Rheum dwarf RnCLA1 gene VIGS silencing system as well as construction method and application thereof
By constructing and applying the TRV2-RnCLA1 vector, specific silencing of the dwarf rhubarb RnCLA1 gene is achieved, solving the problem of difficulty in verifying the function of the dwarf rhubarb gene in the prior art, and providing a fast, simple and low-cost gene function verification method.
Patent Information
- Application Number
- CN202510455385.9
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- Filing Date
- 2025-04-11
- Publication Date
- 2025-07-11
AI Technical Summary
The lack of specific nucleotide fragments and effective VIGS silencing systems for silencing the RnCLA1 gene in the prior art, making it difficult to quickly, easily and at low cost to verify the function of the RnCLA1 gene.
A specific nucleotide fragment for silencing the dwarf rhubarb RnCLA1 gene was constructed and ligated to the pTRV2 vector to form the TRV2-RnCLA1 vector. The dwarf rhubarb plants were infected by Agrobacterium, and the VIGS silencing system was established, which significantly reduced the RnCLA1 gene expression.
The specific silencing of the dwarf rhubarb RnCLA1 gene was successfully achieved, and the leaves showed a significant albinotype, and the RnCLA1 gene expression decreased by 64%, verifying the rapid, simple and low-cost method of the dwarf rhubarb function.
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Figure CN120290565A_ABST
Abstract
Description
Technical Field
[0001] The present invention belongs to the technical field of plant genetic engineering, and more specifically, relates to a VIGS silencing system of Rheum nanum RnCLA1 gene, a construction method thereof, and an application thereof. Background Art
[0002] Rheum nanum Siev. ex Pall. is a perennial dwarf, stout herb in the family Polygonaceae and the genus Rheum, with a height of up to 35 cm; the root is straight or curved, long cylindrical, and the top of the rhizome is surrounded by multiple layers of brown membranous stipules, and the stipules are smooth and hairless. The leaves are leathery, reniform-circular or nearly circular, with a broad round apex, a rounded or very shallow cordate base, palmate veins, yellow-green on the upper surface of the leaf, lighter in color on the lower surface, the petiole is short and thick, the inflorescence grows from the top of the rhizome, the flowers are densely clustered, and the bracts are scale-like; the pedicels are relatively thick, the perianth segments are nearly fleshy, yellowish-white, often with purplish-red smears, the disc is annular, slightly thickened and fleshy; the fruit is reniform-circular, red, and the longitudinal veins are close to the edge of the wing. The seeds are ovate, flowering from May to June, and fruiting from July to September. Rheum likes a cool and dry climate and is highly cold-resistant. It is prone to root rot under high temperature and humid conditions. It is suitable to be planted in sandy loam with deep soil layer and good drainage. Neutral and slightly alkaline soils are preferred. Rheum has strong vitality, and the germination ability of seeds can be maintained for 3 - 4 years. Only leaf clusters are formed in the year of sowing or the second year. It turns green in early April every year, flowers from May to June in the third year, and the fruits mature from June to October.
[0003] Virus-induced gene silencing (VIGS) is an RNA silencing technology specifically induced by virus replication and transcription. This technology inhibits the expression of endogenous genes in plants through recombinant viruses inserted with target gene fragments. Compared with transgenic technology, VIGS has the advantages of simplicity, high efficiency, no need to obtain transgenic plants, short cycle, ability to silence gene families, low cost, etc., and plays an important role in the research of plant functional genomics. At present, the VIGS technology has been successfully applied in many plants, such as tobacco, tomato, etc., but there is no report on its research in Rheum nanum. The VIGS research on Rheum nanum is still in a blank state, but its biological characteristics and medicinal value provide potential directions for future gene function research. Summary of the Invention
[0004] In view of the above problems existing in the prior art, the technical problems to be solved by the present invention are to provide a specific nucleotide fragment for silencing the RnCLA1 gene of Rheum nanum. Another technical problem to be solved by the present invention is to provide a VIGS silencing system for the RnCLA1 gene of Rheum nanum. Another technical problem to be solved by the present invention is to provide a method for constructing a VIGS silencing system for the RnCLA1 gene of Rheum nanum. The technical problem to be solved by the present invention is also to provide the application of the VIGS silencing system for the RnCLA1 gene of Rheum nanum, which is used to quickly, simply and low-cost verify the gene function of Rheum nanum.
[0005] To solve the above technical problems, the technical solutions adopted by the present invention are as follows:
[0006] A specific nucleotide fragment for silencing the RnCLA1 gene of Rheum nanum, and the sequence is as shown in SEQ ID NO.1.
[0007] A method for constructing a VIGS silencing system for the RnCLA1 gene of Rheum nanum, comprising: ligating the specific nucleotide fragment for silencing the RnCLA1 gene of Rheum nanum as shown in SEQ ID NO.1 to the pTRV2 vector to construct the vector TRV2-RnCLA1, that is, the VIGS silencing vector for the RnCLA1 gene of Rheum nanum.
[0008] The application of the VIGS silencing system for the RnCLA1 gene of Rheum nanum in inhibiting the expression of the RnCLA1 gene of Rheum nanum, comprising the following steps:
[0009] 1) Construct a VIGS silencing vector for the RnCLA1 gene of Rheum nanum, that is, the vector TRV2-RnCLA1;
[0010] 2) Transform the TRV1 and the recombinant plasmid containing the TRV2-RnCLA1 vector fragment into Agrobacterium competent cells respectively, and culture the Agrobacterium of TRV2-RnCLA1 after cultivation;
[0011] 3) Mix the Agrobacterium liquid of TRV2-RnCLA1 and the Agrobacterium liquid of TRV1 according to a ratio to prepare an infection solution to infect the Rheum nanum plants;
[0012] 4) Cultivate, screen and obtain the Rheum nanum lines with significantly reduced expression level of the RnCLA1 gene of Rheum nanum.
[0013] The ratio of the above mixing is 1:1.
[0014] The OD 600 of the above infection solution is 1.5.
[0015] The application of the VIGS silencing system for the RnCLA1 gene of Rheum nanum in identifying the function of the RnCLA1 gene of Rheum nanum.
[0016] Application of VIGS silencing system of Rheum nanum RnCLA1 gene in regulating leaf color of Rheum nanum
[0017] Compared with the prior art, the beneficial effects of the present invention are as follows:
[0018] 1) The specific nucleotide fragment for silencing the RnCLA1 gene of Rheum nanum disclosed in the present invention has a sequence as shown in SEQ ID NO.1. After constructing the vector TRV2-RnCLA1 with the specific nucleotide fragment for silencing the RnCLA1 gene of Rheum nanum and transforming the competent cells of Agrobacterium tumefaciens, an infection solution was prepared to infect the Rheum nanum plants; the Rheum nanum plants with significantly reduced expression level of the RnCLA1 gene of Rheum nanum were cultivated, screened and obtained. The results showed that: compared with the wild type (WT) and the blank control of pTRV1 + pTRV2, the leaves of the Rheum nanum plants injected with the pTRV1 + pTRV2-RnCLA1 infection solution showed obvious albino phenotypes, while the leaves of the blank control of pTRV1 + pTRV2 were normal in color and did not show albino phenotypes.
[0019] 2) The VIGS silencing vector of pTRV2-RnCLA1 was constructed for the first time in the present invention, and Rheum nanum was subjected to silencing treatment. After infecting with the pTRV2-RnCLA1 silencing vector, the expression level of RnCLA1 in the infected plants decreased by 64%. The results showed that the VIGS system could be successfully applied to Rheum nanum. Description of the Drawings
[0020] Figure 1 It is a leaf phenotype diagram of the pTRV2-RnCLA1 silencing line;
[0021] Figure 2 It is a detection result diagram after the plants are infected with the virus;
[0022] Figure 3 It is a diagram of the change in the relative expression level of the RnCLA1 gene in the pTRV2-RnCLA1 silencing line. Detailed Embodiments
[0023] To make the objectives, technical solutions and advantages of the present invention clearer, the present invention will be further described below in conjunction with specific embodiments. In the following embodiments, if not otherwise specified in detail, the technical means used are all conventional means well known to those skilled in the art. For the molecular biology experimental methods not specifically described, they can be referred to the methods listed in "Molecular Cloning Experiment Guide" (Third Edition) by J. Sambrook or the conventional methods in the art, or carried out according to the kits and product manuals.
[0024] The materials used in this application are Rheum nanum, which are planted on the campus of Xinjiang Agricultural University.
[0025] Example 1
[0026] 1. Extraction of total RNA and reverse transcription
[0027] The total RNA of the young leaves of Rheum dwarfum was extracted using the TIANGEN polysaccharide and polyphenol plant total RNA extraction kit, and 1 μg of total RNA was reverse transcribed into cDNA using a reverse transcription kit (Vazyme). All experimental steps were carried out according to the instructions.
[0028] 2. Cloning of the silencing fragment of R. dwarfi Rheum RnCLA1
[0029] Using the cDNA of Rheum officinale as a template, PCR amplification was performed using specific primers that added homology arms to the RnCLA1 silencing fragment. The amplified product was purified and stored at -20°C for later use. The primer sequences are as follows:
[0030] Forward Primer:
[0031] 5'-gcctccatggggatccCCATAGTTCTAATAGCATCTCTGCTGGC-3';
[0032] Reverse Primer:
[0033] 5'-agacgcgtgagctcggtaccGACCGTCAACAGGGCCG-3'.
[0034] PCR amplification conditions were: 10 μL high-fidelity enzyme Mix, 7 μL ddH2O, 1 μL forward primer, 1 μL reverse primer, and 1 μL cDNA.
[0035] The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s, annealing at 58°C for 30 s, and extension at 72°C for 60 s for a total of 30 cycles; and extension at 72°C for 5 min.
[0036] The specific nucleotide fragment of 443 bp used for silencing R. dwarfi Rheum RnCLA1 was obtained by sequencing, and its sequence is shown as SEQ ID NO.1.
[0037] Example 2
[0038] 1. Construction of viral vector
[0039] The pTRV2 viral vector was double-digested with BamHI and KpnI. The digestion reaction system was: 10 μL 200 ng / μL plasmid, 2 μL rCutSmart TM Buffer, 1 μL BamHI, 1 μL KpnI, 6 μL ddH2O. Incubate at 37℃ for 1 hour. Cut the enzyme digestion product into a gel to recover the linear plasmid and store at -20℃ for later use.
[0040] Ligate the PCR amplification product with the linear pTRV2 plasmid. Transfer it into Escherichia coli, coat it on an LB plate medium containing 50 mg / L kanamycin antibiotic, and incubate it upside down at 37 °C for 12 - 24 h. Pick a single colony into 1 mL of liquid LB containing 50 mg / L kanamycin antibiotic and incubate it at 37 °C with shaking at 200 r / min for 12 h. Screen for positive clones by colony PCR. After sequencing verification, obtain the recombinant vector of pTRV2-RnCLA1. Extract the plasmid and store it at -20 °C for later use.
[0041] The ligation system is as follows: 4 μL of purified RnCLA1 fragment product, 1 μL of linearized vector, 2 μL of CEⅡ buffer, 1 μL of Novazyme ExnaseⅡ enzyme, and 2 μL of ddH2O.
[0042] 2. Prepare the infiltration solution
[0043] Transform the plasmids of pTRV1, pTRV2, and pTRV2-RnCLA1 into Agrobacterium tumefaciens GV3101 respectively, coat them on an LB plate medium containing two antibiotics, 50 mg / L kanamycin and 50 mg / L rifampicin, and incubate them upside down at 28 °C for 24 - 36 h. After single colonies grow, pick a single colony into 1 mL of liquid LB containing 50 mg / L kanamycin + 50 mg / L rifampicin antibiotics and incubate it at 28 °C with shaking at 200 r / min for 24 h. Conduct colony PCR identification to obtain the positive Agrobacterium strains containing the plasmids of pTRV1, pTRV2, and pTRV2-RnCLA1 respectively. Transfer them into the induction LB medium (50 mg / L kanamycin + 50 mg / L rifampicin + 10 mM morpholineethanesulfonic acid + 20 μM acetosyringone) at a volume ratio of 1:50 for induction culture for 24 - 48 h. After induction culture, centrifuge at 6000 r / min for 15 min to collect the bacterial cells. Use sterile water as the stock solution, and add 200 μM acetosyringone, 10 mM magnesium chloride, and 10 mM morpholineethanesulfonic acid to prepare a suspension, resuspend the bacterial cells, and adjust OD 600 = 1.5 to obtain the VIGS transformation bacterial solution.
[0044] Mix the pTRV1 transformation bacterial solution with the pTRV2 transformation bacterial solution and the pTRV2-RnCLA1 transformation bacterial solution at a ratio of 1:1 (pTRV1 + pTRV2, pTRV1 + pTRV2-RnCLA1) to obtain the infiltration solution. Let it stand at room temperature in the dark for 3 h for infiltration.
[0045] 3. Phenotypic changes of the gene-silenced lines
[0046] Select Rheum nanum seedlings without true leaves. Use a syringe without a needle to aspirate an appropriate amount of the mixed transformed bacterial solution, and inject the infection solution into the cotyledons of 18-day-old seedlings. After culturing in the dark at 20 °C for 1 day, transfer them to a light incubator for continued culturing under the conditions of 16 h day (24 °C) / 8 h night (22 °C), with a light intensity of 600 μmol·m -2 ·s -1 , and a humidity of 70%. Observe the phenotypic changes of the seedlings during this period.
[0047] The results are as Figure 1 shown. Compared with the wild type (WT) and the blank control of pTRV1 + pTRV2, the leaves of Rheum nanum plants injected with the pTRV1 + pTRV2-RnCLA1 infection solution showed obvious albino phenotypes, while the leaves of the blank control of pTRV1 + pTRV2 were normal in color and did not show albino phenotypes.
[0048] 4. Silencing detection
[0049] Once the albino phenomenon appears in the pTRV2-RnCLA1 line, immediately pick the new leaves of the pTRV1 + pTRV2-RnCLA1 line, the pTRV1 + pTRV2 line, and the line without Agrobacterium injection (WT) for virus detection. The primer sequences are as follows:
[0050] TRV2-qF: 5’-AGGAAGTGGCTTGACGAC-3’,
[0051] TRV2-qR: 5’-TCCCCTATGGTAAGACAATGAG-3’.
[0052] The results are as Figure 2 shown. No TRV virus spread was detected in the new leaves of the WT line, and specific fragments of the virus were detected in the pTRV1 + pTRV2-RnCLA1 line and the pTRV1 + pTRV2 line, and the sizes were consistent with the expected values.
[0053] 5. Gene expression detection
[0054] Extract the RNA from the new leaves of the pTRV2-RnCLA1 line, the pTRV2 line, and the line without Agrobacterium injection, and perform reverse transcription. Use Rheum nanum β-actin as the internal reference, and the primer sequences are as follows:
[0055] RnCLA1-qF: 5’-GCCTTCTTGTTTCCGTTACCC-3’,
[0056] RnCLA1-qR: 5’-CCGCCCTCTTCCTATCTCA-3’;
[0057] β-actin-qF: 5’-AGGGTCCAATGCTTTATC-3’,
[0058] β-actin-qR: 5’-CAGTCTTCTCCACCACAA-3’.
[0059] The qPCR amplification system was as follows: 10 μL of 2×SYBR Premix Ex TaqTM, 0.4 μL of 10 μM upstream primer, 0.4 μL of 10 μM downstream primer, 2 μL of 200 ng / μL cDNA, 0.4 μL of ROX Reference Dye II, and 6.8 μL of ddH2O.
[0060] The PCR amplification program was: pre-denaturation at 95°C for 3 min; denaturation at 95°C for 10 s and annealing at 60°C for 30 s, for a total of 30 cycles.
[0061] The results were as Figure 3 shown. After infecting with the pTRV2-RnCLA1 silencing vector, the expression level of RnCLA1 in the infected plants decreased by 64%. In summary, the silencing system provided by the present invention can identify the RnCLA1 gene in Rheum nanum.
[0062] The above description is illustrative rather than restrictive to the present invention. Those of ordinary skill in the art understand that many modifications, variations or equivalents can be made without departing from the spirit and scope defined by the appended claims, and all of them will fall within the protection scope of the present invention.
Claims
1. A specific nucleotide fragment for silencing the Rheum nanum RnCLA1 gene, with the sequence shown in SEQ ID NO.
1.
2. Method for constructing VIGS silencing system of RnCLA1 gene of Rheum nanum, characterized in that, Comprising: The specific nucleotide fragment for silencing the Rheum nanum RnCLA1 gene shown in SEQ ID NO.1 is ligated to the pTRV2 vector to construct the vector TRV2-RnCLA1.
3. The VIGS silencing system of the Rheum nanum RnCLA1 gene constructed by the construction method described in claim 2.
4. The application of the VIGS silencing system of the Rheum nanum RnCLA1 gene described in claim 3 in inhibiting the expression of the Rheum nanum RnCLA1 gene.
5. The application according to claim 4, wherein Comprising the following steps: 1) Construct a VIGS silencing vector for the Rheum nanum RnCLA1 gene, namely the vector TRV2-RnCLA1; 2) Transform the Agrobacterium competent cells with TRV1 and the recombinant plasmid containing the TRV2-RnCLA1 vector fragment respectively, and obtain the Agrobacterium of TRV2-RnCLA1 after cultivation; 3) Mix the Agrobacterium liquid of TRV2-RnCLA1 and the Agrobacterium liquid of TRV1 in a ratio and configure an infection solution to infect the Rheum nanum plants; 4) Cultivate, screen and obtain the Rheum nanum lines with a significantly reduced expression level of the Rheum nanum RnCLA1 gene.
6. The application according to claim 5, wherein The ratio of the mixing is 1:
1.
7. The application according to claim 5, wherein The OD of the infection solution 600 is 1.
5.
8. The application of the VIGS silencing system of the Rheum nanum RnCLA1 gene described in claim 3 in identifying the function of the Rheum nanum RnCLA1 gene.
9. The application of the VIGS silencing system of the Rheum nanum RnCLA1 gene described in claim 3 in regulating the leaf color of Rheum nanum.