A CbuPDS gene VIGS silencing system for Catalpa bungei and its construction method

Abstract

The present invention relates to the field of plant molecular biology, and particularly relates to a CbuPDS gene VIGS silencing system for Catalpa bungei and a construction method thereof. The present invention uses the young leaves of the most easily obtained tissue culture seedlings of Catalpa bungei as materials, and establishes a gene silencing transformation system based on the young leaves of Catalpa bungei tissue culture seedlings that can trigger a relatively long duration. Agrobacterium carrying pTRV1 and Agrobacterium carrying the pTRV2-CbuPDS gene silencing vector are used to infect Catalpa bungei by leaf injection method. The newly grown leaves still show photobleaching symptoms 2 months after inoculation. Real-time fluorescence quantitative PCR is used to verify the leaves with photobleaching symptoms, and the relative expression level of the CbuPDS gene is significantly reduced. The leaves with photobleaching symptoms of Catalpa bungei can also improve the ornamental value of the leaves of Catalpa bungei, provide more landscape elements for urban landscaping, and at the same time provide gene resources for the cultivation of new Catalpa bungei varieties with both ornamental and timber uses.

Description

Technical Field

[0001] The present invention belongs to the technical field of plant genetic engineering, and particularly relates to a CbuPDS gene VIGS silencing system of Catalpa bungei and a construction method thereof. Background Art

[0002] Catalpa bungei is a deciduous tree of the genus Catalpa in the family Bignoniaceae. It is widely distributed in China and suitable for planting in different regions. Catalpa bungei has large and numerous single flowers, which are arranged in terminal corymbose racemes, and has a long flowering period. Therefore, it has been an excellent garden ornamental tree species in China since ancient times; its trunk is straight and the wood is hard, and it is also a precious timber tree species. With the exploration of the application values of Catalpa bungei in ornamental, timber, medicinal and other aspects, the demand for Catalpa bungei has also increased accordingly.

[0003] Virus-induced gene silencing (VIGS) is a post-transcriptional gene silencing technology, which is currently widely used in the research of plant genetic engineering and gene function identification. Its principle is that plants will initiate the RNA silencing mechanism when resisting virus infection. This technology does not rely on genetic transformation and has a short experimental period. Therefore, establishing an efficient transformation system is helpful for plant molecular biology and breeding research. However, since the bacterial activity, plant cell state, environmental factors, inserted fragments and infection methods during the infection process will all affect the gene silencing efficiency, this also increases the difficulty and unpredictability of plant transient transformation work.

[0004] The existing plant VIGS technology has been mostly applied to herbaceous model plants such as Arabidopsis thaliana and tobacco. It is urgently needed to be carried out in woody plants, but there are few successful cases and the transformation efficiency is low. This is mainly caused by the differences between herbaceous and woody plants, such as the highly heterozygous genotype background of materials and gene function differentiation. This severely restricts the research progress of forest tree molecular biology. Therefore, the establishment and application of this system can provide important technical support for identifying the functions of genes controlling important traits of forest trees, carrying out forest tree molecular breeding and cultivating new Catalpa bungei varieties with both ornamental and timber uses, etc.

[0005] He et al. (2022) disclosed a VIGS method using the vacuum infiltration method. The Agrobacterium containing the target gene was resuspended with a buffer solution containing 10 mM MgCl 2 、200 μM acetosyringone and 10 mM MES (pH 5.6), and then the small stem segments of Lilium were immersed in the infection solution and placed under -50 kPa vacuum for 10 minutes. After washing 3 times with distilled water, it was placed in a solution containing 30 g L -1 sucrose and 6 g L -1on MS medium with agar (pH 5.8). However, this method cannot be applied to long-term culture after inoculation, and there is no phenotypic change after testing on tissue-cultured Catalpa bungei seedlings. Li Fangjun et al. (2014) disclosed a VIGS of cotyledon injection-infiltration method, adjusted the bacterial liquid concentration to OD 600 = 1.5, filled the lower surface of cotton cotyledons with the bacterial liquid using a 1 ml syringe. However, Catalpa bungei has self-incompatibility, a low seed setting rate, and a low seed germination rate. It is difficult to obtain cotyledons by seed germination for experiments.

[0006] Under the guidance of molecular biology theory, to verify gene function, ontogenetic transformation needs to be carried out. However, Catalpa bungei has problems such as difficult genetic transformation, low differentiation efficiency, and a long time required to obtain positive transgenic seedlings. Although there have been reports on using the VIGS system to verify gene function, due to the difficult seed germination of Catalpa bungei, difficult acquisition of cotyledons, and unique leaf material of Catalpa bungei, it is very difficult to obtain positive transformed seedlings through the reported methods, resulting in the failure of a large amount of preliminary work. Summary of the Invention

[0007] To solve the above problems, the present invention provides a VIGS silencing system for the CbuPDS gene of Catalpa bungei and a construction method thereof.

[0008] First, the present invention provides a CbuPDS gene of Catalpa bungei, and its nucleotide sequence is shown in SEQ ID No.1.

[0009] Secondly, the present invention provides a CbuPDS gene fragment for VIGS, and its nucleotide sequence is shown in SEQ ID No.2.

[0010] The present invention also provides a method for constructing a VIGS silencing system for the CbuPDS gene of Catalpa bungei, including the following steps:

[0011] 1) Clone a 400 - 600 bp gene fragment of the CbuPDS gene of Catalpa bungei and ligate it to a VIGS vector;

[0012] 2) Transform the VIGS vector obtained by ligation in step 1) into Agrobacterium, mix it with Agrobacterium transformed with an RNAi vector of Tobacco rattle virus (TRV), and inject and infect the young leaves of tissue-cultured Catalpa bungei seedlings with Agrobacterium.

[0013] Wherein, the gene fragment at least contains the 400 - 600 bp CbuPDS gene fragment.

[0014] Wherein, the primer sequences for cloning the gene fragment are shown in SEQ ID No.2 and SEQ ID No.4.

[0015] Among them, the VIGS vector is the tobacco rattle virus (TRV) gene silencing expression vector pTRV2.

[0016] The present invention provides a VIGS vector, a host cell, and an engineered bacterium containing the CbuPDS gene fragment for the VIGS gene.

[0017] The present invention also provides a method for inducing a variegated leaf trait in Catalpa bungei. The VIGS vector carrying the CbuPDS gene fragment and the RNAi vector of tobacco rattle virus (TRV) are transformed into the leaves of Catalpa bungei by leaf injection to induce VIGS of the CbuPDS gene in Catalpa bungei.

[0018] In a specific embodiment of the present invention, after mixing the Agrobacterium carrying the vector and the Agrobacterium carrying the RNAi vector of tobacco rattle virus (TRV), the mixture is injected into the back of the second and third rounds of leaves on the upper part of the stem of the Catalpa bungei tissue culture seedlings until the leaves are filled with water stains. After the injection, the Catalpa bungei tissue culture seedlings are cultured in the dark for 2 days.

[0019] In a preferred embodiment of the present invention, a supplementary injection can be performed 2 weeks after the first injection.

[0020] Aiming at the poor infection effect of the prior art on Catalpa bungei, the present invention first provides a Catalpa bungei PDS gene. Another technical problem to be solved by the present invention lies in the construction method of the VIGS silencing system of the Catalpa bungei PDS gene. Based on the difficulty of Catalpa bungei seed germination at the present stage, the present invention selects the leaves of the Catalpa bungei tissue culture seedlings that are easily obtained as experimental materials, and provides the application of the VIGS silencing system of the Catalpa bungei PDS gene, which can quickly and simply verify the gene function of Catalpa bungei at low cost.

[0021] The Agrobacterium carrying pTRV1 and the Agrobacterium carrying the pTRV2-CbuPDS gene silencing vector are used to infect Catalpa bungei by leaf injection. The newly grown leaves 2 months after inoculation still show photobleaching symptoms. However, when the method reported by predecessors is used to operate on the leaves of Catalpa bungei tissue culture seedlings, no photobleaching phenotype appears in the new leaves.

[0022] Real-time fluorescence quantitative PCR is used to verify the leaves showing photobleaching symptoms, and the relative expression level of the CbuPDS gene is significantly reduced. The leaves of Catalpa bungei with photobleaching symptoms can also improve the ornamental value of the leaves of Catalpa bungei, provide more landscape elements for urban landscaping, and provide gene resources for the cultivation of new Catalpa bungei varieties with both ornamental and timber uses. Description of the Drawings

[0023] Figure 1 Shown is the sequence alignment of the homologous proteins of tomato and Catalpa bungei PDS.

[0024] Figure 2The electrophoretogram of the cloned CbuPDS gene of Catalpa bungei is shown as follows.

[0025] Figure 3 The identification of the CbuPDS gene of Catalpa bungei by bacterial liquid PCR is shown as follows.

[0026] Figure 4 The cloning of the CbuPDS gene vector construction of Catalpa bungei is shown as follows.

[0027] Figure 5 The bacterial liquid PCR of the CbuPDS gene vector construction of Catalpa bungei is shown as follows.

[0028] Figure 6 The agrobacterium liquid PCR of the CbuPDS gene of Catalpa bungei is shown as follows.

[0029] Figure 7 The photo-bleaching phenotype diagram after infecting the tissue culture seedlings of Catalpa bungei by the leaf injection method is shown as follows (A is TRV2-empty; B is TRV2-CbuPDS). C is the relative expression level of the CbuPDS gene of Catalpa bungei in the corresponding materials (lowercase letters indicate significant differences at the 0.05 level).

[0030] Figure 8 The infiltration of the tissue culture seedlings of Catalpa bungei by the vacuum infiltration method is shown as follows. Specific implementation mode

[0031] The following examples are used to illustrate the present invention, but are not used to limit the scope of the present invention. Unless otherwise specified, the examples are carried out under conventional experimental conditions or according to the conditions recommended in the manufacturer's instructions.

[0032] Example 1 Cloning of the CbuPDS gene of Catalpa bungei

[0033] Taking the sequence of the PDS homologous gene CaPDS of tomato in NCBI (gene accession number: NM_001324813.1) as the reference sequence, the PDS protein of Catalpa bungei was retrieved in the proteome file of the Catalpa bungei genome database (assembled and not yet published) by using Two Sequence files in TBtools. A total of 6 sequences were obtained, named CbuPDS-1, CbuPDS-2, CbuPDS-3, CbuPDS-4, CbuPDS-5 and CbuPDS-6 respectively. The amino acid sequence similarities of these Catalpa bungei CbuPDSs with tomato CaPDS were only 7.53%, 27.52%, 34.54%, 36.77%, 26.92% and 14.75% respectively. Through the amino acid sequence alignment results ( Figure 1 ), it shows that the PDS gene is not conserved in Catalpa bungei and tomato.

[0034] In this experiment, EASYspin Plant RNA Rapid Extraction Kit (Aidelai, Beijing) was used to extract RNA from Catalpa ovata leaves, and the specific operations were carried out according to the instructions. Trace DNA in total RNA was removed using the Recombinant DNase I (RNase-free) Kit (TaKaRa, Japan). The first strand of cDNA was synthesized using the PrimeScript RT reagent Kit (TaKaRa, Japan). The CbuPDS-4 gene with a complete sequence was selected, and specific primers (CbuPDS-F and CbuPDS-R) were designed in its 5'UTR and 3'UTR regions, respectively. Using the Catalpa ovata leaf cDNA as a template, a high-fidelity enzyme PCR was selected to amplify the Catalpa ovata CbuPDS gene.

[0035]

[0036] By 1% gel electrophoresis ( Figure 2 ), select the band with the correct size of the predicted target sequence for gel excision and recovery, connect to the pTOPO vector, transform into Escherichia coli DH5α, and spread the bacterial solution on LB solid medium (containing 50 μg / mL Car + ) on the plate, blow dry and seal the plate, invert in a 37°C incubator for 12-14 hours for overnight screening and culture. Pick a single clone and perform bacterial liquid PCR identification ( Figure 3 ), and the bacterial solution with the correct band size was sent for sequencing. Through homologous cloning, this experiment successfully isolated the cDNA sequence of the CbuPDS gene of Catalpa ovata, which is 1095 bp long and is shown in SEQ ID No.1.

[0037] Example 2 Construction of plant transgenic vector pTRV2-CbuPDS of Catalpa ovata CbuPDS gene

[0038] The restriction sites Xba I and Sac I were introduced at both ends of the conservative region (400-600 bp) of the CbuPDS gene to perform homologous recombination with the pTRV2 vector. The plasmid containing the CbuPDS gene of Catalpa ovata was used as a template to design primers (TRV2-PDS_F and TRV2-PDS_R) for PCR amplification ( Figure 4 ).

[0039]

[0040] Gel electrophoresis confirmed that the target fragment was single, so it was purified and recovered and stored at -20°C for future use.

[0041] Extract the pTRV2 plasmid, select Xba I and Sac I restriction enzymes (NEB), and perform double digestion in a 37°C water bath. Purify and recover the linear plasmid of pTRV2. Use the ClonExpress II One Step Cloning Kit (Vazyme, Nanjing) for homologous recombination to ligate the target fragment to the pTRV2 vector. Immediately transform the ligation product into Escherichia coli DH5α and screen it on an LB solid medium (containing 50 μg / mL Kan + ). After picking positive monoclonal colonies, expand the culture in an LB liquid medium (containing 50 μg / mL Kan + ), and identify it by colony PCR ([[]] Figure 5 [[]]). Select the bacterial solution with the correct band size for sample submission and sequencing. Name the plasmid with correct sequencing as pTRV2-CbuPDS.

[0042] Extract pTRV1, pTRV2 (as a negative control), and the correctly sequenced pTRV2-CbuPDS plasmid respectively. Transfer the above plasmids into Agrobacterium tumefaciens EHA105 competent cells and culture them inverted at 28°C for 2 days on an LB solid medium (containing 50 μg / mL Kan + and 25 μg / mL Rif + ). Pick monoclonal colonies and expand the culture in an LB liquid medium (containing 50 μg / mL Kan + and 25 μg / mL Rif + ) (28°C, 200 rpm, 14 hours). After detecting by colony PCR ([[]] Figure 6 [[]]), the bacterial solution with the correct band size on gel electrophoresis proves that the pTRV2-CbuPDS gene silencing vector has been successfully transferred into Agrobacterium tumefaciens. After preparing it into glycerol bacteria, store it at -80°C for later use.

[0043] Example 3 Infection of Catalpa bungei with the pTRV2-CbuPDS Gene Silencing Vector

[0044] 1 Preparation of Catalpa bungei tissue culture seedlings:

[0045] Select the apical buds of Catalpa bungei tissue culture seedlings for subculture and perform rooting culture. After the root length reaches 3 - 5 cm (about 1 month), transplant them into the substrate (peat soil: perlite = 3:1) for acclimatization (light, 16 hours, 23°C; dark, 8 hours, 20°C). The total acclimatization time is 21 days. First, wrap them completely with a transparent cover for 7 days to simulate the culture bottle environment; then gradually open the cover to increase the exchange with external air, and ensure normal photosynthesis of the plants during this period. After completely removing the cover, normal water and fertilizer management can be carried out. When it has been transplanted into the substrate for 28 days, the formal experiment can begin.

[0046] 2 Transient transformation of Catalpa bungei tissue culture seedlings ​​​​

[0047] (1) Day 1:

[0048] Agrobacterium tumefaciens containing pTRV1, pTRV2, and pTRV2-CbuPDS plasmids were streaked and activated on LB solid medium (containing 50 μg / mL Kan + and 25 μg / mL Rif + ) and incubated upside down in an incubator at 28 °C for 2 days.

[0049] (2) Day 3:

[0050] Material preparation: Tissue culture seedlings need to be watered thoroughly.

[0051] Bacterial liquid preparation: Single colonies were picked and cultured in 3 mL of LB liquid medium (28 °C, 210 rpm, 12 hours). The cultured bacterial liquid was taken and transferred to 100 mL of liquid LB medium (added with MES buffer, acetosyringone solution, Rif, and Kan) for further expansion culture (28 °C, 210 rpm, 14 - 16 hours) until OD600 = 0.6 - 0.8. The specific ratios are as follows:

[0052]

[0053] (3) Day 4:

[0054] The expanded bacterial liquid was centrifuged at 4 °C and 5000 rpm for 15 minutes to collect the bacterial cells, and then the bacterial cells were resuspended in infiltration buffer until OD600 = 1.0, and shaken at 28 °C and 150 rpm in the dark for 2 hours. The composition of the infiltration buffer is as follows:

[0055] Composition of the infiltration buffer:

[0056]

[0057] The resuspensions of pTRV1 and pTRV2-empty, pTRV2-CbuPDS were uniformly mixed at a ratio of 1:1, and the mixed resuspension was injected into the back of the 2nd - 3rd round of leaves on the upper part of the stem of Catalpa bungei tissue culture seedlings using a 1 mL sterile syringe until the leaves were filled with water stains. After injection, the Catalpa bungei tissue culture seedlings need to be cultured in the dark for 2 days. To ensure that the tissue culture seedlings are in a high-humidity state, an appropriate amount of water can be sprayed inside the cover. After 2 days, the light is restored, and normal watering and fertilization are carried out. Two weeks after injection, a supplementary injection is required.

[0058] (4) Phenotype observation

[0059] Approximately 2 months after the first injection, the leaves newly grown from the stem tip of the Catalpa bungei tissue culture seedlings infiltrated by the leaf injection method showed photo-bleaching phenomenon, and as the leaves grew, the white area gradually expanded ( Figure 7), indicating that the basic principle and experimental method of this experiment are feasible, and the virus-induced VIGS system has been successfully constructed on tissue culture seedlings of Catalpa bungei.

[0060] Example 4 Verification by Real-Time Fluorescent Quantitative PCR

[0061] Taking the leaves of Catalpa bungei injected with pTRV1 and pTRV2-empty bacterial solution as the empty vector control, and taking the leaves of two line numbers TRV2-CbuPDS#2 and TRV2-CbuPDS#3 of Catalpa bungei showing photobleaching symptoms as the experimental group, the leaf RNA was extracted respectively and reverse transcribed to obtain cDNA.

[0062] Furthermore, taking the Catalpa bungei Cbuactin gene as the internal reference gene, by designing primers in the gene-specific region, the relative expression levels of the Catalpa bungei CbuPDS gene in the leaves of different groups were detected by real-time fluorescent quantitative qRT-PCR technology.

[0063] The quantitative results showed that the relative expression levels of the CbuPDS gene in the leaves of Catalpa bungei TRV2-CbuPDS#2 and TRV2-CbuPDS#3 showing photobleaching symptoms were significantly lower than those of the pTRV2-empty group (P<0.05), indicating that the VIGS system of Catalpa bungei was successfully constructed ( Figure 7 ).

[0064]

[0065] Comparative Example 1 Infiltration of Tissue Culture Seedlings of Catalpa bungei by Vacuum Infiltration Method

[0066] Materials: The tissue culture seedlings of Catalpa bungei had been rooting for nearly a month. After the root length reached 3-5 cm, they were taken out of the culture bottle, the culture medium was washed off, and they were placed in a humid environment for standby.

[0067] Specific method:

[0068] (1) Agrobacterium cells containing pTRV1, pTRV2-empty, and pTRV2-CbuPDS were collected respectively and resuspended in an infiltration buffer containing 10 mM MgCl 2 , 200 μM acetosyringone and 10 mM MES (pH 5.6).

[0069] (2) Before infection, the resuspensions containing pTRV1 and pTRV2 and TRV2-CbuPDS were stored in the dark at room temperature for 4 hours at a ratio of 1:1 (v / v).

[0070] (3) The whole tissue culture seedlings were immersed in the infiltration mixture containing pTRV1 and pTRV2 and TRV2-CbuPDS, and then placed under -50 kPa vacuum for 5 minutes.

[0071] (4) Wash the infected tissue culture seedlings with distilled water three times, for 3 minutes each time, and then grow them under a light / dark cycle of 22°C, 16 hours / 20°C, 8 hours. Observe the color of the newly grown leaves subsequently.

[0072] Results: From the day of vacuum infection to 2 months later, the leaves newly grown from the stem tips remained green ( Figure 8 ), and there was no photo-bleaching manifestation in the whole plant, indicating that this method is not applicable to tissue culture seedlings of Catalpa bungei.

[0073] Although the present invention has been described in detail above with general descriptions and specific embodiments, on the basis of the present invention, some modifications or improvements can be made, which are obvious to those skilled in the art. Therefore, these modifications or improvements made without departing from the spirit of the present invention all fall within the scope of protection required by the present invention.

Claims

1. A catalpa tree CbD The gene, whose nucleotide sequence is shown in SEQ ID No.

1.

2. A method for VIGS CbD The gene fragment has a nucleotide sequence as shown in SEQ ID No.

2.

3. A method for constructing a catalpa tree CbD A method for a VIGS gene silencing system, characterized in that: The steps include: 1) Cloning the catalpa tree described in claim 1 CbD Gene fragments of 400-600 bp were connected to the VIGS vector; 2) The VIGS vector obtained by ligation in step 1) is transformed into Agrobacterium, mixed with Agrobacterium transformed with RNAi vector of tobacco rattle virus (TRV), and injected into tender leaves of tissue culture seedlings of Catalpa ovata by Agrobacterium.

4. The method according to claim 3, characterized in that The gene fragment at least comprises the gene fragment according to claim 2.

5. The method according to claim 3, characterized in that The primer sequences for cloning the gene fragment are shown in SEQ ID No. 2 and SEQ ID No.

4.

6. The method according to claim 3, characterized in that The VIGS vector is a tobacco rattle virus (TRV) gene silencing expression vector pTRV2.

7. Containing the composition according to claim 2 CbD VIGS vectors and / or engineered bacteria containing gene fragments.

8. A method for inducing mosaic traits in leaves of Catalpa ovata, characterized in that: Will carry the CbD The VIGS vector of the gene fragment and the RNAi vector of tobacco rattle virus (TRV) were transformed into catalpa leaves by leaf injection to induce catalpa CbD Genes show VIGS.

9. The method according to claim 8, characterized in that Will carry the CbD After mixing Agrobacterium carrying the VIGS vector of the gene fragment and Agrobacterium carrying the RNAi vector of tobacco rattle virus (TRV), the mixture was injected into the back of the 2nd to 3rd round of leaves on the upper part of the stem of the Catalpa ovata tissue culture seedlings until the leaves were full of water stains. After the injection, the Catalpa ovata tissue culture seedlings were cultured in the dark for 2 days.

10. The method according to claim 9, characterized in that Two weeks after the injection, give another injection.

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