CN110938650B - mRNA variable shearing-luciferase report system and application thereof - Google Patents

Abstract

The invention provides an mRNA variable splicing-luciferase reporter system and application thereof. The invention also provides a transgenic tobacco seed of the variable shearing-luciferase report system, which utilizes the agrobacterium-mediated transient overexpression technology to express exogenous genes on the transgenic tobacco leaf; or by utilizing an agrobacterium-mediated gene silencing technology, silencing endogenous genes of tobacco on the transgenic tobacco leaf, and judging whether the gene coding protein to be detected participates in the variable shearing process of plant mRNA (messenger ribonucleic acid) by detecting the change of luciferase activity. The invention provides an important technical means for rapidly screening whether the exogenous gene or the plant endogenous gene regulates and controls the plant mRNA variable shearing process.

Description

mRNA variable shearing-luciferase report system and application thereof

Technical Field

The invention belongs to the field of plant molecular biology, and particularly relates to a luciferase report system and application thereof, in particular to a luciferase report system for specifically screening whether a gene to be tested participates in a plant mRNA variable shearing process and application thereof, which can be used for rapidly screening whether an exogenous expression gene or a plant endogenous gene participates in the plant mRNA variable shearing process.

Background

The variable splicing refers to a process that different mRNAs are generated from the same mRNA precursor (pre-mRNA) through different splicing modes, and during the growth and development of eukaryotes, the pre-mRNA of most genes needs to be capped by nucleotides at the 5 'end and added with polyadenylic acid at the 3' endA series of processes such as tail cleavage and intron cleavage to generate mature mRNA^1,2. The variable shearing of RNA plays an important role in regulating and controlling the growth, development and other life activities of organisms, and the variable shearing not only can increase the complexity of gene expression in higher organisms^3,4Moreover, some by-products of the variable splicing process can also process various non-coding RNAs to participate in the processes of growth and development of organisms and the like^5,6. At present, in-vitro variable shear system constructed by using cell extracts of mammalian cells, yeast and drosophila in the field of variable shear research provides great help for researching assembly and formation of spliceosome and function and action mechanism of shear regulatory protein^7-9. However, the lack of relevant mRNA variable splicing systems in the field of plant variable splicing research to date has severely limited the functional studies of splicing regulatory proteins in plants and the mechanism of action of mRNA variable splicing in plants. Therefore, the development of a novel in-plant mRNA variable splicing reporter system is an effective way for accelerating the research on the variable splicing action mechanism of plants.

Reference to the literature

1.Black,D.L.Mechanisms of alternative pre-messenger RNA splicing.Annual review of biochemistry 72,291-336,doi:10.1146/annurev.biochem.72.121801.161720(2003).

2.Reddy,A.S.&Shad Ali,G.Plant serine/arginine-rich proteins:roles in precursor messenger RNA splicing,plant development,and stress responses.Wiley interdisciplinary reviews.RNA 2, 875-889,doi:10.1002/wrna.98(2011).

3.Roy,B.,Haupt,L.M.&Griffiths,L.R.Review:Alternative Splicing(AS)of Genes As An Approach for Generating Protein Complexity.Current genomics 14,182-194,doi:Doi 10.2174/1389202911314030004(2013).

4.Frankiw,L.,Baltimore,D.&Li,G.Alternative mRNA splicing in cancer immunotherapy. Nature reviews.Immunology,doi:10.1038/s41577-019-0195-7(2019).

5.Yang,L.Splicing noncoding RNAs from the inside out.Wiley interdisciplinary reviews.RNA 6,651-660,doi:10.1002/wrna.1307(2015).

6.Zhang,Y.et al.The Biogenesis of Nascent Circular RNAs.Cell reports 15,611-624, doi:10.1016/j.celrep.2016.03.058(2016).

7.Lin,R.J.,Newman,A.J.,Cheng,S.C.&Abelson,J.Yeast mRNA splicing in vitro.The Journal of biological chemistry 260,14780-14792(1985).

8.Rio,D.C.Accurate and efficient pre-mRNAsplicing in Drosophila cell-free extracts. Proceedings of the National Academy of Sciences of the United States of America 85,2904- 2908,doi:10.1073/pnas.85.9.2904(1988).

9.Hernandez,N.&Keller,W.Splicing of in vitro synthesized messenger RNA precursors in HeLa cell extracts.Cell 35,89-99,doi:10.1016/0092-8674(83)90211-8(1983).

Disclosure of Invention

The invention aims to provide an mRNA variable splicing-luciferase reporter system and application thereof.

Another objective of the invention is to provide an expression vector of the mRNA variable splicing-luciferase reporter system and a construction method thereof.

Another object of the present invention is to provide a method for breeding a transgenic plant having the mRNA variable excision-luciferase reporter system.

The purpose of the invention can be realized by the following technical scheme:

a mRNA variable splicing-luciferase reporter system comprising a variable splicing region sequence of plant RLPK and a luciferase reporter LUC sequence.

Preferably, the sequence of the variable splicing region of the plant RLPK is SEQ ID NO.1, the sequence of the luciferase reporter gene LUC is SEQ ID NO.2, and the sequence of the mRNA variable splicing-luciferase reporter system (RLPK-LUC) is SEQ ID NO. 3.

A plant transgenic vector of an mRNA variable shearing-luciferase reporter system is obtained by connecting the variable shearing region sequence of the plant RLPK and the luciferase reporter gene LUC sequence into a plant expression vector pCAMBIA1300 together to obtain the plant transgenic vector pCAMBIA1300 RLPK-LUC. The construction process of the plant transgenic vector comprises the following steps:

(1) extracting genomic DNA of tomato, amplifying variable shearing region sequence of plant RLPK by using primers SEQ ID NO.5 and SEQ ID NO.6 to obtain nucleotide sequence shown as SEQ ID NO. 1;

(2) amplifying a luciferase reporter gene LUC sequence by using primers SEQ ID NO.7 and SEQ ID NO.8 to obtain a nucleotide sequence shown as SEQ ID NO. 2;

(3) the variable splicing region sequence of plant RLPK and the luciferase reporter gene LUC sequence are connected into a plant expression vector pCAMBIA1300 together by using a multi-fragment recombination method to obtain a plant transgenic vector pCAMBIA1300, RLPK-LUC of the mRNA variable splicing-luciferase reporter system. The whole sequence of the plant transgenic vector of the mRNA variable shearing-luciferase reporting system is SEQ ID NO. 4.

A method for culturing transgenic plant with mRNA variable shearing-luciferase reporter system includes such steps as stably transforming the transgenic plant carrier to obtain transgenic plant with mRNA variable shearing-luciferase reporter system stably over-expressed by resistance screening and luciferase activity verification. The transgenic plant is preferably the nicotiana benthamiana.

The mRNA variable shearing-luciferase report system, the plant transgenic vector or the transgenic plant cultivated by the method are applied to rapidly screen whether the exogenous gene and the plant endogenous gene participate in the process of regulating and controlling the mRNA variable shearing of the plant.

A gene function research method based on variable shearing change utilizes an agrobacterium-mediated transient expression technology to overexpress exogenous genes on blades of a transgenic plant of an mRNA variable shearing-luciferase report system, or utilizes an agrobacterium-mediated gene silencing technology to silence endogenous genes of the plant on the blades of the transgenic plant of the mRNA variable shearing-luciferase report system, and judges whether genes to be detected participate in the mRNA variable shearing regulation and control process of the plant or not by detecting the change of luciferase activity, so that whether the genes to be detected participate in the mRNA variable shearing process of the plant or not is rapidly screened.

The invention utilizes an mRNA variable shearing-luciferase report system to identify whether a gene to be detected participates in the mRNA variable shearing regulation and control process of plants, and specifically comprises the following steps;

(1) extracting genomic DNA of tomato, and amplifying a variable shearing region sequence of RLPK by using primers (SEQ ID NO.5 and SEQ ID NO.6) to obtain a nucleotide sequence shown as SEQ ID NO. 1.

(2) Luciferase reporter gene LUC sequence was amplified using primers (SEQ ID NO.7 and SEQ ID NO.8) to obtain the nucleotide sequence shown as SEQ ID NO. 2.

(3) The variable splicing region sequence of RLPK and the luciferase reporter gene LUC sequence are connected into a plant expression vector pCAMBIA1300 together by using a multi-fragment recombination method to obtain the sequence of the variable splicing-luciferase reporter system shown as SEQ ID NO.3 and a plant transgenic vector pCAMBIA1300, RLPK-LUC.

(4) The mRNA variable shearing-luciferase reporter system is over-expressed in the tobacco (N.benthamian) by using a stable transformation method, and a stable transgenic tobacco plant of the variable shearing-luciferase reporter system is obtained by a resistance screening and luciferase activity verification method.

(5) And after the tobacco grows up, further harvesting the stable transgenic tobacco seeds of the variable shearing-luciferase reporter system.

(6) The method comprises the steps of overexpressing an exogenous gene or silencing a plant endogenous gene on tobacco of a report system by using an agrobacterium injection method, selecting a leaf of an injection point after 2 days, adding the leaf into a 96-well enzyme label plate (containing a luciferase substrate Luciferin with the final concentration of 1 mM), placing the plate in the dark at normal temperature for 5-10 minutes, detecting the enzyme activity of luciferase in a Promega GloMax 96 micropore plate luminescence detector, and judging whether the gene to be detected participates in the variable shearing process of plant mRNA (messenger ribonucleic acid) according to the change of the enzyme activity value.

According to the method provided by the invention, the sequence subjected to variable shearing in the RLPK gene is connected with the sequence of the luciferase reporter gene LUC to construct a luciferase reporter system capable of effectively screening and identifying whether the gene to be detected is involved in the plant mRNA variable shearing process, so that the problem of identifying whether the gene to be detected is involved in the plant mRNA variable shearing process is effectively solved, and a high-efficiency reporter system tool and a screening method are provided for discovering and researching the gene function.

The invention has the advantages of

The invention provides a transgenic plant of an mRNA variable shearing-luciferase report system, which overexpresses an exogenous gene on a tobacco leaf of the report system by utilizing an agrobacterium-mediated transient expression technology or silences a plant endogenous gene on the tobacco leaf of the report system by utilizing an agrobacterium-mediated gene silencing technology, and judges whether a gene to be detected participates in the variable shearing process of the plant or not through the change of luciferase activity. The invention can realize the rapid screening of whether the gene to be tested participates in the variable shearing process of plant mRNA.

Drawings

FIG. 1 shows the sequencing of transcriptome results that the tomato RLPK gene is variably sheared during the infection process of Phytophthora infestans.

FIG. 2 is a real-time fluorescent quantitative verification that the tomato RLPK gene is subjected to variable shearing in the phytophthora infestans process: the ratio of the expression amounts (RLPK.1/RLPK.2) of different transcripts of the tomato RLPK gene is verified by real-time fluorescent quantitative PCR (polymerase chain reaction) to be obviously changed in the process that two phytophthora infestans infect tomatoes.

FIG. 3 schematic of the construction of the variable shear-luciferase reporter system: the transcript RLPK.1 can be read through, and the protein translation is normal, so that the enzyme activity of luciferase can be utilized to carry out quantitative detection; the transcript RLPK.2 has a stop codon appearing in advance, cannot be read through and cannot detect the enzyme activity of luciferase; the occurrence of variable cleavage can be judged based on the level of the detected luciferase activity.

FIG. 4 detection of luciferase Activity in transgenic plants of the reporter System: the luciferase activity of the transgenic plants of the RLPK-LUC reporter system is detected by using an instrument.

FIG. 5 is a flow chart of the use of the variable shear-luciferase reporter system.

FIG. 6 screening of virulence factors in Phytophthora infestans for their involvement in the variable cleavage process of plant mRNA using a reporter system: a variable shearing-luciferase report system is utilized to screen a toxic factor participating in mRNA variable shearing in Phytophthora infestans, and whether the toxic factor participates in the variable shearing process of plant mRNA can be judged according to the activity of luciferase.

FIG. 7 uses a reporter system to investigate whether plant endogenous genes are involved in plant mRNA variable splicing: a report system is utilized to research whether the endogenous gene in the tobacco participates in the variable shearing of plant mRNA, and whether the endogenous gene participates in the variable shearing process of the plant mRNA can be judged according to the activity of luciferase.

Detailed Description

The following examples are for better understanding of the present invention, but are not intended to limit the present invention. The experimental methods used in the following examples are all conventional experimental methods unless otherwise specified. The test materials and the like used in the following examples are available from conventional biochemicals, unless otherwise specified. The primers involved in the examples of the present invention were synthesized by Nanjing Kingsrei Biotechnology Ltd.

Example 1 verification of the changes in the ratio of expression levels between different transcripts of the tomato RLPK Gene during Phytophthora infestans

Total RNA extraction of infection samples: tomato leaves are taken as materials, phytophthora infestans 88069 and T30-4 are used for infecting tomatoes, and samples which are infected for 0, 1, 2 and 3 days are selected to extract total RNA. The total RNA was extracted using an Invitrogen RNA extraction kit (cat. No.12183018A) according to the instructions, and the RNA concentration was measured using a NanoDrop spectrophotometer.

Reverse transcription to generate the first strand: mu.g of RNA was used as a template in accordance with PrimeScript of Takara^TMThe reverse transcription kit (RR047A) uses the instruction to perform cDNA synthesis, and finally the volume is determined to 20 mu L system, and the appropriate amount of reverse transcription product is used for the subsequent real-time fluorescent quantitative PCR detection.

Rlpk.1 primer sequence:

an upstream primer: SEQ ID NO.9

(5’-ACCTTCTTGTGCAGAGAATG-3’)

A downstream primer: SEQ ID NO.10

(5’-ATAGTAAGAACGCCATAGTAG-3’),

Rlpk.2 primer sequence:

an upstream primer: SEQ ID NO.11

(5’-CTACTATGGCGGTACGTATTTG-3’)

A downstream primer: SEQ ID NO.12

(5’-GTGGCAATGAACGCGGATTCT-3’),

20 μ L of the reaction system

Premix Ex Taq 10. mu.l, ROX Reference Dye 0.4. mu.l, upstream primers 0.4. mu.l each, cDNA 2. mu.l, ddH₂O6.8 μ l; real-time PCR reaction procedure: pre-denaturation at 95 ℃ for 30 sec; then, the mixture was circulated at 95 ℃ for 5sec, 60 ℃ for 34 sec. The cycle number of amplification, the melting curve, and the Ct value of each sample are automatically given by the instrument.

The results show that the tomato RLPK gene undergoes variable shearing during phytophthora infestations (fig. 1), and the ratio of expression levels between different transcripts of the RLPK gene (rlpk.1/rlpk.2) changes significantly during phytophthora infestations 88069 and T30-4 in tomato (fig. 2).

Example 2 construction of variable cleavage-luciferase reporter System:

extracting genomic DNA of tomato, designing a primer according to the variable splicing sequence of RLPK to amplify the variable splicing sequence region of RLPK, designing a primer according to the LUC sequence of luciferase gene to amplify the luciferase gene, and constructing a variable splicing-luciferase reporter system (figure 3).

pCAMBIA1300 RLPK upstream primer: SEQ ID NO.5

5’-ACGGGGGACGAGCTCGGTACCATGTGTAGCATCCACTACTA-3’

pCAMBIA1300 RLPK downstream primer: SEQ ID NO.6

5’-ATGTTTTTGGCGTCTTCCATTATACTTATTCCACCGTAC-3’

pCAMBIA1300 LUC upstream primer: SEQ ID NO.7

5’-ATGGAAGACGCCAAAAACAT-3’

pCAMBIA1300 LUC downstream primer: SEQ ID NO.8

5’-ATACGAACGAAAGCTCTGCAGTTACACGGCGATCTTTCCGC-3’

The variable cleavage sequence region of RLPK and the sequence of luciferase gene LUC were amplified with primers SEQ ID NO.5, SEQ ID NO.6 and SEQ ID NO.7, SEQ ID NO.8, respectively. The PCR program is pre-denaturation at 98 ℃ for 5 min, denaturation at 98 ℃ for 30sec, annealing at 58 ℃ for 30sec, extension at 72 ℃ for 1.5 min, 30 cycles, and final extension at 72 ℃ for 5 min; the PCR product was separated by electrophoresis on a 1% agarose gel, the sizes of the variable cleavage sequence region of RLPK and the luciferase reporter gene LUC were 260bp and 1653bp, respectively, and the objective band was recovered by cutting the gel and then recovered by using a DNA purification kit (code. No.9762) of TaKaRa. The recovered product was ligated to a KpnI and PstI double-digested pCAMBIA1300 vector according to the protocol of the Vazyme's Multi-fragment ligation Kit (Clonexpress Multi S One Step Cloning Kit, C113) to obtain pCAMBIA1300: RLPK-LUC plasmid.

The ligation products were transformed into competent cells of Escherichia coli JM109, and subsequently the transformed competence was spread on Carna-resistant LB plates (containing 50. mu.g/mL of Carna), cultured in an incubator at 37 ℃ for 12 to 16 hours, followed by colony PCR verification of positive clones using vector primers (SEQ ID NO.13 and SEQ ID NO.14) and shake cultivation. The plasmid pCAMBIA1300, RLPK-LUC from which positive clones were extracted was subjected to sequencing by southern Jing King Shirui Biotech Co., Ltd, according to the procedure of Takara plasmid extraction kit (code. No.9760), and the sequencing primers had the sequences of SEQ ID No.13 and SEQ ID No. 14. The plasmid with correct sequencing is delivered to Wuhan double helix biotechnology limited company to perform stable transformation on Nicotiana benthamian (N.benthamian), and finally, a transgenic tobacco plant of pCAMBIA1300: RLPK-LUC with stable overexpression is obtained (figure 4).

Example 3 Using the variable excision-luciferase reporter System, it was investigated whether the genes to be tested are involved in the variable excision Process of plants

According to the operation scheme of FIG. 5, Agrobacterium transformed with the virulence factor of Phytophthora infestans was picked and inoculated into a test tube (liquid LB: Carna concentration)50. mu.g/mL, rifampicin concentration 50. mu.g/mL), cultured in a shaker (28 ℃,200 rpm) for 12-16 hours, and centrifuged at 4000g for 2 minutes to collect the cells. Followed by tobacco buffer (composition: 10mM MgCl)₂10mM MES, pH 5.6,200. mu.M AS) was resuspended 2 times to a final concentration OD₆₀₀0.5. Selecting tobacco growing for 4-6 weeks, injecting prepared virulence factor bacterial liquid and control empty carrier bacterial liquid onto tobacco leaves, injecting 8 different tobacco leaves into each sample, and culturing the injected tobacco in a greenhouse (24 ℃/16h illumination and 22 ℃/8h darkness). After 2 days, the tobacco leaves at the injection point position are selected by a puncher for luciferase activity determination, three repeats (24 repeats in total for 8 leaves) at each injection point are taken and placed in a 96-hole enzyme label plate, a luciferase substrate (Luciferin; catalogue No. 7903; Biovision, USA) is added for reaction for 5-10 minutes, and then the obtained product is placed in a Promega Glo96 micropore plate luminescence detector for detecting the activity of the luciferase, and three independent experiments are carried out. As a result, it was found that the activities of the luciferases in the Pi04089 and Pi15038 treated samples were decreased compared with the control GFP treatment, indicating that Pi04089 and Pi15038 are involved in the process of variable cleavage of plant mRNA (FIG. 6).

The silencing fragments of NbSR30 and NbRS2Z32 were inserted into PK7GWIWG2D (II) vector to form silencing vectors for RNAi NbSR30 and RNAi NbRS2Z32 according to previous literature reports (Huang J, Gu L, Zhang Y, Yan T, Kong G, Kong L, Guo B, Qiu M, Wang Y, Jung M, et al 2017.An oomycete plant pathogen reagent pre-mRNA hybridization to subvert immunity. nat Commn 8(1): 2051). According to the operation scheme of FIG. 5, Agrobacterium transformed with RNAi: NbSR30 and RNAi: NbRS2Z32 was picked up, inoculated into a test tube (liquid LB: spectinomycin concentration 50. mu.g/mL, rifampicin concentration 50. mu.g/mL) for culture, cultured in a shaker (28 ℃,200 rpm) for 12-16 hours, and centrifuged at 4000g for 2-3 minutes to collect the cells. Followed by tobacco buffer (10 mM MgCl)₂10mM MES, pH 5.6,200. mu.M AS) was resuspended 2 times to a final concentration OD₆₀₀0.5. Selecting tobacco growing for 4-6 weeks, injecting the prepared bacterial liquid of RNAi NbSR30 and RNAi NbRS2Z32 and the bacterial liquid of control RNAi RFP onto tobacco leaves, injecting 8 different tobacco leaves into each sample, and injectingThe tobacco was cultured in a greenhouse (24 ℃/16h light and 22 ℃/8h dark). After 2 days, the tobacco leaves at the injection point position are selected by a puncher for luciferase activity determination, three injection points are repeatedly placed into a 96-hole enzyme label plate at each injection point, luciferase substrates are added for reaction for 5-10 minutes, then the reaction product is placed into a Promega GloMax 96 micropore plate luminescence detector for detecting the activity of luciferase, three independent experiments are carried out totally, and NbSR30 can be preliminarily found to interfere the variable shearing process of the transgenic tobacco (figure 7).

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cagaagacca aagggcaatt gagacttttc aacaaagggt aatatccgga aacctcctcg 540

gattccattg cccagctatc tgtcacttta ttgtgaagat agtggaaaag gaaggtggct 600

cctacaaatg ccatcattgc gataaaggaa aggccatcgt tgaagatgcc tctgccgaca 660

gtggtcccaa agatggaccc ccacccacga ggagcatcgt ggaaaaagaa gacgttccaa 720

ccacgtcttc aaagcaagtg gattgatgtg atatctccac tgacgtaagg gatgacgcac 780

aatcccacta tccttcgcaa gacccttcct ctatataagg aagttcattt catttggaga 840

gaacacgggg gacgagctcg gtaccatgtg tagcatccac tactatggcg gtacgtattt 900

gagatactgg agtgcaataa cttcattaca ctaaacattt tattgaaagg agattgttaa 960

agcaataggt ttaccatttt tagaatccgc gttcattgcc acctatatag ctgtaagcta 1020

tttcttcaag actttcaatg attttacatc ctcttaactt gatttttcct ttgtgattta 1080

cctgttttga cagttcttac tataatgtac ggtggaataa gtataatgga agacgccaaa 1140

aacataaaga aaggcccggc gccattctat ccgctggaag atggaaccgc tggagagcaa 1200

ctgcataagg ctatgaagag atacgccctg gttcctggaa caattgcttt tacagatgca 1260

catatcgagg tggacatcac ttacgctgag tacttcgaaa tgtccgttcg gttggcagaa 1320

gctatgaaac gatatgggct gaatacaaat cacagaatcg tcgtatgcag tgaaaactct 1380

cttcaattct ttatgccggt gttgggcgcg ttatttatcg gagttgcagt tgcgcccgcg 1440

aacgacattt ataatgaacg tgaattgctc aacagtatgg gcatttcgca gcctaccgtg 1500

gtgttcgttt ccaaaaaggg gttgcaaaaa attttgaacg tgcaaaaaaa gctcccaatc 1560

atccaaaaaa ttattatcat ggattctaaa acggattacc agggatttca gtcgatgtac 1620

acgttcgtca catctcatct acctcccggt tttaatgaat acgattttgt gccagagtcc 1680

ttcgataggg acaagacaat tgcactgatc atgaactcct ctggatctac tggtctgcct 1740

aaaggtgtcg ctctgcctca tagaactgcc tgcgtgagat tctcgcatgc cagagatcct 1800

atttttggca atcaaatcat tccggatact gcgattttaa gtgttgttcc attccatcac 1860

ggttttggaa tgtttactac actcggatat ttgatatgtg gatttcgagt cgtcttaatg 1920

tatagatttg aagaagagct gtttctgagg agccttcagg attacaagat tcaaagtgcg 1980

ctgctggtgc caaccctatt ctccttcttc gccaaaagca ctctgattga caaatacgat 2040

ttatctaatt tacacgaaat tgcttctggt ggcgctcccc tctctaagga agtcggggaa 2100

gcggttgcca agaggttcca tctgccaggt atcaggcaag gatatgggct cactgagact 2160

acatcagcta ttctgattac acccgagggg gatgataaac cgggcgcggt cggtaaagtt 2220

gttccatttt ttgaagcgaa ggttgtggat ctggataccg ggaaaacgct gggcgttaat 2280

caaagaggcg aactgtgtgt gagaggtcct atgattatgt ccggttatgt aaacaatccg 2340

gaagcgacca acgccttgat tgacaaggat ggatggctac attctggaga catagcttac 2400

tgggacgaag acgaacactt cttcatcgtt gaccgcctga agtctctgat taagtacaaa 2460

ggctatcagg tggctcccgc tgaattggaa tccatcttgc tccaacaccc caacatcttc 2520

gacgcaggtg tcgcaggtct tcccgacgat gacgccggtg aacttcccgc cgccgttgtt 2580

gttttggagc acggaaagac gatgacggaa aaagagatcg tggattacgt cgccagtcaa 2640

gtaacaaccg cgaaaaagtt gcgcggagga gttgtgtttg tggacgaagt accgaaaggt 2700

cttaccggaa aactcgacgc aagaaaaatc agagagatcc tcataaaggc caagaagggc 2760

ggaaagatcg ccgtgtaact gcagagcttt cgttcgtatc atcggtttcg acaacgttcg 2820

tcaagttcaa tgcatcagtt tcattgcgca cacaccagaa tcctactgag ttcgagtatt 2880

atggcattgg gaaacatgtt tttcttgtac catttgttgt gcttgtaatt tactgtgttt 2940

tttattcggt tttcgctatc gaactgtgaa atggaaatgg atggagaaga gttaatgaat 3000

gatatggtcc ttttgttcat tctcaaatta atattatttg ttttttctct tatttgttgt 3060

gtgttgaatt tgaaaatata agagatatgc aaacattttg ttttgagtaa aaatgtgtca 3120

aatcgtggcc tctaatgacc gaagttaata tgaggagtaa aacacttgta gttgtaccat 3180

tatgcttatt cactaggcaa caaatatatt ttcagaccta gaaaagctgc aaatgttact 3240

gaatacaagt atgtcctctt gtgttttaga catttatgaa ctttccttta tgtaattttc 3300

cagaatcctt gtcagattct aatcattgct ttataattat agttatactc atggatttgt 3360

agttgagtat gaaaatattt tttaatgcat tttatgactt gccaattgat tgacaacatg 3420

catcaagctt ggcactggcc gtcgttttac aacgtcgtga ctgggaaaac cctggcgtta 3480

cccaacttaa tcgccttgca gcacatcccc ctttcgccag ctggcgtaat agcgaagagg 3540

cccgcaccga tcgcccttcc caacagttgc gcagcctgaa tggcgaatgc tagagcagct 3600

tgagcttgga tcagattgtc gtttcccgcc ttcagtttaa actatcagtg tttgacagga 3660

tatattggcg ggtaaaccta agagaaaaga gcgtttatta gaataacgga tatttaaaag 3720

ggcgtgaaaa ggtttatccg ttcgtccatt tgtatgtgca tgccaaccac agggttcccc 3780

tcgggatcaa agtactttga tccaacccct ccgctgctat agtgcagtcg gcttctgacg 3840

ttcagtgcag ccgtcttctg aaaacgacat gtcgcacaag tcctaagtta cgcgacaggc 3900

tgccgccctg cccttttcct ggcgttttct tgtcgcgtgt tttagtcgca taaagtagaa 3960

tacttgcgac tagaaccgga gacattacgc catgaacaag agcgccgccg ctggcctgct 4020

gggctatgcc cgcgtcagca ccgacgacca ggacttgacc aaccaacggg ccgaactgca 4080

cgcggccggc tgcaccaagc tgttttccga gaagatcacc ggcaccaggc gcgaccgccc 4140

ggagctggcc aggatgcttg accacctacg ccctggcgac gttgtgacag tgaccaggct 4200

agaccgcctg gcccgcagca cccgcgacct actggacatt gccgagcgca tccaggaggc 4260

cggcgcgggc ctgcgtagcc tggcagagcc gtgggccgac accaccacgc cggccggccg 4320

catggtgttg accgtgttcg ccggcattgc cgagttcgag cgttccctaa tcatcgaccg 4380

cacccggagc gggcgcgagg ccgccaaggc ccgaggcgtg aagtttggcc cccgccctac 4440

cctcaccccg gcacagatcg cgcacgcccg cgagctgatc gaccaggaag gccgcaccgt 4500

gaaagaggcg gctgcactgc ttggcgtgca tcgctcgacc ctgtaccgcg cacttgagcg 4560

cagcgaggaa gtgacgccca ccgaggccag gcggcgcggt gccttccgtg aggacgcatt 4620

gaccgaggcc gacgccctgg cggccgccga gaatgaacgc caagaggaac aagcatgaaa 4680

ccgcaccagg acggccagga cgaaccgttt ttcattaccg aagagatcga ggcggagatg 4740

atcgcggccg ggtacgtgtt cgagccgccc gcgcacgtct caaccgtgcg gctgcatgaa 4800

atcctggccg gtttgtctga tgccaagctg gcggcctggc cggccagctt ggccgctgaa 4860

gaaaccgagc gccgccgtct aaaaaggtga tgtgtatttg agtaaaacag cttgcgtcat 4920

gcggtcgctg cgtatatgat gcgatgagta aataaacaaa tacgcaaggg gaacgcatga 4980

aggttatcgc tgtacttaac cagaaaggcg ggtcaggcaa gacgaccatc gcaacccatc 5040

tagcccgcgc cctgcaactc gccggggccg atgttctgtt agtcgattcc gatccccagg 5100

gcagtgcccg cgattgggcg gccgtgcggg aagatcaacc gctaaccgtt gtcggcatcg 5160

accgcccgac gattgaccgc gacgtgaagg ccatcggccg gcgcgacttc gtagtgatcg 5220

acggagcgcc ccaggcggcg gacttggctg tgtccgcgat caaggcagcc gacttcgtgc 5280

tgattccggt gcagccaagc ccttacgaca tatgggccac cgccgacctg gtggagctgg 5340

ttaagcagcg cattgaggtc acggatggaa ggctacaagc ggcctttgtc gtgtcgcggg 5400

cgatcaaagg cacgcgcatc ggcggtgagg ttgccgaggc gctggccggg tacgagctgc 5460

ccattcttga gtcccgtatc acgcagcgcg tgagctaccc aggcactgcc gccgccggca 5520

caaccgttct tgaatcagaa cccgagggcg acgctgcccg cgaggtccag gcgctggccg 5580

ctgaaattaa atcaaaactc atttgagtta atgaggtaaa gagaaaatga gcaaaagcac 5640

aaacacgcta agtgccggcc gtccgagcgc acgcagcagc aaggctgcaa cgttggccag 5700

cctggcagac acgccagcca tgaagcgggt caactttcag ttgccggcgg aggatcacac 5760

caagctgaag atgtacgcgg tacgccaagg caagaccatt accgagctgc tatctgaata 5820

catcgcgcag ctaccagagt aaatgagcaa atgaataaat gagtagatga attttagcgg 5880

ctaaaggagg cggcatggaa aatcaagaac aaccaggcac cgacgccgtg gaatgcccca 5940

tgtgtggagg aacgggcggt tggccaggcg taagcggctg ggttgtctgc cggccctgca 6000

atggcactgg aacccccaag cccgaggaat cggcgtgacg gtcgcaaacc atccggcccg 6060

gtacaaatcg gcgcggcgct gggtgatgac ctggtggaga agttgaaggc cgcgcaggcc 6120

gcccagcggc aacgcatcga ggcagaagca cgccccggtg aatcgtggca agcggccgct 6180

gatcgaatcc gcaaagaatc ccggcaaccg ccggcagccg gtgcgccgtc gattaggaag 6240

ccgcccaagg gcgacgagca accagatttt ttcgttccga tgctctatga cgtgggcacc 6300

cgcgatagtc gcagcatcat ggacgtggcc gttttccgtc tgtcgaagcg tgaccgacga 6360

gctggcgagg tgatccgcta cgagcttcca gacgggcacg tagaggtttc cgcagggccg 6420

gccggcatgg ccagtgtgtg ggattacgac ctggtactga tggcggtttc ccatctaacc 6480

gaatccatga accgataccg ggaagggaag ggagacaagc ccggccgcgt gttccgtcca 6540

cacgttgcgg acgtactcaa gttctgccgg cgagccgatg gcggaaagca gaaagacgac 6600

ctggtagaaa cctgcattcg gttaaacacc acgcacgttg ccatgcagcg tacgaagaag 6660

gccaagaacg gccgcctggt gacggtatcc gagggtgaag ccttgattag ccgctacaag 6720

atcgtaaaga gcgaaaccgg gcggccggag tacatcgaga tcgagctagc tgattggatg 6780

taccgcgaga tcacagaagg caagaacccg gacgtgctga cggttcaccc cgattacttt 6840

ttgatcgatc ccggcatcgg ccgttttctc taccgcctgg cacgccgcgc cgcaggcaag 6900

gcagaagcca gatggttgtt caagacgatc tacgaacgca gtggcagcgc cggagagttc 6960

aagaagttct gtttcaccgt gcgcaagctg atcgggtcaa atgacctgcc ggagtacgat 7020

ttgaaggagg aggcggggca ggctggcccg atcctagtca tgcgctaccg caacctgatc 7080

gagggcgaag catccgccgg ttcctaatgt acggagcaga tgctagggca aattgcccta 7140

gcaggggaaa aaggtcgaaa aggtctcttt cctgtggata gcacgtacat tgggaaccca 7200

aagccgtaca ttgggaaccg gaacccgtac attgggaacc caaagccgta cattgggaac 7260

cggtcacaca tgtaagtgac tgatataaaa gagaaaaaag gcgatttttc cgcctaaaac 7320

tctttaaaac ttattaaaac tcttaaaacc cgcctggcct gtgcataact gtctggccag 7380

cgcacagccg aagagctgca aaaagcgcct acccttcggt cgctgcgctc cctacgcccc 7440

gccgcttcgc gtcggcctat cgcggccgct ggccgctcaa aaatggctgg cctacggcca 7500

ggcaatctac cagggcgcgg acaagccgcg ccgtcgccac tcgaccgccg gcgcccacat 7560

caaggcaccc tgcctcgcgc gtttcggtga tgacggtgaa aacctctgac acatgcagct 7620

cccggagacg gtcacagctt gtctgtaagc ggatgccggg agcagacaag cccgtcaggg 7680

cgcgtcagcg ggtgttggcg ggtgtcgggg cgcagccatg acccagtcac gtagcgatag 7740

cggagtgtat actggcttaa ctatgcggca tcagagcaga ttgtactgag agtgcaccat 7800

atgcggtgtg aaataccgca cagatgcgta aggagaaaat accgcatcag gcgctcttcc 7860

gcttcctcgc tcactgactc gctgcgctcg gtcgttcggc tgcggcgagc ggtatcagct 7920

cactcaaagg cggtaatacg gttatccaca gaatcagggg ataacgcagg aaagaacatg 7980

tgagcaaaag gccagcaaaa ggccaggaac cgtaaaaagg ccgcgttgct ggcgtttttc 8040

cataggctcc gcccccctga cgagcatcac aaaaatcgac gctcaagtca gaggtggcga 8100

aacccgacag gactataaag ataccaggcg tttccccctg gaagctccct cgtgcgctct 8160

cctgttccga ccctgccgct taccggatac ctgtccgcct ttctcccttc gggaagcgtg 8220

gcgctttctc atagctcacg ctgtaggtat ctcagttcgg tgtaggtcgt tcgctccaag 8280

ctgggctgtg tgcacgaacc ccccgttcag cccgaccgct gcgccttatc cggtaactat 8340

cgtcttgagt ccaacccggt aagacacgac ttatcgccac tggcagcagc cactggtaac 8400

aggattagca gagcgaggta tgtaggcggt gctacagagt tcttgaagtg gtggcctaac 8460

tacggctaca ctagaaggac agtatttggt atctgcgctc tgctgaagcc agttaccttc 8520

ggaaaaagag ttggtagctc ttgatccggc aaacaaacca ccgctggtag cggtggtttt 8580

tttgtttgca agcagcagat tacgcgcaga aaaaaaggat ctcaagaaga tcctttgatc 8640

ttttctacgg ggtctgacgc tcagtggaac gaaaactcac gttaagggat tttggtcatg 8700

cattctaggt actaaaacaa ttcatccagt aaaatataat attttatttt ctcccaatca 8760

ggcttgatcc ccagtaagtc aaaaaatagc tcgacatact gttcttcccc gatatcctcc 8820

ctgatcgacc ggacgcagaa ggcaatgtca taccacttgt ccgccctgcc gcttctccca 8880

agatcaataa agccacttac tttgccatct ttcacaaaga tgttgctgtc tcccaggtcg 8940

ccgtgggaaa agacaagttc ctcttcgggc ttttccgtct ttaaaaaatc atacagctcg 9000

cgcggatctt taaatggagt gtcttcttcc cagttttcgc aatccacatc ggccagatcg 9060

ttattcagta agtaatccaa ttcggctaag cggctgtcta agctattcgt atagggacaa 9120

tccgatatgt cgatggagtg aaagagcctg atgcactccg catacagctc gataatcttt 9180

tcagggcttt gttcatcttc atactcttcc gagcaaagga cgccatcggc ctcactcatg 9240

agcagattgc tccagccatc atgccgttca aagtgcagga cctttggaac aggcagcttt 9300

ccttccagcc atagcatcat gtccttttcc cgttccacat cataggtggt ccctttatac 9360

cggctgtccg tcatttttaa atataggttt tcattttctc ccaccagctt atatacctta 9420

gcaggagaca ttccttccgt atcttttacg cagcggtatt tttcgatcag ttttttcaat 9480

tccggtgata ttctcatttt agccatttat tatttccttc ctcttttcta cagtatttaa 9540

agatacccca agaagctaat tataacaaga cgaactccaa ttcactgttc cttgcattct 9600

aaaaccttaa ataccagaaa acagcttttt caaagttgtt ttcaaagttg gcgtataaca 9660

tagtatcgac ggagccgatt ttgaaaccgc ggtgatcaca ggcagcaacg ctctgtcatc 9720

gttacaatca acatgctacc ctccgcgaga tcatccgtgt ttcaaacccg gcagcttagt 9780

tgccgttctt ccgaatagca tcggtaacat gagcaaagtc tgccgcctta caacggctct 9840

cccgctgacg ccgtcccgga ctgatgggct gcctgtatcg agtggtgatt ttgtgccgag 9900

ctgccggtcg gggagctgtt ggctggctgg tggcaggata tattgtggtg taaacaaatt 9960

gacgcttaga caacttaata acacattgcg gacgttttta atgtactgaa ttaacgccga 10020

attaattcgg gggatctgga ttttagtact ggattttggt tttaggaatt agaaatttta 10080

ttgatagaag tattttacaa atacaaatac atactaaggg tttcttatat gctcaacaca 10140

tgagcgaaac cctataggaa ccctaattcc cttatctggg aactactcac acattattat 10200

ggagaaactc gagcttgtcg atcgacagat ccggtcggca tctactctat ttctttgccc 10260

tcggacgagt gctggggcgt cggtttccac tatcggcgag tacttctaca cagccatcgg 10320

tccagacggc cgcgcttctg cgggcgattt gtgtacgccc gacagtcccg gctccggatc 10380

ggacgattgc gtcgcatcga ccctgcgccc aagctgcatc atcgaaattg ccgtcaacca 10440

agctctgata gagttggtca agaccaatgc ggagcatata cgcccggagt cgtggcgatc 10500

ctgcaagctc cggatgcctc cgctcgaagt agcgcgtctg ctgctccata caagccaacc 10560

acggcctcca gaagaagatg ttggcgacct cgtattggga atccccgaac atcgcctcgc 10620

tccagtcaat gaccgctgtt atgcggccat tgtccgtcag gacattgttg gagccgaaat 10680

ccgcgtgcac gaggtgccgg acttcggggc agtcctcggc ccaaagcatc agctcatcga 10740

gagcctgcgc gacggacgca ctgacggtgt cgtccatcac agtttgccag tgatacacat 10800

ggggatcagc aatcgcgcat atgaaatcac gccatgtagt gtattgaccg attccttgcg 10860

gtccgaatgg gccgaacccg ctcgtctggc taagatcggc cgcagcgatc gcatccatag 10920

cctccgcgac cggttgtaga acagcgggca gttcggtttc aggcaggtct tgcaacgtga 10980

caccctgtgc acggcgggag atgcaatagg tcaggctctc gctaaactcc ccaatgtcaa 11040

gcacttccgg aatcgggagc gcggccgatg caaagtgccg ataaacataa cgatctttgt 11100

agaaaccatc ggcgcagcta tttacccgca ggacatatcc acgccctcct acatcgaagc 11160

tgaaagcacg agattcttcg ccctccgaga gctgcatcag gtcggagacg ctgtcgaact 11220

tttcgatcag aaacttctcg acagacgtcg cggtgagttc aggctttttc atatctcatt 11280

gccccccggg atctgcgaaa gctcgagaga gatagatttg tagagagaga ctggtgattt 11340

cagcgtgtcc tctccaaatg aaatgaactt ccttatatag aggaaggtct tgcgaaggat 11400

agtgggattg tgcgtcatcc cttacgtcag tggagatatc acatcaatcc acttgctttg 11460

aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt 11520

tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc 11580

atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc 11640

aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt 11700

ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca 11760

tgttatcaca tcaatccact tgctttgaag acgtggttgg aacgtcttct ttttccacga 11820

tgctcctcgt gggtgggggt ccatctttgg gaccactgtc ggcagaggca tcttgaacga 11880

tagcctttcc tttatcgcaa tgatggcatt tgtaggtgcc accttccttt tctactgtcc 11940

ttttgatgaa gtgacagata gctgggcaat ggaatccgag gaggtttccc gatattaccc 12000

tttgttgaaa agtctcaata gccctttggt cttctgagac tgtatctttg atattcttgg 12060

agtagacgag agtgtcgtgc tccaccatgt tggcaagctg ctctagccaa tacgcaaacc 12120

gcctctcccc gcgcgttggc cgattcatta atgcagctgg cacgacaggt ttcccgactg 12180

gaaagcgggc agtgagcgca acgcaattaa tgtgagttag ctcactcatt aggcacccca 12240

ggctttacac tttatgcttc cggctcgtat gttgtgtgga attgtgagcg gataacaatt 12300

tcacacagga aacagctatg accatgatta cg 12332

<210> 5

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

acgggggacg agctcggtac catgtgtagc atccactact a 41

<210> 6

<211> 39

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 6

atgtttttgg cgtcttccat tatacttatt ccaccgtac 39

<210> 7

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 7

atggaagacg ccaaaaacat 20

<210> 8

<211> 41

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 8

atacgaacga aagctctgca gttacacggc gatctttccg c 41

<210> 9

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 9

accttcttgt gcagagaatg 20

<210> 10

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 10

atagtaagaa cgccatagta g 21

<210> 11

<211> 22

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 11

ctactatggc ggtacgtatt tg 22

<210> 12

<211> 21

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 12

gtggcaatga acgcggattc t 21

<210> 13

<211> 17

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 13

tattgtgaag atagtgg 17

<210> 14

<211> 20

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 14

tgaaactgat gcattgaact 20

Claims (7)

1. A mRNA variable splicing-luciferase reporter system comprising a variable splicing region sequence of plant RLPK and a luciferase reporter sequence; the sequence of the variable shearing region of the plant RLPK is SEQ ID NO.1, the sequence of the luciferase reporter gene LUC is SEQ ID NO.2, and the sequence of the mRNA variable shearing-luciferase reporter system is SEQ ID NO. 3.

2. A plant transgenic vector of the mRNA variable splicing-luciferase reporter system, which is obtained by connecting the variable splicing region sequence of the plant RLPK of claim 1 and the luciferase reporter gene LUC sequence together into a plant expression vector pCAMBIA1300 to obtain the plant transgenic vector pCAMBIA1300: RLPK-LUC.

3. The plant transgenic vector according to claim 2, wherein the construction process comprises the following steps:

(1) extracting tomato genome DNA, and amplifying plant by using primers SEQ ID NO.5 and SEQ ID NO.6RLPKObtaining the nucleotide sequence shown as SEQ ID NO. 1;

(2) amplifying a luciferase reporter gene sequence by using primers SEQ ID NO.7 and SEQ ID NO.8 to obtain a nucleotide sequence shown as SEQ ID NO. 2;

(3) the variable splicing region sequence of RLPK and the luciferase reporter gene LUC sequence in the plant are connected to a plant expression vector pCAMBIA1300 by using a multi-fragment recombination method to obtain a plant transgenic vector pCAMBIA1300 of an mRNA variable splicing-luciferase reporter system, namely RLPK-LUC.

4. A method for breeding transgenic plants of RLPK mRNA variable splicing-luciferase reporter system, which is characterized in that the transgenic vector pCAMBIA1300 RLPK-LUC of claim 2 or 3 is stably transformed on the plants, and the transgenic plants which stably over-express the variable splicing-luciferase reporter system are obtained by a method of resistance screening and luciferase activity verification.

5. The method of claim 4, wherein said transgenic plant is Nicotiana benthamiana.

6. Use of the mRNA variable splicing-luciferase reporter system of claim 1, the plant transgenic vector of claim 2 or 3, or the transgenic plant grown by the method of claim 4 or 5 for rapid screening of whether a test gene modulates the RLPK mRNA variable splicing process in a plant.

7.A gene screening method based on the variable splicing change of RLPK, which is characterized in that an exogenous gene is overexpressed on the leaves of a transgenic plant of the mRNA variable splicing-luciferase reporter system cultivated by the method of claim 4 or 5 by using an Agrobacterium-mediated transient overexpression technology; or silencing endogenous genes of plants in the transgenic plant leaves of the mRNA variable shearing-luciferase reporter system cultured by the method of claim 4 or 5 by utilizing an agrobacterium-mediated gene silencing technology, and judging whether the genes to be detected participate in the variable shearing process of the plants by detecting the change of the luciferase activity so as to realize the rapid screening of whether the genes to be detected participate in the variable shearing process of the plants.

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