Solanum lyratum PDS gene VIGS silencing system as well as construction method and application thereof

Abstract

The invention discloses a solanum lyratum PDS gene VIGS silencing system as well as a construction method and application thereof, and relates to the technical field of plant genetic engineering. A specific nucleotide fragment shown as SEQ ID NO.1 and used for silencing a solanum lyratum PDS gene is connected to a TRV2 vector, a vector TRV2-PDS is obtained through construction, agrobacterium competent cells are transformed, and a solanum lyratum plant is dip-dyed after positive bacterium liquid is mixed according to a proportion; and obtaining the solanum lyratum thunb true leaves with obviously reduced PDS gene expression quantity. Results of embodiments show that after a mixed suspension of TRV2-PDS and TRV1 agrobacterium is injected for 25 days, an obvious whitening phenotype appears, the silence system can successfully silence the solanum lyratum PDS gene, and the expression quantity of the PDS gene is reduced, so that the silence system is used for identifying the function of the solanum lyratum PDS gene, the synthesis mechanism of medicinal components of solanum lyratum can be deeply understood, and the application prospect is wide. And a way is provided for researching the color-related physiological process and quality improvement of the solanum lyratum thunb.

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 Solanum lyratum PDS gene, a construction method thereof and an application thereof. Background Art

[0002] Solanum lyratum Thunb. is a solanaceous plant with important medicinal value in traditional medicine. Modern research shows that it contains a variety of active ingredients and exhibits great application potential in aspects such as anti-tumor and anti-viral. As a key rate-limiting enzyme gene in the carotenoid synthesis pathway, the expression level of phytoene desaturase (PDS) gene directly affects the synthesis of carotenoids, and carotenoids are not only involved in physiological processes such as plant photosynthesis and photoprotection, but also closely related to the color, quality of plants and the formation of medicinal active ingredients. In-depth study of the function of Solanum lyratum PDS gene is of great significance for revealing the synthesis mechanism of medicinal components of Solanum lyratum and improving the quality of Solanum lyratum.

[0003] Virus-induced gene silencing (VIGS) is an RNA silencing technology specifically induced by virus replication and transcription. This technology inhibits the expression of plant endogenous genes through recombinant viruses inserted with target gene fragments. Compared with transgenic technology, VIGS has the advantages of being simple, efficient, not requiring the acquisition of transgenic plants, short cycle, being able to silence gene families, and low cost, and plays an important role in the research of plant functional genomics. At present, the VIGS technology has been successfully applied in various plants such as tobacco and tomatoes, but there is no report on its research in Solanum lyratum. The VIGS research on Solanum lyratum 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] Aiming at 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 Solanum lyratum PDS gene. Another technical problem to be solved by the present invention is to provide a VIGS silencing system of Solanum lyratum PDS gene. Another technical problem to be solved by the present invention is to provide a construction method of the VIGS silencing system of Solanum lyratum PDS gene. Another technical problem to be solved by the present invention is to provide an application of the VIGS silencing system of Solanum lyratum PDS gene, which is used to quickly, simply and low-cost verify the function of Solanum lyratum genes.

[0005] In order to solve the above technical problems, the technical solutions adopted by the present invention are as follows:

[0006] A specific nucleotide fragment for silencing Solanum lyratum PDS gene, the sequence of which is shown in SEQ ID NO.1.

[0007] Construction method of VIGS silencing system for Solanum lyratum Thunb. PDS gene, comprising: ligating the specific nucleotide fragment for silencing Solanum lyratum Thunb. PDS gene shown in SEQ ID NO.1 to the TRV2 vector to construct the vector TRV2-PDS, namely the VIGS silencing system for Solanum lyratum Thunb. PDS gene.

[0008] Application of the VIGS silencing system for Solanum lyratum Thunb. PDS gene in inhibiting the expression of Solanum lyratum Thunb. PDS gene, comprising the following steps:

[0009] 1) Construct the VIGS silencing system for Solanum lyratum Thunb. PDS gene, namely the vector TRV2-PDS;

[0010] 2) Transform the Agrobacterium competent cells with TRV1 and the recombinant plasmid containing the TRV2-PDS vector fragment respectively, and collect the positive bacterial liquid after culturing;

[0011] 3) Mix the TRV2-PDS positive bacterial liquid and the TRV1 positive bacterial liquid in a ratio to prepare an infection solution, and infect Solanum lyratum Thunb. seedlings;

[0012] 4) Cultivate, screen and obtain Solanum lyratum Thunb. lines with significantly reduced expression level of Solanum lyratum Thunb. PDS gene.

[0013] The mixing ratio of the TRV2-PDS positive bacterial liquid and the TRV1 positive bacterial liquid is 1:1.

[0014] The OD of the infection solution 600 is 0.5.

[0015] Application of the VIGS silencing system for Solanum lyratum Thunb. PDS gene in identifying the function of Solanum lyratum Thunb. PDS gene.

[0016] Application of the VIGS silencing system for Solanum lyratum Thunb. PDS gene in regulating the leaf color of Solanum lyratum Thunb.

[0017] Compared with the prior art, the beneficial effects of the present invention are:

[0018] 1) The specific nucleotide fragment for silencing Solanum lyratum Thunb. PDS gene disclosed in the present invention has the sequence shown in SEQ ID NO.1. After transforming the Agrobacterium competent cells with the vector TRV2-PDS constructed by the specific nucleotide fragment for silencing Solanum lyratum Thunb. PDS gene, an infection solution is prepared to infect Solanum lyratum Thunb. plants; cultivate, screen and obtain the true leaves of Solanum lyratum Thunb. with significantly reduced expression level of Solanum lyratum Thunb. PDS gene.

[0019] 2) In the present invention, a VIGS silencing vector of TRV2-PDS was constructed for the first time to silence Solanum lyratum Thunb. Through this silencing system, the PDS gene of Solanum lyratum Thunb. can be successfully silenced, significantly reducing the expression level of the PDS gene, which can be used to identify the function of the PDS gene of Solanum lyratum Thunb. and help to deeply understand the synthesis mechanism of the medicinal components of Solanum lyratum Thunb., etc.; by using this system, the regulation of the leaf color of Solanum lyratum Thunb. can be realized, providing a way for studying the physiological processes related to the color and quality improvement of Solanum lyratum Thunb.; it helps to deeply study the function of the PDS gene of Solanum lyratum Thunb., which is of great significance for revealing the formation mechanism of the medicinal active components of Solanum lyratum Thunb. and improving the quality of Solanum lyratum Thunb., and helps to enhance the medicinal value and economic value of Solanum lyratum Thunb. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] Figure 1 It is a figure for verifying the function of VIGS of Solanum lyratum Thunb. (A is the Solanum lyratum Thunb. plant 25 days after germination when injected with TRV; B is the successful expression of GFP in the injected leaf and systemic leaf 4 days after TRV injection; C is the obvious albino phenotype of the Solanum lyratum Thunb. plant 25 days after TRV injection; D is the detection of the interference effect of the PDS gene of Solanum lyratum Thunb. by semi-qPCR; E is the detection of the interference effect of the PDS gene of Solanum lyratum Thunb. by qPCR. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0021] 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 there is no detailed description, 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: A Laboratory Manual" (Third Edition) by J. Sambrook or the conventional methods in the art, or carried out according to the kits and product manuals.

[0022] The material used in this application is Solanum lyratum Thunb., and the seeds were collected from Jinpen Mountain National Forest Park in Xinfeng, Ganzhou, Jiangxi.

[0023] Example 1

[0024] 1. Extraction of total RNA and reverse transcription

[0025] Collect the albino leaves of Solanum lyratum Thunb., quickly freeze them in liquid nitrogen and then grind them. Take 0.3 mL of the powder into a 2 mL sterilized flat-bottom centrifuge tube, add 1 mL of transZol reagent (purchased from Quanshi Jin, product number: ET101), shake vigorously for 20 s, let it stand at room temperature for 5 min, add 200 μL of RNA Extraction reagent, shake vigorously for 20 s, after standing at room temperature for 5 min, centrifuge at 12000 rpm at 4 °C for 10 min. Take 800 μL of the supernatant into a new 1.5 mL sterilized pointed-bottom centrifuge tube, and add 600 μL of isopropanol. After mixing, let it stand at room temperature for 10 min, centrifuge at 12000 rpm at 4 °C for 10 min. Wash the precipitate with 1 mL of 75% ethanol, dry it in a laminar flow bench and then add 50 μL of sterilized ddH2O to dissolve the RNA. Use the ToloScript All-in-one RT EasyMix for qPCR kit (product number: 22107) to perform reverse transcription of RNA, and operate according to the method in the instruction manual.

[0026] 2. Cloning of the PDS silencing fragment of Solanum lyratum Thunb.

[0027] Download the whole-genome CDS sequence of Solanum lyratum Thunb. from the Solanum lyratum Thunb. genome website (https: / / ngdc.cncb.ac.cn / gwh / Assembly / 37524 / show). After aligning with the known PDS sequence of tomato, the PDS gene number of Solanum lyratum Thunb. is obtained as GWHTCAXC002235. Design PDS primers containing homologous recombination adapters, and use the DNA of Solanum lyratum Thunb. as a template to perform PCR amplification to obtain the PDS fragment. The primer sequences are as follows:

[0028] Forward primer:

[0029] 5’-gaggagaagagcccCATTGAACAGATTTCTTCAGGAGAAACATGGTTCAA AAATGG-3’;

[0030] Reverse primer:

[0031] 5’-cgacgacaagacccAAAGAAGCTTGAGAATATCCACTGGAGTGGCAAACA CAAAAG-3’.

[0032] The PCR amplification conditions are as follows: 25 μL of 2×phanta PCR Buffer, 1 μL of 10 mM dNTP Mix, 2 μL each of 10 μM BS-F and BS-R, 1 μL of 5 U / μL phanta high-fidelity enzyme, 1 μL of 100 ng / μL template DNA, add ddH2O to 50 μL.

[0033] The PCR amplification program was as follows: 95°C for 3 min; 95°C for 20 sec, 55°C for 20 sec, 72°C for 20 sec, for 35 cycles; 72°C for 5 min.

[0034] Sequencing was performed to obtain a 225-bp specific nucleotide fragment for silencing the PDS gene of Solanum lyratum Thunb., and its sequence is shown as SEQ ID NO.1.

[0035] Example 2

[0036] 1. Construction of viral vector

[0037] First, the TRV2 backbone vector was digested with the smaI restriction endonuclease at 25°C. The digested TRV2 fragment and the amplified specific nucleotide fragment for silencing the PDS gene of Solanum lyratum Thunb. were reacted with a homologous recombinase (Tolo Harbour, catalog number: 24305) and then transformed into competent Escherichia coli DH5a strains. Positive monoclonal Escherichia coli colonies were obtained by first-generation Sanger sequencing. After culturing in an LB solution containing the antibiotic kanamycin, plasmids were extracted and then transformed into Agrobacterium tumefaciens GV3101. After identifying positive clones by colony PCR, plasmid sequencing was performed for verification, and the successfully ligated recombinant viral vector TRV2-PDS was obtained.

[0038] 2. Preparation of infection solution

[0039] Agrobacterium tumefaciens GV3101 carrying TRV2-PDS and TRV1 was cultured in an LB solution containing kanamycin and rifampicin. After culturing in a constant-temperature shaker at 28°C for 14 h, the Agrobacterium precipitate was obtained by centrifugation at 4000 rpm at room temperature, and the bacterial suspension was suspended with the prepared buffer (take 30 mL of sterilized ddH2O, sequentially add 0.5 mL of 1 M magnesium chloride, 0.5 mL of 1 M MES, and 0.1 mL of 0.125 M acetosyringone, and then make up the volume to 50 mL with sterilized ddH2O) until the OD 600 = 0.5; TRV1 and TRV2-PDS were mixed at a ratio of 1:1 to form the infection solution.

[0040] 3. Infection of Solanum lyratum Thunb.

[0041] The bacterial suspension was injected into the back of the leaves of Solanum lyratum Thunb. at 25 days old using a syringe, and then placed in an artificial climate chamber with a light intensity of 10000 lx, a temperature of 25°C, and a photoperiod of 16 h light cultivation and 8 h dark cultivation.

[0042] 4. Phenotypic changes of gene-silenced lines

[0043] Three days after infection with Agrobacterium tumefaciens TRV, the expression of green fluorescent protein GFP was observed using a hand-held ultraviolet lamp.

[0044] The results were as Figure 1As shown in A-C, the TRV virus carrying GFP could be observed for GFP expression in the inoculated leaves and systemic leaves at the initial stage of Agrobacterium injection, and obvious albino characteristics appeared 25 days after infection.

[0045] 5. Detection of PDS gene silencing

[0046] Total RNA was extracted from the albino leaves and the leaves of Solanum lyratum that were not inoculated at the same time, and reverse transcription was carried out. Semi-quantitative and quantitative detections were performed using the PDS primers and Actin primers of Solanum lyratum. The primer sequences are shown as follows:

[0047] Actin-F: 5’-TGCTCCACGAGCTGTATTCCCT-3’,

[0048] Actin-R: 5’-TCTCCATATCATCCCAATTGCTA-3’;

[0049] PDS-F: 5’-CTTTTTAGATGGTAATCCTCCTGA-3’,

[0050] PDS-R: 5’-TTCCATCCTCATTCAGCTCAAT-3’.

[0051] The results are as Figure 1 shown in D and E. The expression level of PDS in the leaves of Solanum lyratum injected with TRV2-PDS was significantly lower than that of the control leaves, indicating that the TRV virus carrying PDS successfully silenced the PDS gene of Solanum lyratum.

[0052] The above description is only illustrative and not restrictive for the present invention. Those of ordinary skill in the art understand that many modifications, changes 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 Solanum lyratum Thunb. PDS gene, with the sequence shown in SEQ ID NO.

1.

2. Construction method of VIGS silencing system for PDS gene of Solanum lyratum Thunb., characterized in that, Comprising: The specific nucleotide fragment for silencing the Solanum lyratum Thunb. PDS gene shown in SEQ ID NO.1 is ligated to the TRV2 vector to construct the vector TRV2-PDS, namely the VIGS silencing system of the Solanum lyratum Thunb. PDS gene.

3. The VIGS silencing system of the Solanum lyratum Thunb. PDS gene constructed by the construction method described in claim 2.

4. Application of the VIGS silencing system of the Solanum lyratum Thunb. PDS gene described in claim 3 in inhibiting the expression of the Solanum lyratum Thunb. PDS gene.

5. The application according to claim 4, characterized in that, Comprising the following steps: 1) Construct the VIGS silencing system of the Solanum lyratum Thunb. PDS gene, namely the vector TRV2-PDS; 2) Transform the Agrobacterium competent cells with TRV1 and the recombinant plasmid containing the TRV2-PDS vector fragment respectively, and collect the positive bacterial liquid after cultivation; 3) Prepare an infection solution by mixing the TRV2-PDS positive bacterial liquid and the TRV1 positive bacterial liquid in a ratio, and infect the Solanum lyratum Thunb. seedlings; 4) Cultivate, screen and obtain Solanum lyratum Thunb. lines with a significantly reduced expression level of the Solanum lyratum Thunb. PDS gene.

6. The application according to claim 5, wherein The ratio of mixing the TRV2-PDS positive bacterial liquid and the TRV1 positive bacterial liquid is 1:

1.

7. The application according to claim 5, wherein The OD of the infection solution 600 is 0.

5.

8. Application of the VIGS silencing system of the Solanum lyratum Thunb. PDS gene described in claim 3 in identifying the function of the Solanum lyratum Thunb. PDS gene.

9. Application of the VIGS silencing system of the Solanum lyratum Thunb. PDS gene described in claim 3 in regulating the leaf color of Solanum lyratum Thunb.

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