainattB-PCR product, stored at-20 ℃.

(2) Obtaining vectors for connecting target genes

By bands containing endogenous flanking sequences upstream and downstream of the gene of interestattB-Circular RNA overexpression frameworks of linker sequences, i.e.attB-Replacement of Gateway by PCR product for cloning Universal entry vector pDONR207attP-And (4) obtaining a site, thereby obtaining an entry vector carrying a gentamicin resistance gene and connected with a target gene.

The specific method comprises the following steps: mix 0.5. mu.L in a 200. mu.LEP tubeattB-PCR product, 0.5. mu.L pDONR207 vector (150 ng/. mu.L) and 0.25. mu.L BP clone (Invitrogen, No. 11789-020), the liquid was mixed with the bottom of a flick tube, and the reaction was carried out in a metal bath at 25 ℃ for 12 hours. And then transforming the ligation products into DH-5 alpha competent cells, thermally shocking for 60 s at 45 ℃, quickly putting back on ice for standing for 2 min, adding the cells into LB liquid culture medium without antibiotics, recovering the cells for 1h in a shaking table at the temperature of 37 ℃ and the rpm of 200, centrifugally concentrating bacterial liquid, uniformly coating the bacterial liquid on LB solid culture medium with the final concentration of 0.03 mg/mL gentamicin resistance, and culturing the cells for 12 h in an incubator at the temperature of 37 ℃. Selecting 3 colonies, gently dipping the selected colonies with a 10 μ L pipette tip, and addingEach of the forward and reverse primers (Potri.004G162600) was 0.5. mu.L, 7.5. mu.L of Premix Taq (TaKaRa), and ddH₂And in a PCR system of O6.5 mu L and total 15 mu L, primarily verifying the connection condition of the target fragment, then sequencing the bacteria with correct bands, and carrying out downstream reaction after the sequencing is correct.

(3) Obtaining of recombinant plasmid ligated with target Gene

10 ng-30 ng of the entry ligation plasmid, 0.2. mu.L (30 ng/. mu.L) of pUC19-35s-sGFP final vector and 0.4. mu.L of LR clone (ThermoFisher SCIENTIFIC, No. 11791-020) were mixed in a 200. mu.L tube, and ddH was added₂O to 2 mu L, flicking the tube bottom, mixing the liquid evenly, and reacting in a metal bath at 25 ℃ overnight. The ligation products were then transformed into DH-5. alpha. competent cells for colony validation, and the correct colonies were picked and added to 5 mL LB liquid medium containing a final concentration of 0.05 mg/mL carbenicillin resistance (INALCO, No. 1758-9317) and propagated for 16 h in a shaker at 37 ℃ and 200 rpm. Centrifuging at 4000 rpm for 10 min, removing supernatant until 400 μ L remains, resuspending colony precipitate, dividing the resuspension evenly, spreading on LB solid culture medium containing carbenicillin with final concentration of 0.05 mg/mL, and culturing at 37 deg.C in incubator for 16 h. The bacteria on the two culture media are scraped to 250 mL LB liquid culture medium containing carbenicillin resistance with final concentration of 0.05 mg/mL, expanded and propagated for 18 h in a shaker with 200 rpm at 37 ℃, recombinant plasmids are extracted by using an EndoFree Maxi Plasmid Kit (TIANGEN, No. DP117, China), the concentration of the recombinant plasmids is adjusted to 3 mg/mL, recombinant plasmids connected with target genes are obtained, and the schematic diagram of recombinant Plasmid knot construction is shown in FIG. 4.

Example 4 circular RNA protoplast transformation

(1) Isolation of protoplasts

Protoplast isolation and transformation experiments were based on the description of forest profit et al (Lin)et al2014).

Materials: 2 g of 10 cm secondary xylem stem segments of Chinese white poplar;

enzymolysis: 40 mL of cell wall enzymolysis solution [1.5% (wt/vol) Cellulase R-10, 0.4% (wt/vol) Macerozyme R-10, 20 mM MES (pH 5.7), 0.5M mannitol, 20 mM KCl, 10 mM CaCl₂and 0.1% (wt/vol) BSA]Standing at room temperature in dark placePerforming enzymolysis for 3 hours; 30 mL of MMG solution [ 0.25M mannitol, 4 mM MES (pH 5.7) and 15 mM MgCl were then added₂]Gently shake back and forth for 1.5 min to release protoplasts;

and (3) filtering: filtering protoplasts in the MMG in a new 50 mL tube by using a three-layer 200-mesh nylon net after enzymolysis, centrifuging for 3 min at 500 g, quickly pouring out supernatant, and resuspending the protoplasts by using 1mL of MMG; the number of protoplasts was counted on a hemocytometer (Sigma-Aldrich, No. 3110), and the number of protoplasts was adjusted to 2X 10⁵~8×10⁵Each per milliliter.

(2) Transformation of protoplasts

PEG-mediated transformation of protoplasts: the protoplast, the recombinant plasmid ligated to the target gene, and PEG solution [40% (wt/vol) PEG 4000, 0.2M mannitol and 100 mM CaCl ] were mixed gently in an EP tube at a ratio of 10:1:11₂]Flicking the tube bottom, mixing the liquid uniformly, and incubating at room temperature for 10 min; 2 volumes of WI solution [ 0.25M mannitol, 4 mM MES (pH 5.7), 20 mM KCl ] were added]The tube was gently inverted and the solution was mixed seven times, centrifuged at 500 g for 3 min, the supernatant aspirated, and protoplasts were resuspended by adding 10 ml of WI solution. Blank control group: adding equal amount of ddH₂O in place of the recombinant plasmid; the negative control group was supplemented with an equal amount of plasmid not carrying the gene of interest.

Protoplast culture: dark incubation was performed for 12 h in petri dishes soaked with 1mL 1% (wt/vol) BSA. The downstream experiments were performed after the transformation efficiency reached 15% (preliminary determination of circular RNA overexpression by fluorescence observation) (fig. 5, fig. 6).

(3) Method for efficiently counting protoplast transformation efficiency

Experimental equipment: hemocytometer (Sigma-Aldrich, No. 3110), Zeiss fluorescence microscope.

The method comprises the following steps: sucking 10 mu L of protoplast of the transformed target gene on a blood counting chamber, and inversely covering on a Zeiss fluorescent microscope objective table; firstly, adjusting an eyepiece and an aperture to clearly see lines on a counting plate in a bright field, then adjusting to clearly see protoplasts and ensuring a proper visual field, and preferably observing under a 10-fold or 20-fold lens; taking 16 grids at four corners of the counting plate as a fixed observation range (the range preferably does not exceed the visual field range of an ocular lens and is not too small), adjusting a light source to a GFP (green fluorescent protein) level, turning off and on a bright field in turn, and counting the total number of protoplasts and the number of the protoplasts with fluorescence; and (3) observing data within the range of 4 16 grids by only moving the object stage up, down, left and right without changing any parameter, and then calculating the average conversion efficiency. If the experiment is to be made more stringent, a second 10. mu.L of protoplast transformed with the gene of interest is aspirated onto a counting plate and the same procedure is performed.

EXAMPLE 5 construction of an over-expressed circular RNA System

(1) Total RNA extraction of poplar secondary xylem protoplast transformed with recombinant plasmid: total RNA was extracted using the RNAprep Pure Plant Kit (Polysaccharides & Polyphenonics-rich) (TIANGEN). The concentration of RNA was measured using a NanoDrop 2000 spectrophotometer and the RNA was stored for a long period at-80 ℃.

(2) RT-PCR: step 1) 0.5. mu.g of extracted RNA was measured, and a reverse transcription experiment was performed using Random primer according to PrimeScript ™ II 1st Strand cDNA Synthesis Kit (TaKaKa).

(3) Circular RNA semi-quantitative PCR: diluting the cDNA obtained in the step 2) by 5 times to be used as a template, and taking EF1 alpha as an internal reference primer. Using 5 XcDNA with different volume gradients as template, 1. mu.L of each of the "back-to-back" forward and reverse primers, 25. mu.L of Premix Taq (TaKaRa), plus ddH₂O to 50. mu.L, internal reference leveling was performed in 35 cycles at 94 ℃ for 45s, 50 ℃ for 35s, and 72 ℃ for 20 s, and volumes of cDNA with uniform brightness of the EF 1. alpha. band were searched (P: 0.7. mu.L, EV: 3. mu.L, OX: 1.7. mu.L). After the internal reference was leveled, 1. mu.L each of "back-to-back" forward and reverse primers, 25. mu.L of Premix Taq (TaKaRa), cDNA (P: 0.7. mu.L, EV: 3. mu.L, OX: 1.7. mu.L) and ddH were added₂O to 50. mu.L, 50 PCR cycles at 94 ℃ for 45s, 50 ℃ for 35s, and 72 ℃ for 20 s, resulted in overexpression of circular RNA (Potri.004G162600) (FIG. 7). Successfully constructs an overexpression system of the poplar circular RNA.

Example 6 overexpression of circular RNA enhances expression of host genes

Taking the host gene of the Potri.004G162600 gene as an example, linear primers of the host are designed, and the primer sequences are as follows:

SEQ ID NO:46：Ptr-linear-1-F: 5’-TTCGCTCTGCTTGCAATGTG-3’

SEQ ID NO:47：Ptr-linear-1-R: 5’-GCTTAGGCGGCTGATTTTGG-3’

semi-quantitative PCR of host genes was performed based on the over-expression circular RNA system. Positive and negative primers 1. mu.L each, Premix Taq (TaKaRa) 25. mu.L, cDNA (P: 0.7. mu.L, EV: 3. mu.L, OX: 1.7. mu.L) and ddH₂O to 50. mu.L, 50 cycles of PCR were performed at 94 ℃ for 45s, 50 ℃ for 35s, and 72 ℃ for 20 s. The results are shown in FIG. 8, indicating that the selective splicing event occurred in the indicated host gene and that the expression of the host gene was enhanced due to the overexpression of the circular RNA.

The above description is only a preferred embodiment of the present invention, and all equivalent changes and modifications made in accordance with the claims of the present invention should be covered by the present invention.

SEQUENCE LISTING

<110> Fujian agriculture and forestry university

<120> construction method and application of poplar secondary xylem protoplast circular RNA overexpression system

<130> 47

<160> 47

<170> PatentIn version 3.3

<210> 1

<211> 359

<212> DNA

<213> Potri.004G162600

<400> 1

ctttctttta ctgcgagata gatggcagtt cgctctgctt gcaatgtgat atgatagtgc 60

atgttggtgg taaaaggacc catgggagat atctcctgtt gaggcagaga gttgagtttc 120

caggggataa acctggctgt acagaggaac aaggacagca accgcttgat gataatgaaa 180

caagaaggga ccaaaatcag ccgcctaagc tcacagctag agaaaatcaa cagaaccaca 240

gggcctctcc agttccaatg gtagagaata acactgatag tgacgggaaa atggacaaca 300

aattgattga tcttaatgcc aggcctcagc gtgtacatgg gaaaaatcca actaaccag 359

<210> 2

<211> 341

<212> DNA

<213> Potri.009G164300

<400> 2

ctttatttac agtaacaggc agacctgcag ctacactgga aatgtttcct tcatggccaa 60

tgagattcca agaaacccca agaggaagtt caaggtcagg aggggagagt actgactcag 120

gatcagcatt aaacactctt tcaagcaaag ctgaagccca tctggaacca gaatctccca 180

tcagtaaaaa agtttcttct tcagatcatt acaatcaggc tttttatcag aagcatctac 240

agtttcaaga acagcaacaa gtagatatgg ctaatgatac atcaagaaca ggggggccct 300

ctcaacagaa tcaatcacct gccaaatctc cccaagaaaa g 341

<210> 3

<211> 426

<212> DNA

<213> Potri.006G145100

<400> 3

gaatcatcta ttgattggaa actcatttct gatattgatg gcagctaaac agtaatctca 60

aaggcgagcc accaagaatc tttgattgct taaggatttt cttcaaattc aggatatata 120

tatagataaa ctagatggct ttacgaattc agaacttgct gaagaaaaac tttgatgaac 180

cacactttac tgtcagttgc ccttcatggt ggaattcaaa tgaacagcac ttttcaccat 240

ctttatccaa gaacataaac tttaaagtag actcttcacc ccaaccttat catgaagcaa 300

agcagttagg acttaatctt ccggaccagg aatcgtcctc aactctgtca attggtcaat 360

ctcacaatga aatgagtgcc atgggaggga ccaattcaca aggtcaatgt atttcgtctg 420

aatccg 426

<210> 4

<211> 987

<212> DNA

<213> Potri.002G257900

<400> 4

agagaatatg aagaattcaa agtaaggatc aacgcgttgg tgtcaaaggc ccaaaagaaa 60

ccagaagaag gatgggtgat gcaggatggt accccatggc ctggaaacat cacacgtgat 120

catcctggaa tgattcaggt atatctagga agtgagggtg cgctcgacgt ggaaggcaag 180

gagcttccga ggcttgtgta tgtttcccgt gagaaacgac ctggatataa ccaccacaag 240

aaagcaggtg ccatgaatgc tctgattcga gtctcagcag tgctcaccaa tgcacctttt 300

atgttgaatt tggattgtga ccattacatc aataacagca aggctgtaag agaagccatg 360

tgctttttga tggatcccca acttgggaag aagctctgct atgtccagtt tccgcagagg 420

tttgatggta ttgatcgcca tgatagatat gctaatcgca atgttgtgtt ctttgatata 480

aacatgaaag gtctagatgg ggttcaaggg ccagtatatg ttggtactgg atgtgtcttc 540

aacaggcagt ccttgtatgg ctatgatcct ccagtgtctg agaagagacc caagatgaca 600

tgcgattgct ggccttcatg gtgttgctgt tgctgtggtg gttcaaggaa aaagtctaag 660

aagaaagggc aaagaagtct tcttggagga ctatacccca tgaaaaagaa aatgatgggg 720

aagaagtaca caaggaaagc atctgcacca gtctttgatc ttgaagagat tgaagaaggg 780

cttgaaggct acgaagagtt ggagaaatca tcactcatgt cacaaaaaag tttcgagaaa 840

cggtttgggc aatcaccggt atttattgcc tctaccctca tggaaaatgg tggcctgcct 900

gaaggaacta actctcaatc acacattaag gaagccattc atgttataag ttgcgggtat 960

gaagaaaaaa ccgaatgggg taaagag 987

<210> 5

<211> 322

<212> DNA

<213> Potri.007G107200

<400> 5

gatttcttat tcattaaaaa tcacatggtg caacccttgt tgctaagaac ttggagtcat 60

taagttgagc aatgggtggc tcaccgggtt ctgttgaagc taggcataga ttatctgcat 120

cttttgaaga tttatacaaa agaaggttaa caagaagtaa ggttaaaggt gttgaaaaac 180

ccttcaacat ccccatccaa gatagaagtt cttgttgcaa gtttccacta atcaaattta 240

tccttgtggt cattatcggt ggcactattg tatcactctt atattctcca gatgtggatc 300

aactatctca ctcaggttca ag 322

<210> 6

<211> 365

<212> DNA

<213> Potri.009G006500

<400> 6

gattattaaa ggatttgaag gtatacatat atgagttgcc atcaaaatac aacacggact 60

ggctagcaaa tgagaggtgc agcaaccatt tgtttgcatc agaagttgcc attcataaag 120

cactatcaaa cagtcttgat atacggacgt ttgacccata cgaagctgat ttcttctttg 180

ttcctgttta cgtgtcctgc aatttcagca ccgttaatgg gttccctgca attggtcatg 240

caaggtcctt attatcctct gcggtgcagc tcatttcctc taactatcca ttttggaacc 300

gctctcaagg atctgatcat gtctttgttg cctctcacga ttacggcgct tgtttccatg 360

ccatg 365

<210> 7

<211> 602

<212> DNA

<213> Potri.001G088600

<400> 7

atttatcttc aactgcatca actcggaatt ctggattgac acagtcccat gatttaactc 60

aactgagtgt taccggtgaa ggcaaaagtt atgaaaatca cgtcatgtgg catcaaaaac 120

agactgatgt taatggccat agcaactcta tgtcaaggac tcaaactgaa gggggctggt 180

tagcttctcc ccatgttaat gtttctcagc atccgtttca ggatgccatg gaggatagca 240

aaagtgtttc cccgtggcct gtattctcag gctattcaac tcccctctca tccaagtcaa 300

agaatgacgc aatccttgac ccatctgata aggggaggaa atctgaggtg cctactagtt 360

atcgattatt tggcattgat cttgtaaatc attcctcaag ttcaactcct attgaagtgc 420

ctgcacagct gatgagtata tgtagtggtg ccactgaagg acatgttcta ggtgccctgt 480

ctgcagttga ttcagaccaa aaatctgaag tatccaagga acagaaacca gaacagttac 540

agaaatcacc aaaagaaatt cagagcaagc aaagttctac ttccactaga agtcgcacga 600

ag 602

<210> 8

<211> 458

<212> DNA

<213> Potri_002G045800

<400> 8

gaaaccaatg ctgtaaagcc ggaagttttt tcttcgttac ctgaagatca aaggctagct 60

gcaagaaata tgtcgttggg aacacaatct tttcataatg gtactgatca aggcatttca 120

atggcacaaa gcggggcaga tggtgtatca ctcggcattc cacctttact tactacacag 180

agaaaatcag atgatgaatc actcggcatt ccagctttac ctactacaca gagaaaatca 240

gatgatgaat cactcggcat tccagcttta cctactacac agagaaaatc agatggtgga 300

tcacttggca ttccagcttt acctattaca cagagaaaac aagaggttca agccaggcaa 360

acaaccagtg gatcatcaag ctcggatgat gatgaccttg aaggagacac aggaaccaat 420

gaaaatatgg atcctgctgt tgtgaaacgt gcgaggag 458

<210> 9

<211> 621

<212> DNA

<213> Potri_003G001000

<400> 9

gtccattggg atgaaccttc ttccatcttg cgtccagaga gagtatcacc atgggatttg 60

gaacctcttg ttgcaactac tccttcgaac tcccaaccta tgcagaggaa caagcggcca 120

cggccatctg tcttgccctc accaacagcc aatctttctg cacttggtat gtggaaacct 180

tcggttgagt cttcagcttt ctcatatggt gaatcacaac gtggacgaga cccttatcca 240

tcacccaatt tctctaccac tgcaaaggcc aactctctta gcttctgtgg caatagtcaa 300

gtgaccagtg tttcgccgaa ttcaatgtat cggcctaacc aagtggaaag tgtcacagat 360

tcttttgctc cagttgtaaa caaagatttg ggagaaagga gacagggtac tgggattggc 420

tacagacttt tcgggattca acttattgac aatttcaatg cagaaggaac ttcaccagtg 480

gttactgtgt ctggaacagt gggcaatgat cgcccggttg tgtctttaga ggctgagtct 540

gatcagcatt ctgagcctga gaaatcatgt ctgagatctc atcaggagtt gcaaagtagg 600

caaatcagga gctgcacaaa g 621

<210> 10

<211> 422

<212> DNA

<213> Potri_003G142100

<400> 10

gttgtggaga ggatgatctg tatacggagt tatggaaggc atgtgcaggt ccacttgtgg 60

atgttcccaa gcgtggagaa agagtgttct attttcctca gggtcatatg gaacagttag 120

aggcatcaac aaatcaagaa ttgaatcaga gggttccttt gtttaatctg ccttcgaaga 180

ttctctgtcg tgttattaat actcagctgc tggcggaaca agaaacagat gaggtttatg 240

cgcagattac tttactgcct gaatcagatc aaattgaaac tacaagtcct gatccatgtc 300

catctgagcc tccaagacct acggtccact ctttctgcaa ggttttaact gcctctgata 360

cgagcaccca cggtggattt tctgttcttc gtaaacatgc ctctgaatgc cttcctccac 420

tg 422

<210> 11

<211> 269

<212> DNA

<213> Potri_018G022700

<400> 11

ggttacaaag aaaaaattgt tgacattaga gaggaaattc tgcgaaagag aagagcagga 60

aagcttcctg gggacacaac ctcagtctta aaagcttggt ggcaatcaca ttccaagtgg 120

ccatatccta ccgaggaaga caaggcaaga ttggtgcagg aaacgggctt gcaattaaag 180

cagataaata attggttcat caatcaaagg aagaggaact ggcacagtaa tccttcaacc 240

tcaacagtct tgaaaagcaa acgcaaaag 269

<210> 12

<211> 621

<212> DNA

<213> Potri_019G053200

<400> 12

aatgagatgg agtgtgctta ttacttaaga acaggacagt gcaagtttgg tagcacttgt 60

aaatttcatc atcctcaacc agctaatgtg atggttcctt tgcgtggctc tcctgtttac 120

cctactgtca attctccaac tacccctggt cagcaatctt atccaggagg acttgcaaca 180

aactggtcaa gagcatcttt tattaccagc ccacgctggc aagctccttc aaattataca 240

cctttgattc tacctcaggg agtcgtatca gtccctggct ggaatgcata cagtggtcag 300

gttgggtcgg tatcatctcc agagagtcag caacaaacag gaaacagtca gatatatgga 360

acctcacgcc agaatgaatc agtcaatgca ggatctcaag ggacattttc tccataccgt 420

tctgattctg tccccatggg attttatgcc ttgcaaagag agagtgtttt tcctgagaga 480

cctggtcagc ctgaatgcca gttttacatg aagaccggag actgtaagtt tggtgcagtt 540

tgtagattcc accatccaag ggagaggcta attcctgctc cagactgtgt cttgagtgca 600

atagggctgc ctttacgtcc g 621

<210> 13

<211> 414

<212> DNA

<213> Potri_019G053200

<400> 13

tactacttga agactggaac ttgcaagttt ggagccacgt gcaagtttca tcaccctaga 60

gacaaggctg gggtttctgg aagagtgtct ttaaatattt tgggctatcc acttcgactg 120

aatgagatgg agtgtgctta ttacttaaga acaggacagt gcaagtttgg tagcacttgt 180

aaatttcatc atcctcaacc agctaatgtg atggttcctt tgcgtggctc tcctgtttac 240

cctactgtca attctccaac tacccctggt cagcaatctt atccaggagg acttgcaaca 300

aactggtcaa gagcatcttt tattaccagc ccacgctggc aagctccttc aaattataca 360

cctttgattc tacctcaggg agtcgtatca gtccctggct ggaatgcata cagt 414

<210> 14

<211> 20

<212> DNA

<213> Potri.004G162600-F

<400> 14

gcctctccag ttccaatggt 20

<210> 15

<211> 20

<212> DNA

<213> Potri.004G162600-R

<400> 15

gcagagcgaa ctgccatcta 20

<210> 16

<211> 20

<212> DNA

<213> Potri.009G164300-F

<400> 16

caagcaaagc tgaagcccat 20

<210> 17

<211> 20

<212> DNA

<213> Potri.009G164300-R

<400> 17

tccagtgtag ctgcaggtct 20

<210> 18

<211> 20

<212> DNA

<213> Potri.006G145100-F

<400> 18

cttccggacc aggaatcgtc 20

<210> 19

<211> 25

<212> DNA

<213> Potri.006G145100-R

<400> 19

tgccatcaat atcagaaatg agttt 25

<210> 20

<211> 20

<212> DNA

<213> Potri.002G257900-F

<400> 20

catggaaaat ggtggcctgc 20

<210> 21

<211> 20

<212> DNA

<213> Potri.002G257900-R

<400> 21

ggtaccatcc tgcatcaccc 20

<210> 22

<211> 21

<212> DNA

<213> Potri.007G107200-F

<400> 22

aacccttcaa catccccatc c 21

<210> 23

<211> 20

<212> DNA

<213> Potri.007G107200-R

<400> 23

gagccaccca ttgctcaact 20

<210> 24

<211> 20

<212> DNA

<213> Potri.009G006500-F

<400> 24

atgggttccc tgcaattggt 20

<210> 25

<211> 20

<212> DNA

<213> Potri.009G006500-R

<400> 25

tttgctagcc agtccgtgtt 20

<210> 26

<211> 20

<212> DNA

<213> Potri.001G088600-F

<400> 26

gttctaggtg ccctgtctgc 20

<210> 27

<211> 20

<212> DNA

<213> Potri.001G088600-R

<400> 27

tgccttcacc ggtaacactc 20

<210> 28

<211> 22

<212> DNA

<213> Potri_002G045800-F

<400> 28

aaaacaagag gttcaagcca gg 22

<210> 29

<211> 20

<212> DNA

<213> Potri_002G045800-R

<400> 29

aacttccggc tttacagcat 20

<210> 30

<211> 20

<212> DNA

<213> Potri_003G001000-F

<400> 30

tcgcccggtt gtgtctttag 20

<210> 31

<211> 20

<212> DNA

<213> Potri_003G001000-R

<400> 31

ggttgggagt tcgaaggagt 20

<210> 32

<211> 21

<212> DNA

<213> Potri_003G142100-F

<400> 32

gtccatctga gcctccaaga c 21

<210> 33

<211> 21

<212> DNA

<213> Potri_003G142100-F

<400> 33

cctgcacatg ccttccataa c 21

<210> 34

<211> 21

<212> DNA

<213> Potri_018G022700-F

<400> 34

cgggcttgca attaaagcag a 21

<210> 35

<211> 20

<212> DNA

<213> Potri_018G022700-R

<400> 35

gaggttgtgt ccccaggaag 20

<210> 36

<211> 20

<212> DNA

<213> Potri_019G053200-F

<400> 36

gcaatagggc tgcctttacg 20

<210> 37

<211> 20

<212> DNA

<213> Potri_019G053200-R

<400> 37

ctgaccaggg gtagttggag 20

<210> 38

<211> 20

<212> DNA

<213> Potri_019G053200-F

<400> 38

agtcgtatca gtccctggct 20

<210> 39

<211> 20

<212> DNA

<213> Potri_019G053200-R

<400> 39

cagtcgaagt ggatagccca 20

<210> 40

<211> 24

<212> DNA

<213> EF1α-F

<400> 40

attgacaggc ggtctggtaa ggaa 24

<210> 41

<211> 24

<212> DNA

<213> EF1α-R

<400> 41

aaacgaccaa gtggaggata cgct 24

<210> 42

<211> 24

<212> DNA

<213> 18S rRNA-F

<400> 42

ttaacgagga tccattggag ggca 24

<210> 43

<211> 24

<212> DNA

<213> 18S rRNA-R

<400> 43

acccaaccca aagtccaact acga 24

<210> 44

<211> 54

<212> DNA

<213> Ptr-trans-130-F

<400> 44

ggggacaagt ttgtacaaaa aagcaggctt cgtatatgca ggcctgtctt tgct 54

<210> 45

<211> 57

<212> DNA

<213> Ptr-trans-130-R

<400> 45

ggggaccact ttgtacaaga aagctgggtc ctaaacagaa gacggacaca attttgt 57

<210> 46

<211> 20

<212> DNA

<213> Ptr-linear-1-F

<400> 46

ttcgctctgc ttgcaatgtg 20

<210> 47

<211> 20

<212> DNA

<213> Ptr-linear-1-R

<400> 47

gcttaggcgg ctgattttgg 20

Claims (8)

1. A method for constructing a poplar secondary xylem protoplast circular RNA overexpression system is characterized by comprising the following steps of:

(1) molecular biological verification of circular RNA: designing back-to-back primers of the corresponding circular RNA genes according to the circular RNA gene sequence obtained by transcriptome sequencing, carrying out PCR amplification and sequencing, and verifying the authenticity of the circular RNA genes through experiments;

(2) construction of circular RNA overexpression recombinant plasmid: PCR cloning poplar circular RNA host gene segment, PCR cloning partial sequence of obtained circular RNA host gene as target segment, constructing circular RNA over-expression recombinant plasmid by using Gateway method, and taking Potri.004G162600 circular RNA gene as host gene;

(3) transforming poplar secondary xylem protoplast by PEG mediated circular RNA overexpression recombinant plasmid;

(4) molecular biological detection of the circular RNA over-expression in the transformed recombinant plasmid protoplast;

the circular RNA gene obtained by sequencing the transcriptome is Potri.004G162600, the position of the circular RNA gene in a chromosome is Chr04:18369599-18370055, and the sequence of the circular RNA gene is shown as SEQ ID NO 1; the back-to-back primers are as follows: according to the sequence of the circular RNA gene obtained by transcriptome sequencing, back-to-back primers are designed, and the specific sequences of the primers are shown as SEQ ID NO. 14-SEQ ID NO. 15.

2. The method for constructing the poplar secondary xylem protoplast circular RNA overexpression system according to claim 1, wherein the PCR cloning of poplar circular RNA host genes comprises: the sequence fragment of the poplar circular RNA host gene is cloned by PCR by using poplar Genomic DNA as a template and back-to-back primers of corresponding circular RNA genes as primers and using high-fidelity Taq enzyme.

3. The method for constructing the circular RNA overexpression system of the poplar secondary xylem protoplast according to claim 1, wherein the Gateway method for constructing the circular RNA overexpression recombinant plasmid comprises the following steps: construction of a band comprising endogenous flanking sequences upstream and downstream of the Gene of interestattB-A circular RNA overexpression framework of a linker sequence; pDONR207 was used as the entry vector for Gateway cloning; using pUC19-35s-sGFP as a final vector; the band containing endogenous flanking sequences upstream and downstream of the target geneattB-The primer sequences of the joint are shown as SEQ ID NO.44 and SEQ ID NO. 45.

4. The method for constructing the circular RNA overexpression system of the poplar secondary xylem protoplast according to claim 1, wherein the PEG-mediated recombinant plasmid is used for transforming the poplar secondary xylem protoplast, and the method comprises the following steps:

(1) separating protoplasts: adding 40 mL of cell wall enzymolysis liquid into 2 g of the secondary xylem stem segments of the populus trichocarpa, and performing enzymolysis for 3 h at room temperature in a dark place; centrifuging for 3 min at 500 g, discarding the upper layer enzymolysis solution, adding 30 mL MMG solution into the precipitate, and slightly shaking back and forth for 1.5 min to release protoplast; then filtering the protoplast in the MMG by using a three-layer 200-mesh nylon net, centrifuging for 3 min at 500 g in a new 50 mL tube, quickly pouring out the supernatant, and adding 1mL of MMG to resuspend the protoplast; counting the number of the protoplasts by using a blood counting plate,and adjusting the number of protoplasts to 2 × 10⁵~8×10⁵Each per milliliter;

(2) PEG-mediated recombinant plasmid transformation of protoplasts: protoplasts, plasmid ligated to the target gene and PEG solution were gently mixed in EP tube at a ratio of 10:1:11, ddH₂O and no load are used as controls, and the incubation is carried out for 10 min at room temperature; adding 2 times of WI solution, mixing the solution by soft inversion for seven times, and centrifuging for 3 min at 500 g; removing the supernatant by suction, and adding 10 mL of WI solution to resuspend the protoplasts; spread on a petri dish soaked with 1mL of BSA at a mass-volume percentage concentration of 1% for 12 h in the dark.

5. The method for constructing the circular RNA overexpression system of the poplar secondary xylem protoplast according to claim 4, wherein the cell wall enzymolysis liquid comprises the following steps: cellulase R-10 with the mass volume percentage concentration of 1.5 percent, Macerozyme R-10 with the mass volume percentage concentration of 0.4 percent, 20 mM MES, 0.5M mannitol, pH 5.7, 20 mM KCl, 10 mM CaCl₂BSA with the mass volume percentage concentration of 0.1 percent;

the MMG solution: 0.25M mannitol, 4 mM MES, pH 5.7, 15 mM MgCl₂；

The PEG solution: PEG 4000 with the mass volume percentage concentration of 40%, 0.2M mannitol and 100 mM CaCl₂；

The WI solution: 0.25M mannitol, 4 mM MES, pH 5.7, 20 mM KCl.

6. The method for constructing the circular RNA overexpression system of the protoplast of the secondary xylem of poplar as claimed in claim 1, wherein the method comprises the following steps: the molecular biological detection of the circular RNA over-expression condition in the transformed recombinant plasmid protoplast comprises the steps of extracting Total RNA of the transformed circular RNA protoplast, carrying out RT-PCR reverse transcription to obtain cDNA, and carrying out semi-quantitative PCR detection on the over-expression condition of the circular RNA.

7. The application of the poplar secondary xylem protoplast circular RNA overexpression system is characterized in that: the system for overexpression of circular RNA constructed according to the construction method of claim 1, wherein the overexpression of circular RNA regulates the expression of host genes.

8. The use of the circular RNA overexpression system of the protoplast of secondary xylem of poplar according to claim 7, wherein the circular RNA overexpression system comprises: constructing a poplar protoplast overexpression circular RNA system by taking Potri.004G162600 as a host gene, and detecting the expression regulation and control effect of circular RNA on the host gene; designing a linear primer of the host gene, wherein the sequence of the primer is as follows: 46, 47; semi-quantitative PCR detection shows that the over-expression of the circular RNA enhances the expression of a host gene Potri.004G162600.

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