A VIGS silencing system of Ilex dabieshanensis IdChlH gene and its construction method and application

Abstract

The present invention discloses a VIGS silencing system of IdChlH gene of Ilex dabieshanensis and its construction method and application, and belongs to the technical field of plant genetic engineering. The IdChlH gene of Ilex dabieshanensis described in the present invention has a sequence as shown in SEQ ID NO.1, and the silencing system has a silencing fragment gene sequence as shown in SEQ ID NO.2. By constructing a pTRV2 virus silencing expression vector containing a specific fragment of the IdChlH gene, the leaves of Ilex dabieshanensis are infected by Agrobacterium-mediated method, and the endogenous IdChlH gene of Ilex dabieshanensis is induced to be silenced, which effectively reduces the expression level of the IdChlH gene of Ilex dabieshanensis, thereby causing the leaves to have a yellow phenotype. The present invention constructs a VIGS technical system of Ilex dabieshanensis for the first time. The VIGS technical system is simple, rapid and efficient, and provides a powerful tool for large-scale gene function research on Ilex dabieshanensis.

Description

Technical Field

[0001] The invention belongs to the technical field of plant genetic engineering, and more specifically relates to a VIGS silencing system of Ilex dabieshanensis IdChlH gene and a construction method and application thereof. Background Art

[0002] Virus-induced gene silencing (VIGS) is a reverse genetics technology developed based on the plant's defense mechanism against RNA viruses. This technology uses a recombinant viral vector carrying a target gene fragment to specifically silence the expression of endogenous genes in plants through post-transcriptional gene silencing, thereby achieving rapid identification of gene function. Compared with traditional transgenic technology, VIGS technology has the advantages of short cycle, high silencing efficiency, simple operation and low cost, and has become one of the effective technical means in the field of functional genomics research. This technology has been widely used in gene function research such as plant growth and development, disease and pest resistance, adverse stress, signal transduction, and metabolic regulation.

[0003] A variety of plant viruses have been developed into VIGS vectors, among which tobacco rattle virus (TRV) is currently the most widely used and most effective VIGS virus vector due to its wide host range and relatively mild symptoms of virus infection. TRV-VIGS vectors have been successfully applied to a variety of plants such as Solanaceae (tomato, pepper, eggplant, tobacco), Arabidopsis, poppy, cotton, petunia, strawberry, etc., but are mostly limited to herbaceous plants and are currently less used in woody plants. In VIGS, a reporter gene with an obvious silenced phenotype is required to determine whether VIGS is effective. The magnesium ion chelatase H subunit gene (ChlH) is a key enzyme gene for chlorophyll synthesis. The silencing or deletion of the ChlH gene in plants will significantly affect the accumulation of chlorophyll, resulting in a yellow phenotype in the silent leaves. Therefore, the ChlH gene is an effective reporter gene for the evaluation of the VIGS system. In addition, environmental conditions, such as temperature and humidity, also play an important role in the efficiency of the VIGS system.

[0004] Ilex dabieshanensis, also known as small bitter tea, is an evergreen broad-leaved tree of the Aquifoliaceae family. It is an important gardening tree species with high ornamental and economic value, combining ornamental, medicinal and tea-drinking uses. With the development of high-throughput sequencing technology, a large number of gene expression sequence tags can be obtained. However, due to the long juvenile period of forest plants such as Ilex dabieshanensis and the difficulty of genetic transformation, the progress of gene function research has been limited. VIGS technology is expected to break through the above bottleneck and quickly identify the gene function of this type of woody plants. Summary of the invention

[0005] In view of the above problems existing in the prior art, the technical problem to be solved by the present invention is to provide a VIGS silencing system for the IdChlH gene of Ilex dabieshanensis. Another technical problem to be solved by the present invention is to provide a construction method for the VIGS silencing system for the IdChlH gene of Ilex dabieshanensis. The last technical problem to be solved by the present invention is to provide a specific application of the VIGS silencing system for the IdChlH gene of Ilex dabieshanensis.

[0006] In order to solve the above technical problems, the technical solution adopted by the present invention is as follows:

[0007] A IdChlH gene from Ilex dabieshanensis, the nucleotide sequence of which is shown in SEQ ID NO.1.

[0008] A specific nucleotide fragment for silencing the IdChlH gene of Ilex dabieshanensis, the nucleotide sequence of which is shown in SEQ ID NO.2.

[0009] A VIGS silencing system for the IdChlH gene of Ilex dabieshanensis: the specific nucleotide fragment for silencing the IdChlH gene of Ilex dabieshanensis according to claim 2 is inserted into a VIGS silencing vector.

[0010] Furthermore, the VIGS silencing vector is a tobacco rattle virus TRV2 vector.

[0011] The method for constructing the VIGS silencing system of the IdChlH gene of Ilex bieshanensis comprises the following steps:

[0012] 1) Design primers for amplifying the specific nucleotide fragment that silences the IdChlH gene of Ilex dabieshanensis, and add homologous sequences (15-20 bp) and restriction sites at both ends of the linearized pTRV2 vector at the upstream and downstream primers respectively;

[0013] 2) amplifying the target gene using high-fidelity PCR using the primers designed in step 1);

[0014] 3) The PCR amplification product obtained in step 2) was homologously recombined with the pTRV2 vector plasmid that had been digested with enzymes to obtain the VIGS silencing system pTRV2-IdChlH of the Ilex dabieshanensis IdChlH gene.

[0015] Furthermore, the amplification primers for the specific nucleotide fragment silencing the IdChlH gene of Ilex dabieshanensis are as follows:

[0016] IdChlH-BamHI-F:

[0017] 5′-GAAGGCCTCCATGGGGATCCGCGCTGGATCCACAATCTATTC-3′,

[0018] IdChlH-SacI-R:

[0019] 5′-CGAGACGCGTGAGCTCCTCTAACCTCAACGCCAAGCGC-3′.

[0020] The application of the VIGS silencing system of the IdChlH gene of Ilex dabieshanensis in identifying the Ilex dabieshanensis gene.

[0021] Furthermore, the application comprises the following steps:

[0022] (1) pTRV2-IdChlH was transformed into Agrobacterium GV3101 competent cells by freeze-thaw method, and PCR identification was positive;

[0023] (2) Mixing Agrobacterium bacterial cultures containing pTRV1 and pTRV2-IdChlH at a volume ratio of 1:1 to prepare an infection solution;

[0024] (3) Leaf injection infection method: first use the needle of a syringe to gently puncture the back of the leaf to create a micro-wound, and then use a syringe without the needle to inject the infection solution into the wound on the back of the leaf;

[0025] (4) The infected plants were placed in a dark place for 12-24 hours and then switched to 23°C with a 16h / 8h light / dark cycle;

[0026] (5) Observe the phenotypic changes of gene-silenced plants and / or detect the expression level of the silenced target gene by fluorescence quantitative PCR.

[0027] Compared with the prior art, the present invention has the following beneficial effects:

[0028] (1) The present invention first constructed the VIGS silencing system of the IdChlH gene of Ilex dabieshanensis, and obtained a system capable of silencing the IdChlH gene of Ilex dabieshanensis;

[0029] (2) The present invention can carry out systematic diffusion and propagation in Ilex dabieshanensis plants by introducing BamHI and SacI restriction sites into specific primers and using the VIGS silencing system of the IdChlH gene induced by TRV virus;

[0030] (3) The VIGS silencing system constructed by the present invention can effectively reduce the expression of the IdChlH gene in Ilex dabieshanensis, affecting the synthesis of chlorophyll, making the silenced leaves present a yellow phenotype, and can make the VIGS silencing efficiency reach more than 85%;

[0031] (4) The establishment of the VIGS silencing system for Ilex dabieense provides an effective technical means to study the gene function of Ilex dabieense, and also increases the number of woody plant species that can use VIGS technology. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 The electrophoresis diagram is the PCR amplification result of the specific fragment of IdChlH gene; M: 2000bp marker;

[0033] Figure 2 This is the electrophoresis diagram of PCR detection of Agrobacterium tumefaciens culture solution of pTRV2-IdChlH recombinant vector; M: 2000bp marker; 1-7: pTRV2-IdChlH Agrobacterium tumefaciens culture solution Agrobacterium single clone;

[0034] Figure 3 The phenotype of the leaves of Dabieshan Holly seedlings infected with the pTRV2-IdChlH recombinant vector, A: pTRV2-IdChlH silencing effect, B: negative control, C: blank control, D: leaf phenotype;

[0035] Figure 4 The expression level of IdChlH gene was detected by qRT-PCR (CK: uninfected plants; pTRV2: empty-infected plants; pTRV2-IdChlH: pTRV2-IdChlH silenced plants). DETAILED DESCRIPTION

[0036] The present invention is further described below in conjunction with specific examples. The experimental methods in the following examples without specifying specific conditions are generally based on well-known methods in the art.

[0037] Example 1

[0038] 1. Obtaining IdChlH gene specific fragment

[0039] 1.1 Obtaining the full-length sequence of the IdChlH gene

[0040] The amino acid sequence of Arabidopsis thaliana AtChlH (AT5G13630) was obtained from the Tair website. The homologous gene sequence of the third-generation full-length transcriptome library of Ilex dabieshanensis owned by our research group was searched through local BlastP to obtain the full-length sequence of the IdChlH gene, and its CDS sequence is shown in SEQ ID NO.1.

[0041] 1.2 Cloning of IdChlH gene-specific fragment

[0042] Total RNA was extracted from leaves of Ilex dabieshanensis (picked in October 2021) using a rapid universal plant RNA extraction kit (Beijing Huayueyang), and reverse transcribed into cDNA using a cDNA reverse transcription kit (TaKaRa). Specific primers were designed using the full-length sequence of the IdChlH gene (SEQ ID NO.1) as a template, and 15 bp homologous sequences and restriction sites (BamHI and SacI) at both ends of the linearized pTRV2 vector were added to the upstream and downstream primers, respectively. The primer sequences are as follows:

[0043] IdChlH-BamHI-F:

[0044] 5′-GAAGGCCTCCATGGGGATCCGCGCTGGATCCACAATCTATTC-3′

[0045] IdChlH-SacI-R:

[0046] 5′-CGAGACGCGTGAGCTCCTCTAACCTCAACGCCAAGCGC-3′

[0047] Using leaf cDNA as template, PCR amplification was performed using KOD FX high-fidelity PCR enzyme, and sequencing obtained a 442 bp IdChlH gene-specific fragment ( Figure 1 ), the nucleotide sequence is shown in SEQ ID NO.2. The PCR reaction system is: 2×PCR buffer for KOD FX 25μL, 1.5μL of upstream and downstream primers, 10μL of dNTP (2mM), 1μL of cDNA template, 1μL of KOD Fx enzyme, and ddH2O to 50μL. The reaction procedure is: 94℃ pre-denaturation for 2min, then 98℃ for 10s, 55℃ for 30s, 68℃ for 1min / 1kb, 35 cycles, 72℃ extension for 7min, and storage at 4℃. The PCR product was detected by electrophoresis and recovered by gel cutting. After sequencing verification, the IdChlH gene silencing fragment of Ilex dabieshanensis was obtained.

[0048] 2. Construction of recombinant viral vector pTRV2-IdChlH

[0049] use II One Step Cloning Kit (Novozyme) was used to homologously recombine the PCR product obtained above with the pTRV2 vector plasmid that had been double-digested with BamHI and SacI, and transformed into competent Escherichia coli DH5α. The single clone PCR bacterial solution was picked for detection and sequencing verification, and the recombinant plasmid pTRV2-IdChlH with correct sequencing was extracted.

[0050] Plasmids pTRV1, pTRV2 and recombinant plasmid pTRV2-IdChlH were transformed into Agrobacterium GV3101 competent cells by freeze-thaw method, and single clones were selected for PCR bacterial liquid detection ( Figure 2 ), select the positive clones verified by PCR, shake the bacteria, preserve the bacteria in glycerol, and store them at -80℃ for later use.

[0051] 3. Infected plants

[0052] 3.1 Material preparation

[0053] Cut the semi-lignified new shoots of the current year into 5-8 cm long cuttings, dip the cuttings in 2000ppm IBA for 6-10 seconds, and insert them into a matrix prepared by mixing peat, rice husk ash and perlite in a ratio of 2:2:1. Keep the matrix humidity moderate and place it in a full light spray environment. Generally, it will take root and survive in 30-40 days. Select the rooted seedlings with consistent growth for infection experiments.

[0054] 3.2 Preparation of infection solution

[0055] Positive Agrobacterium monoclones containing the correct pTRV1, pTRV2 and pTRV2-IdChlH plasmids were picked from LB (containing 50ug / mL kanamycin and 50ug / mL rifampicin) plates and inoculated into 2mL LB liquid medium (containing 50μg / mL kanamycin and 50μg / mL rifampicin) respectively, and cultured at 28℃ and 200rpm for 16h. Then, they were transferred to the induction LB liquid medium (containing 50μg / mL kanamycin, 50μg / mL rifampicin, 10mM MES, 20μM acetosyringone) at 2% content and cultured at 28℃ and 200rpm for about 12h until the bacterial solution OD 600 The cell density was 1.0-2.0, centrifuged at 6000 rpm for 10 min, the supernatant was discarded, and the cells were collected and resuspended in appropriate amount of resuspension solution (10 mM MgCl2, 10 mM MES, 200 μM acetosyringone, pH 5.6, prepared on the spot) to adjust the final OD 600 The resuspension was placed in the dark at room temperature for 3-4 hours, and then the Agrobacterium containing the pTRV1 and pTRV2 empty vectors, and the pTRV1 and pTRV2-IdChlH recombinant plasmids were mixed at a ratio of 1:1 (v / v) and prepared for inoculation.

[0056] 3.3 Injection infection of Ilex dabieshanensis leaves

[0057] First, use the syringe needle to gently puncture the back of the leaf to create a micro-wound, then use a 1mL syringe to absorb an appropriate amount of infection solution, pull out the needle, and inject the infection solution from the wound on the back of the leaf.

[0058] 3.4 Plant culture after infection

[0059] The infected Ilex dabieshanensis plants were cultured in the dark for 24 hours and then switched to 23°C, 16h / 8h light / dark cycle, and relative humidity of 60%-70%.

[0060] 4. Observation of Silenced Plant Phenotypes

[0061] After 20 days of infection, the newly grown leaves of the plants injected with the pTRV2-IdChlH vector solution showed obvious yellowing phenotype ( Figure 3 ), the seedling yellowing rate was above 85%, and this phenotype could last for 1-2 months, indicating that the IdChlH gene was successfully silenced, while there was no change in the leaves of the plants infected with the pTRV2 empty vector (negative control) and the uninfected plants (blank control).

[0062] 5.qRT-PCR was used to detect the expression level of IdChlH.

[0063] The blank control group, negative control group and the 2nd to 3rd yellowing leaves of plants infected with pTRV2-IdChlH were selected, and the total RNA of the leaves was extracted and reverse transcribed into cDNA. The gene region after the IdChlH silencer fragment was selected to design the fluorescent quantitative primer qIdChlH-F / R, and β-TUB was used as the internal reference gene to detect the expression of the IdChlH gene by RT-PCR. The results showed that compared with the blank control and negative control, the expression level of the endogenous IdChlH gene in the leaves of plants infected with pTRV2-IdChlH was significantly reduced ( Figure 4 ), indicating that this system can be effectively used to identify gene functions in Ilex dabieshanensis.

[0064] The fluorescent quantitative primers involved are:

[0065] qIdChlH-F: 5′-GTACCTAAGCCGGAAGTCCTTT-3′,

[0066] qIdChlH-R: 5′-GGTCGGGTCTGAATCAAAGTAG-3′;

[0067] β-TUB-F: 5′-CCAACAATGTGAAGTCAAGT-3′,

[0068] β-TUB-R: 5′-CTTACTCTCCTGAACATCTCT-3′.

[0069] The above contents are only preferred embodiments of the present invention, but the present invention is not limited thereto. All equivalent improvements and changes made within the scope of application of the present invention shall fall within the protection scope of the present invention.

Claims

1. A VIGS silencing vector for the IdChlH gene of Ilex dabieshanensis, characterized in that: The VIGS silencing vector is inserted with a specific nucleotide fragment for silencing the Ilex dabieshanensis IdChlH gene; the nucleotide sequence of the Ilex dabieshanensis IdChlH gene is shown in SEQ ID NO.1; the nucleotide sequence of the specific nucleotide fragment is shown in SEQ ID NO.

2.

2. The VIGS silencing vector of the Ilex dabieshanensis IdChlH gene according to claim 1, characterized in that: The VIGS silencing vector is a tobacco rattle virus TRV2 vector.

3. The method for constructing the VIGS silencing vector of the IdChlH gene of Ilex dabieshanensis according to claim 2, characterized in that: The following steps are involved: 1) Design primers for amplifying the specific nucleotide fragment that silences the IdChlH gene of Ilex dabieshanensis, and add homologous sequences and restriction sites at the ends of the linearized pTRV2 vector at the upstream and downstream primers respectively; 2) Amplify the target gene using high-fidelity PCR using the primers designed in step 1); 3) The PCR amplification product obtained in step 2) was homologously recombined with the pTRV2 vector plasmid that had been digested with enzymes to obtain the VIGS silencing vector pTRV2-IdChlH of the Ilex dabieshanensis IdChlH gene.

4. The method for constructing the VIGS silencing vector of the Ilex dabieshanensis IdChlH gene according to claim 3, characterized in that: The specific primers for amplifying the nucleotide fragment of the silencing IdChlH gene of Ilex dabieshanensis are as follows: IdChlH-BamHI-F: 5′-GAAGGCCTCCATGGGGATCCGCGCTGGATCCACAATCTATTC-3′, IdChlH-SacI-R: 5′-CGAGACGCGTGAGCTCCTCTAACCTCAACGCCAAGCGC-3′.

5. Use of the VIGS silencing vector of the Ilex dabieshanensis IdChlH gene according to claim 2 in silencing the Ilex dabieshanensis IdChlH gene.

6. The use according to claim 5, characterized in that: The steps include: (1) pTRV2-IdChlH was transformed into Agrobacterium competent cells by freeze-thaw method, and PCR identification was positive; (2) Mixing the Agrobacterium culture solutions containing pTRV1 and pTRV2-IdChlH in a certain volume ratio to prepare an infection solution; (3) Inject the prepared infection solution into the back of Ilex dabieshanensis leaves by leaf injection method; (4) After the infected plants are cultured in the dark for a period of time, they are switched to a light / dark cycle; (5) Observe the phenotypic changes of gene-silenced plants and / or detect the expression level of the silenced target gene by fluorescence quantitative PCR.

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