Turnip root expansion related gene BrrRSN1 and application thereof

Abstract

The invention belongs to the field of plant genetic engineering, and provides an amino acid sequence of a protein coded by a turnip root expansion related gene BrrRSN1, a CDS sequence of the turnip root expansion related gene BrrRSN1, a DNA full-length sequence of the turnip root expansion related gene BrrRSN1 and application of the turnip root expansion related gene BrrRSN1. According to the invention, turnip root expansion character related genes are excavated and subjected to functional analysis, a molecular mechanism for regulating root expansion formation by BrrRSN1 is analyzed, and more gene resources are provided for further enriching understanding of a root expansion regulation network of cruciferae root vegetable crops and high-yield breeding of root expansion crops such as radishes and root mustard; and a theoretical basis is provided for realizing directional transformation of vegetable crop storage organs.

Description

Technical Field

[0001] The invention belongs to the field of plant genetic engineering, and particularly relates to a turnip root enlargement-related gene BrrRSN1 and an application thereof. Background Art

[0002] The formation of enlarged organs is a common phenomenon in plant evolution. Among vegetable crops, enlarged, fleshy taproots are the most common edible organs. These taproots are rich in water, carbohydrates, protein, and vitamins. These taproots are modified organs of plant roots or hypocotyls, which evolved during evolution to adapt to terrestrial environments. These modified roots provide nutrients and energy, enabling plants to survive in harsh environments. In agricultural production, as the primary edible organ, fleshy roots directly influence crop yield and quality.

[0003] Turnip (Brassica rapa L.) is a Brassica vegetable from the same family as Chinese cabbage. It is an ancestor of Chinese cabbage and has enlarged, fleshy roots. Along with mustard greens, radish, and rutabaga, it belongs to the same family of cruciferous root vegetables with edible fleshy roots. As its high medicinal and feed value continues to be discovered, turnip is becoming increasingly popular among consumers.

[0004] The enlarged, fleshy taproots of Brassica rapa (Brassica rapa) are a classic model for studying the formation of plant storage organs. Currently, research on the molecular mechanisms of plant root development has primarily focused on the model plants Arabidopsis thaliana, tobacco, maize, and rice, with related studies also reported in potato and sweet potato. However, the molecular mechanisms controlling root enlargement in Brassica rapa remain largely unknown. Summary of the Invention

[0005] In order to solve the above problems existing in the prior art, the present invention provides a turnip root enlargement-related gene BrrRSN1 and its application.

[0006] The present invention provides the amino acid sequence of the protein encoded by the turnip root enlargement-related gene BrrRSN1, as shown in Sequence 1 in the sequence table;

[0007] or: having a homology of greater than or equal to 95% with sequence 1 in the sequence listing;

[0008] Or: an amino acid sequence having the same function as the protein encoded by the BrrRSN1 gene obtained by substitution, and / or deletion, and / or addition of one or more amino acid residues based on sequence 1 in the sequence listing.

[0009] The present invention also provides a CDS sequence of the turnip root enlargement-related gene BrrRSN1, as shown in Sequence 2 in the sequence table;

[0010] Or: having a homology of greater than or equal to 95% with sequence 2 in the sequence listing;

[0011] Or: A nucleotide sequence encoding a protein having the same function as the protein encoded by the BrrRSN1 gene obtained by substitution, and / or deletion, and / or addition of one or more nucleotides based on sequence 2 in the sequence listing.

[0012] The present invention also provides the full-length DNA sequence of the turnip root enlargement-related gene BrrRSN1, as shown in Sequence 3 in the sequence table;

[0013] or: having a homology of greater than or equal to 95% with sequence 3 in the sequence listing;

[0014] Or: A nucleotide sequence encoding a protein having the same function as the protein encoded by the BrrRSN1 gene obtained by substitution, and / or deletion, and / or addition of one or more nucleotides based on sequence 3 in the sequence listing.

[0015] Compared with the prior art, the present invention has the following beneficial effects:

[0016] This study identified genes associated with root enlargement in turnips and conducted functional analysis, revealing the molecular mechanisms by which they regulate root enlargement. This study will further enrich our understanding of the root enlargement regulatory network in cruciferous root vegetables, providing more genetic resources for high-yield breeding of root-enlargement crops such as radish and root mustard, and providing a theoretical basis for the targeted modification of storage organs in vegetable crops. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 These are photos of root tissues of the two parents at five different stages of the root enlargement process in Example 2; from left to right, they are 20 days (RS1), 30 days (RS2), 40 days (RS3), 50 days (RS4), and 60 days (RS5) after sowing.

[0018] Figure 2 This is a bar graph of the BrrRSN1 gene expression levels at different stages of root development of the two parents in Example 2.

[0019] Figure 3 The following are photos of the root phenotype of Arabidopsis thaliana 5 weeks after sowing in Example 3 (Figure A), a bar graph of root thickness (Figure B), and a bar graph of stem thickness (Figure C).

[0020] Figure 4 Root phenotype photos of the CK group plants and the pTY-BrrRSN1 group plants in Example 4 (Figure A), gene expression level bar graph (Figure B), and root thickness bar graph (Figure C).

[0021] Figure 5This is a map of the vector pTY-S. DETAILED DESCRIPTION

[0022] To make the technical solutions, objectives and advantages of the present invention more clear, the present invention is further described in detail below through specific embodiments. It should be understood that the specific embodiments described herein are only used to illustrate and explain the present invention and are not intended to limit the present invention.

[0023] In a first aspect, the present invention provides an amino acid sequence of a protein encoded by the turnip root enlargement-related gene BrrRSN1, the amino acid sequence of which is shown in Sequence 1 in the sequence listing;

[0024] Or: the amino acid sequence composition has greater than or equal to 95% homology with Sequence 1 in the sequence listing;

[0025] Or: an amino acid sequence having the same function as the protein encoded by the BrrRSN1 gene obtained by substitution, and / or deletion, and / or addition of one or more amino acid residues based on sequence 1 in the sequence listing.

[0026] In a second aspect, the present invention provides a CDS sequence of the turnip root enlargement-related gene BrrRSN1, the nucleotide sequence of which is shown in Sequence 2 in the sequence listing;

[0027] Or: the nucleotide sequence composition of the CDS sequence has greater than or equal to 95% homology with Sequence 2 in the sequence listing;

[0028] Or: A nucleotide sequence encoding a protein having the same function as the protein encoded by the BrrRSN1 gene obtained by substitution, and / or deletion, and / or addition of one or more nucleotides based on sequence 2 in the sequence listing.

[0029] In a third aspect, the present invention provides a full-length DNA sequence of the turnip root enlargement-related gene BrrRSN1, the nucleotide sequence of which is shown in Sequence 3 in the sequence listing;

[0030] Or: the nucleotide sequence composition of the gene has greater than or equal to 95% homology with Sequence 3 in the sequence listing;

[0031] Or: A nucleotide sequence encoding a protein having the same function as the protein encoded by the BrrRSN1 gene obtained by substitution, and / or deletion, and / or addition of one or more nucleotides based on sequence 3 in the sequence listing.

[0032] In a fourth aspect, the present invention provides PCR amplification primers for amplifying the full-length DNA sequence of the turnip root enlargement-related gene BrrRSN1 (sequence 3 in the sequence listing), comprising:

[0033] Forward primer: 5′-ATGGGAAGTTCATCTTTACC-3′;

[0034] Reverse primer: 5′-CTAGAAAAGGTTTGAGTAAC-3′.

[0035] In a fifth aspect, the present invention provides PCR amplification primers for amplifying the CDS sequence of the turnip root enlargement-related gene BrrRSN1 (sequence 2 in the sequence listing), comprising:

[0036] Forward primer: 5′-ATGGGAAGTTCATCTTTACC-3′;

[0037] Reverse primer: 5′-CTAGAAAAGGTTTGAGTAAC-3′.

[0038] In a sixth aspect, the present invention provides an application of the turnip root enlargement-related gene BrrRSN1, comprising:

[0039] By overexpressing the BrrRSN1 gene, the root enlargement trait of turnip was enhanced;

[0040] Alternatively, the root enlargement trait of turnip can be suppressed by inhibiting the expression of BrrRSN1 gene or silencing the BrrRSN1 gene, thereby reducing the content and / or activity of BrrRSN1 protein in the plant.

[0041] The above-mentioned BrrRSN1 gene overexpression operation includes: constructing an overexpression vector of the BrrRSN1 gene CDS sequence (the framework vector can be pCAMBIA2300 vector or other plant expression vectors), and then transferring the overexpression vector into plants to obtain transgenic plants overexpressing the BrrRSN1 gene.

[0042] The above-mentioned BrrRSN1 gene silencing operation includes: designing a BrrRSN1 gene silencing sequence, constructing a BrrRSN1 gene silencing vector (the framework vector can be a pTY-S vector or other gene silencing vectors), and then transferring the gene silencing vector into turnip to obtain transformed plants.

[0043] The BrrRSN1 gene silencing sequence includes a target sequence and a reverse complementary sequence of the target sequence.

[0044] Among them, the target sequences include:

[0045] 5'-TTAGATTCCATCCAACAGATCAGGAGCTCATAGGCTATTA-3'.

[0046] Preferably, specific nucleotide sequences are added to the upstream and downstream ends of the BrrRSN1 gene silencing sequence. The specific nucleotide sequences are preferably nucleotide sequences upstream and downstream (preferably 15 bp each) of the SnaBI restriction site of the linearized pTY-S vector.

[0047] Preferably, the BrrRSN1 gene silencing sequence with the specific nucleotide sequence is shown as Sequence 5 in the sequence listing.

[0048] Unless otherwise specified, the various reagents, materials, etc. used in the following examples are all products that can be obtained from commercial channels; unless otherwise specified, the various tests and detection methods used in the following examples are all conventional tests and detection methods in the field, which can be obtained from textbooks, reference books or academic journals.

[0049] Example 1

[0050] This example is used to illustrate the discovery of the BrrRSN1 gene and BrrRSN1 protein.

[0051] 1. An F2 segregating population was constructed using the turnip inbred line "MM" with expanded roots and the cabbage inbred line "Bai Yang (BY)" with non-expanded roots as parents.

[0052] Through BSA-seq and QTL analysis, a major QTL locus controlling root expansion was initially located at the front end of chromosome A01. Based on this initial localization, KASP molecular markers were designed based on sequence differences between the two parents for fine mapping, ultimately pinpointing the candidate gene to an interval of approximately 227 kb.

[0053] By performing functional annotation, gene sequencing and expression analysis on candidate genes within the 227kb interval, a gene related to the root enlargement trait of turnip was finally identified and named BrrRSN1 gene.

[0054] The Brassica rapa germplasm involved in the present invention is all from the Beijing Crop Germplasm Resource Bank (Vegetables), (Contact number: 010-81127107), and anyone can freely obtain the above varieties in order to achieve the purpose of the present invention. Among them, the turnip inbred line "MM" (i.e., "European turnip line MM" in the literature) is an inbred line with significantly enlarged roots from Western Europe. The Chinese cabbage inbred line "Bai Yang" (i.e., "Chinese cabbage line BY" in the literature) is an inbred line with normal roots from Taiwan, China. Both inbred lines are recorded in the following documents: Yu Shuancang, Zhang Fenglan, Zhao Xiang, Yu Yangjun, Zhang Deshuang, Zhao Xiuyun, Wang Weihong. An improved Brassicarapa genetic linkage map and locus-specific variations in a doubled haploid population. Plant Mol Biol Rep, 2013, 31: 558-568.

[0055] 2. The gene sequence corresponding to BrrRSN1 was searched online using the Brassica Database (BRAD). Specific primers were designed using Primer Premier 5 based on the nucleotide sequence given in the database. The primer sequences were as follows:

[0056] BrrRSN1-cdsF: 5'-ATGGGAAGTTCATCTTTACC-3';

[0057] BrrRSN1-cdsR: 5'-CTAGAAAAGGTTTGAGTAAC-3'.

[0058] 3. Use the young leaves of turnip MM as materials, quickly put them into liquid nitrogen and grind them into powder, and use the CTAB method to extract genomic DNA.

[0059] 4. Using the obtained turnip MM DNA as a template, the above-mentioned specific primers BrrRSN1-cdsF / BrrRSN1-cdsR were used to generate the nucleotide sequence of the nucleotide sequence of the turnip MM. The high-fidelity enzyme (Tks Gflex TM DNA Polymerase, Takara, Japan) was used for PCR amplification.

[0060] The PCR amplification reaction system (50 μL) includes: Tks Gflex TMDNA Polymerase 1μL, Forwardprimer 3.75μL, Reverse primer 3.75μL, 2×Gflex PCR Buffer (Mg 2+ , dNTP plus) 25 μL, turnip genomic DNA (30-50 ng / μL) 5 μL, ddH2O to 50 μL.

[0061] The PCR amplification reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s, for 35 cycles; and extension at 72°C for 5 min.

[0062] The PCR amplification product was identified by agarose gel electrophoresis and then sequenced to obtain the full-length DNA sequence of the BrrRSN1 gene, as shown in Sequence 3 in the sequence listing.

[0063] 5. Young leaves of turnip MM were used as tissue material and quickly placed in liquid nitrogen to be ground into powder. Total RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen, Beijing, China). The obtained total RNA was then used as a template for reverse transcription using the TaKaRa Primer ScriptTM RTreagent kit provided by Japan (TaKaRa Primer ScriptTM RTreagent kit, Japan) to obtain cDNA.

[0064] 6. Using cDNA as template, the above-mentioned specific primers BrrRSN1-cdsF / BrrRSN1-cdsR were used to generate the protein using high-fidelity enzyme (Tks Gflex TM DNA Polymerase, Takara, Japan) was used for PCR amplification.

[0065] The PCR amplification reaction system (50 μL) includes: Tks Gflex TM DNA Polymerase 1μL, Forwardprimer 3.75μL, Reverse primer 3.75μL, 2×Gflex PCR Buffer (Mg 2+ , dNTP plus) 25 μL, cDNA (30-50 ng / μL) 5 μL, ddH2O to 50 μL.

[0066] The PCR amplification reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s, for 35 cycles; and extension at 72°C for 5 min.

[0067] The PCR amplification product was identified by agarose gel electrophoresis and then sequenced to obtain the CDS sequence of the BrrRSN1 gene, as shown in Sequence 2 in the sequence listing.

[0068] The protein encoded by the CDS sequence of the BrrRSN1 gene is shown in Sequence 1 in the sequence listing.

[0069] Example 2

[0070] This example is used to illustrate the expression analysis of the BrrRSN1 gene at different stages of turnip root development.

[0071] 1. Test materials: Root tissues of the turnip inbred line "MM" and the cabbage inbred line "Bai Yang (BY)" at five different stages of root enlargement ( Figure 1 ).

[0072] 2. After the test materials were collected, they were quickly frozen in liquid nitrogen and total RNA was extracted using the RNAprep Pure Polysaccharide and Polyphenol Plant Total RNA Extraction Kit (Tiangen, Beijing, China). The obtained total RNA was used as a template and the reverse transcription kit (TaKaRa PrimeScript TM RT reagent kit, Japan) was used for reverse transcription to obtain cDNA.

[0073] 3. Using cDNA as template and BrrGAPDH gene as internal reference gene, the expression level of BrrRSN1 at different stages of root development of the two parents was detected by real-time fluorescence quantitative PCR.

[0074] The primers used to detect the expression level of BrrRSN1 gene are as follows:

[0075] Forward primer qRTBrrRSN1-F: 5′-TCACCAATGCGTGCCAATA-3′;

[0076] Reverse primer qRTBrrRSN1-R: 5′-GAAGCTCCTTCGACAGCT-3′.

[0077] The primers used to detect the expression level of BrrGAPDH gene are as follows:

[0078] Forward primer BrrGAPDH-F: 5′-CAGGTTTGGAATTGTCGAGG-3′;

[0079] Reverse primer BrrGAPDH-R: 5′-GAGCTGTGGAAGCACCTTTC-3′.

[0080] The PCR amplification reaction system (10 μL) includes: 5 μL of SYBR Green I Master, 0.5 μL of Forward primer, 0.5 μL of Reverse primer, 1 μL of cDNA (200 ng / μL), and 3 μL of ddH2O.

[0081] The PCR amplification reaction procedure was as follows: pre-denaturation at 95°C for 5 min; cycling: denaturation at 95°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 10 s, for 45 cycles; melting curve analysis: 95°C for 5 sec, followed by a temperature increase from 65°C to 97°C, with a step increase of 0.5°C, and fluorescence acquisition.

[0082] 4. The expression levels of BrrRSN1 in different stages of root development of the two parents are shown in Figure 4. Figure 2 The results showed that the expression level of BrrRSN1 in turnip MM was significantly higher than that in cabbage BY at all stages of root expansion.

[0083] Example 3

[0084] This example is used to describe the functional verification of the BrrRSN1 gene: an experiment on overexpression of the BrrRSN1 gene in Arabidopsis thaliana.

[0085] 1. Construction of BrrRSN1 overexpression vector

[0086] 1. Using the cDNA of MM obtained in Example 1 as a template, the following specific primers p2300-BrrRSN1-F and p2300-BrrRSN1-R were used to generate the MM. TM DNA Polymerase, Takara, Japan) was used for PCR amplification to obtain a CDS sequence with restriction enzyme cleavage sites and homology arms, the nucleotide sequence of which is shown as Sequence 4 in the sequence listing.

[0087] p2300-BrrRSN1-F:

[0088] 5'-GGTACCCGGGGATCCTCTAGAATGGGAAGTTCATCTTTACC-3';

[0089] p2300-BrrRSN1-R:

[0090] 5'-CTCGCCCTTGCTCACCATGTCGACGAAAAGGTTTGAGTAACC-3'.

[0091] The PCR amplification reaction system (50 μL) includes: Tks Gflex TMDNA Polymerase 1μL, Forwardprimer 3.75μL, Reverse primer 3.75μL, 2×Gflex PCR Buffer (Mg 2+ , dNTP plus) 25 μL, turnip cDNA (30-50 ng / μL) 5 μL, ddH2O to 50 μL.

[0092] The PCR amplification reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s, for 35 cycles; and extension at 72°C for 5 min.

[0093] 2. The pCAMBIA2300 plasmid vector (a double-stranded circular plasmid, the full sequence of which is shown in Sequence 6 in the sequence listing) was double-digested with XbaI and SalI, and then identified by agarose gel electrophoresis and the large fragment (about 10 kb, linearized pCAMBIA2300 plasmid vector) was recovered.

[0094] The enzyme digestion system (50 μL) includes: 2 μL of XbaI endonuclease (NEB), 2 μL of SalI endonuclease (NEB), 5 μL of 10× cutsmart buffer, 5 μg of plasmid pCAMBIA2300, and ddH2O to make up to 50 μL.

[0095] The above enzyme digestion reaction procedure: digest at 37℃ for 3-4h.

[0096] 3. The CDS sequence obtained in step 1 was ligated with the double-enzyme-digested plasmid (i.e., the large fragment, linearized pCAMBIA2300 plasmid vector) obtained in step 2 by homologous recombination (i.e., the two were ligated) to obtain a circular plasmid, i.e., the BrrRSN1 overexpression vector.

[0097] The ligation reaction system was as follows: 5 μL of 2×Seamless Cloning mix (Beijing Biomed), a molar ratio of 3:1 between the CDS sequence and the vector after enzyme digestion (i.e., the large fragment, linearized pCAMBIA2300 plasmid vector) (total volume of 5 μL).

[0098] The reaction procedure of the ligation reaction was: 50°C for 15 min, followed by 2 min on ice.

[0099] Sequencing confirmed that the only difference between the BrrRSN1 overexpression vector and the pCAMBIA2300 empty vector is that the pCAMBIA2300 empty vector

[0100]

[0101] This fragment was replaced with the DNA molecule shown in Sequence 4 in the sequence listing.

[0102] 2. Obtaining transgenic Arabidopsis plants

[0103] 1. Introduce the BrrRSN1 overexpression vector obtained in step 1 into Agrobacterium GV3101 to obtain recombinant Agrobacterium. Transform recombinant Agrobacterium into Arabidopsis thaliana ecotype Columbia using the floral dip method. Sow the harvested seeds in screening medium (solid 1 / 2 MS medium containing 50 μg / mL kanamycin). After two weeks, transfer the transgenic Arabidopsis plants that have grown normally in the screening medium to nutrient soil for cultivation.

[0104] 2. After culturing in nutrient soil for 2 weeks, DNA of T1 generation plants was extracted and PCR amplified using the primer pair consisting of p2300-BrrRSN1-F and p2300-BrrRSN1-R. Plants with an amplified product of 915 bp were positive transgenic plants.

[0105] 3. Self-pollinate the T1 generation plants, harvest the seeds, and cultivate the seeds into plants, which are the T2 generation plants.

[0106] The PCR amplification reaction system (10 μL) includes: 2× Taq Master Mix (Nanjing Novozymes) 5 μL, forward primer 0.4 μL, reverse primer 0.4 μL, genomic DNA (30-50 ng / μL) 1 μL, and ddH2O to 10 μL.

[0107] The PCR amplification reaction procedure was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 15 s, annealing at 60°C for 15 s, and extension at 72°C for 15 s, for 35 cycles; and extension at 72°C for 5 min.

[0108] Note: If a T1 generation plant and its self-pollinated T2 generation plant are both transgenic plants, then the T1 generation plant is a homozygous transgenic plant.

[0109] 3. Preparation of Empty Vector Transgenic Arabidopsis Plants (Control)

[0110] The BrrRSN1 overexpression vector was replaced with the pCAMBIA2300 empty vector, and the operations of steps 1 to 3 were referred to obtain empty vector-transfected plants as a control of transgenic plants.

[0111] IV. Observation of root enlargement traits in overexpressing Arabidopsis plants

[0112] Test seeds: seeds of the p2300-BrrRSN1-1 line (seeds of T2 generation plants), seeds of the p2300-BrrRSN1-2 line (seeds of T2 generation plants), seeds of empty vector-transformed plants (seeds of T2 generation plants), and seeds of Columbia ecotype Arabidopsis thaliana (WT seeds).

[0113] First, T2 transgenic seeds were sown in a screening medium (solid 1 / 2 MS medium containing 50 μg / ml kanamycin). Two weeks later, the normally growing Arabidopsis plants in the medium were cultured in nutrient soil under a 16-hour light cycle (25°C) and an 8-hour dark cycle (22°C).

[0114] Five weeks after sowing, the root and stem phenotypes of the transgenic Arabidopsis plants were observed. Figure 3 As shown, compared with the Columbia ecotype (WT), all transgenic Arabidopsis plants showed a phenotype of thickened roots and stem bases (e.g. Figure 3 The root and stem diameters of the transgenic plants were nearly twice as large as those of the Colombian ecology (Figures A, B, and C). Figure 3 Five weeks after sowing, plants grown from seeds transplanted with the empty vector showed no significant increase in root and stem thickness compared to the Columbia ecotype. These results indicate that BrrRSN1 is a positive regulator of the root enlargement phenotype.

[0115] Example 4

[0116] This example describes the functional verification of the BrrRSN1 gene: a gene silencing experiment of the BrrRSN1 gene in turnip.

[0117] 1. Construction of gene silencing vector

[0118] 1. Design a BrrRSN1 gene silencing sequence with specific nucleotide sequences at both upstream and downstream ends:

[0119] A 40bp sequence was selected from the exon of the BrrRSN1 gene as the target sequence, and the target sequence and its reverse complementary sequence were used as the gene silencing sequence. Then, a specific nucleotide sequence was added upstream and downstream of the gene silencing sequence. The specific nucleotide sequence was: 15bp nucleotide sequences upstream and downstream of the SnaBI restriction site of the linearized pTY-S vector (wherein, 15bp upstream of the SnaBI restriction site of the linearized pTY-S vector was added upstream of the gene silencing sequence, and 15bp downstream of the SnaBI restriction site of the linearized pTY-S vector was added downstream of the gene silencing sequence), forming a BrrRSN1 gene silencing sequence with a specific nucleotide sequence, as shown in Sequence 5 in the sequence listing of the sequence listing.

[0120] The nucleotide sequence of the above target sequence is:

[0121] 5'-TTAGATTCCATCCAACAGATCAGGAGCTCATAGGCTATTA-3'.

[0122] 2. Using the pTY-S vector as the base vector, digest it with the restriction endonuclease SnaBI to obtain a linearized pTY-S vector.

[0123] The pTY-S vector was modified from the TYMV virus and prepared as follows: the promoter and terminator of CAMV35s were searched in the NCBI database, the sequences were synthesized, and ligated to the 5' and 3' ends of the TYMV cDNA, respectively, and then cloned into the pMD-18T (Takara) vector; the map of the pTY-S vector is shown in Figure 5 shown.

[0124] The pTY-S plasmid is described in the literature, "Efficient virus-induced genesilencing in Arabidopsis using a 'one-step' TYMV-derived vector," and in Chinese patent application No. 201710789234.2, "A TYMV virus-induced endogenous gene silencing method in Cruciferae and its application." The vector is available from the Beijing Academy of Agriculture and Forestry Sciences. Anyone may freely obtain the vector for the purposes of the present invention.

[0125] 3. Synthesize the double-stranded DNA molecules of the above-mentioned interfering sequence and clone the above-mentioned interfering sequence into the linearized pTY-S vector by homologous recombination. That is, the BrrRSN1 gene silencing sequence and its upstream and downstream specific nucleotide sequences are ligated with the linearized pTY-S vector to construct the silencing expression vector pTY-BrrRSN1, and perform plasmid sequencing verification.

[0126] 4. The plasmid successfully verified by sequencing was transformed into stbl3 competent cells. After expansion culture, the plasmid was extracted using the PlasmidGiga Kit (Omega, D6920-01) to obtain the recombinant plasmid pTY-BrrRSN1.

[0127] 5. Dilute the recombinant plasmid with ddH2O to a concentration of 300-500 ng / μL, which is the recombinant plasmid pTY-BrrRSN1 solution. Dilute the pTY-S plasmid with ddH2O to a concentration of 300-500 ng / μL, which is the pTY-S solution.

[0128] 2. Vaccination

[0129] The true leaves of the turnip inbred line "MM" that had been cultured for 3 to 4 weeks and were at the three-leaf, one-heart stage or the four-leaf, one-heart stage were inoculated.

[0130] The specific inoculation method is as follows: First, sprinkle green silicon carbide evenly on the true leaves of the plants and gently rub to create wounds. Then, inoculate the plants in groups: 1) pTY-BrrRSN1 group: 10 μL of the recombinant plasmid pTY-BrrRSN1 solution is dropped onto the wound, rubbed until absorbed, and then allowed to stand for 2 minutes. Rinse with distilled water for 15 seconds, gently wipe with soft absorbent paper to absorb excess water, and continue incubation in the dark at 22-25°C for 24 hours. 2) pTY-S group and CK group: 10 μL of pTY-S solution and 10 μL of distilled water are used instead of the pTY-BrrRSN1 solution for inoculation, respectively.

[0131] After inoculation, the seedlings were transferred to a culturing environment with a temperature of 22-25°C and a photoperiod of 16 hours light / 8 hours dark.

[0132] 3. Root Phenotypic Observation and BrrRSN1 Expression Analysis

[0133] 1. Two weeks after the inoculation, the root phenotype of turnips should be regularly observed after the inoculated plants have grown new leaves. The root survey results are as follows: Figure 4 As shown; among them, pTY-S represents the plants of the pTY-S group, and pTY-BrrRSN1-60#, pTY-BrrRSN1-91# and pTY-BrrRSN1-95# represent three different plants of the pTY-BrrRSN1 group. Figure 4 As shown in Figure A, compared with the CK group (WT), the root diameter of the pTY-BrrRSN1 group was thinner and the root expansion phenotype was significantly inhibited. Figure 4 As shown in Figure C, the root diameter of the pTY-BrrRSN1 group was significantly thinner than that of the CK group. Compared with the CK group, the root shape phenotype and root diameter of the pTY-S group did not change significantly.

[0134] 2. Total RNA was extracted from root tissues of pTY-BrrRSN1-1, pTY-BrrRSN1-2, and pTY-BrrRSN1-3 and reverse transcribed into cDNA. PCR amplification was performed using the cDNA as a template with the primer pair qRTBrrRSN1-F and qRTBrrRSN1-R to measure BrrRSN1 gene expression (BrrGAPDH was used as an internal reference gene).

[0135] The primers used to detect the BrrGAPDH gene are as follows:

[0136] Forward primer BrrGAPDH-F: 5′-CAGGTTTGGAATTGTCGAGG-3′;

[0137] Reverse primer BrrGAPDH-R: 5′-GAGCTGTGGAAGCACCTTTC-3′.

[0138] The PCR amplification reaction system (10 μL) includes: 5 μL of SYBR Green I Master, 0.5 μL of Forward primer, 0.5 μL of Reverse primer, 1 μL of cDNA (200 ng / μl), and 3 μL of ddH2O.

[0139] The PCR amplification reaction procedure was as follows: pre-denaturation at 95°C for 5 min; cycling: denaturation at 95°C for 10 s, annealing at 60°C for 10 s, and extension at 72°C for 10 s, for 45 cycles; melting curve analysis: 95°C for 5 sec, followed by a temperature increase from 65°C to 97°C, with a step increase of 0.5°C, and fluorescence acquisition.

[0140] like Figure 4 As shown in Figure B, compared with the CK group, the expression level of the BrrRSN1 gene in the pTY-BrrRSN1 group was significantly reduced, indicating that the BrrRSN1 gene in the pTY-BrrRSN1 group was silenced, and further indicating that the reduced expression of the BrrRSN1 gene led to the suppression of the root enlargement phenotype of turnip. Compared with the CK group, the expression level of the BrrRSN1 gene in the pTY-S group was not significantly different.

[0141] The foregoing description is merely a preferred embodiment of the present invention and is not intended to limit the present invention. Those skilled in the art will readily appreciate that various modifications and variations of the present invention are possible. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention are intended to be within the scope of protection of the present invention.

Claims

1. Amino acid sequence of the protein encoded by the turnip root enlargement-related gene BrrRSN1: As shown in Sequence 1 in the sequence listing; or: having a homology of greater than or equal to 95% with sequence 1 in the sequence listing; Or: an amino acid sequence having the same function as the protein encoded by the BrrRSN1 gene obtained by substitution, and / or deletion, and / or addition of one or more amino acid residues based on sequence 1 in the sequence listing.

2. CDS sequence of the turnip root enlargement-related gene BrrRSN1: As shown in Sequence 2 in the sequence listing; or: having a homology of greater than or equal to 95% with sequence 2 in the sequence listing; Or: A nucleotide sequence encoding a protein having the same function as the protein encoded by the BrrRSN1 gene obtained by substitution, and / or deletion, and / or addition of one or more nucleotides based on sequence 2 in the sequence listing.

3. Full-length DNA sequence of the turnip root enlargement-related gene BrrRSN1: As shown in Sequence 3 in the sequence listing; or: having a homology of greater than or equal to 95% with sequence 3 in the sequence listing; Or: A nucleotide sequence encoding a protein having the same function as the protein encoded by the BrrRSN1 gene obtained by substitution, and / or deletion, and / or addition of one or more nucleotides based on sequence 3 in the sequence listing.

4. PCR primers for amplifying the CDS sequence of the turnip root enlargement-related gene BrrRSN1 according to claim 2, comprising: Forward primer: 5′-ATGGGAAGTTCATCTTTACC-3′; Reverse primer: 5′-CTAGAAAAGGTTTGAGTAAC-3′.

5. PCR primers for amplifying the full-length DNA sequence of the turnip root enlargement-related gene BrrRSN1 according to claim 3, comprising: Forward primer: 5′-ATGGGAAGTTCATCTTTACC-3′; Reverse primer: 5′-CTAGAAAAGGTTTGAGTAAC-3′.

6. Applications of the turnip root enlargement-related gene BrrRSN1, including: By overexpressing the BrrRSN1 gene, the root enlargement trait of turnip was enhanced; Alternatively, the root enlargement trait of turnip can be inhibited by inhibiting the expression of the BrrRSN1 gene or silencing the BrrRSN1 gene.

7. The use of the turnip root enlargement-related gene BrrRSN1 according to claim 6, characterized in that: The operation of overexpressing the BrrRSN1 gene includes: constructing an overexpression vector of the CDS sequence of the BrrRSN1 gene, and then transferring the overexpression vector into turnip to obtain a transformed plant with overexpression of the BrrRSN1 gene.

8. The use of the turnip root enlargement-related gene BrrRSN1 according to claim 6, characterized in that: The BrrRSN1 gene silencing operation includes: designing a BrrRSN1 gene silencing sequence, constructing a gene silencing vector of the BrrRSN1 gene, and then transferring the gene silencing vector into turnip to obtain a transformed plant.

9. The use of the turnip root enlargement-related gene BrrRSN1 according to claim 8, characterized in that: The BrrRSN1 gene silencing sequence includes: a target sequence and a reverse complementary sequence of the target sequence; The target sequence includes: 5'-TTAGATTCCATCCAACAGATCAGGAGCTCATAGGCTATTA-3'; The nucleotide sequences upstream and downstream of the restriction enzyme cleavage site include: The nucleotide sequences upstream and downstream of the SnaBI restriction site of the linearized pTY-S vector; Specific nucleotide sequences are further added to the upstream and downstream ends of the BrrRSN1 gene silencing sequence. The specific nucleotide sequences are the nucleotide sequences upstream and downstream of the restriction enzyme cutting site of the linearized framework vector.

10. The use of the turnip root enlargement-related gene BrrRSN1 according to claim 8, characterized in that: The BrrRSN1 gene silencing sequence added with the specific nucleotide sequence is shown as Sequence 5 in the sequence table.

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