Citrus spinosa PDS gene VIGS silencing system as well as construction method and application thereof

Abstract

The invention discloses a capparis spinosa PDS gene VIGS silencing system as well as a construction method and application thereof, and relates to the technical field of plant genetic engineering. The sequence of the specific nucleotide fragment for silencing the PDS gene of the capparis spinosa disclosed by the invention is as shown in SEQ ID NO. 1. According to the present invention, the specific nucleotide fragment for silencing the capparis spinosa PDS gene is connected to the pTRV2 vector to construct the vector pTRV2: CsPDS, i.e., the VIGS silencing system of the capparis spinosa PDS gene. The result of the embodiment shows that the expression quantity of the PDS gene in the Bensi tobacco leaves silenced by the VIGS silencing system is obviously reduced, so that the white spot whitening phenomenon of the leaves is caused. A VIGS virus silencing system which is low in cost, short in period, easy to operate and high in efficiency of the capparis spinosa is established, and the method can be used for preliminary screening and identification of functional genes of the capparis spinosa.

Description

Technical Field

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

[0002] Capparis spinosa L. is a plant of the Capparis genus of the Capparaceae family (also known as the Capparis family). It is also known as wild watermelon and is mainly distributed in Xinjiang, Tibet, Gansu and other places. The capers in Xinjiang mostly grow in extreme environments such as deserts, Gobi, sandy land, rocky hillsides and foothills. They are environmentally friendly plants with strong vitality and windproof and sand-fixing properties. Capparis spinosa has high medicinal value. Its main active ingredients are alkaloids, flavonoids and glucosinolates. It is a common medicinal material used by the Uyghur people. Modern pharmacological studies have shown that capers have significant anti-inflammatory, analgesic, antioxidant, anti-tumor effects, and can improve hyperglycemia, hyperlipidemia, skin fibrosis and intervertebral disc degeneration. It has good application value and development prospects.

[0003] In order to better study the synthesis and regulation of the active ingredients of Caper, as well as its excellent stress resistance, it is necessary to study the verification of its important functional genes and the mechanism of action. At present, the research on Caper mainly focuses on the chemical analysis of the active ingredients, while there are few reports on molecular biology. Important gene information can be obtained through genome and transcriptome sequencing technology, but the lack of genetic transformation system makes it impossible to verify gene function. Virus-mediated gene silencing (VIGS) is a technology that uses viruses as vectors to infect plants and silence genes at the post-transcriptional level, thereby studying the function of target genes. It has the advantages of high silencing efficiency, rapid identification function, short experimental cycle and low cost. It has been applied to a variety of plants for gene function verification, but this system has not been established and applied in Caper.

[0004] Phytoene desaturase (PDS) is one of the key rate-limiting enzymes in plant carotenoid biosynthesis and photosynthesis. Silencing of this gene will lead to the obstruction of the carotenoid metabolic pathway that produces photoprotective pigments in plants. Chlorophyll molecules lose protection and are degraded by photooxidation, causing the green parts of the plant to exhibit a photobleached phenotype. Therefore, the PDS gene is often used as a reporter gene in the plant VIGS gene silencing system to reflect the gene silencing efficiency. 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 specific nucleotide fragment for silencing the PDS gene of Caper. Another technical problem to be solved by the present invention is to provide a VIGS silencing system for the PDS gene of Caper. Another technical problem to be solved by the present invention is to provide a method for constructing the VIGS silencing system for the PDS gene of Caper. Another technical problem to be solved by the present invention is to provide an application of the VIGS silencing system for the PDS gene of Caper, which is used for rapid, simple and low-cost verification of the gene function of Caper.

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

[0007] A specific nucleotide fragment for silencing the PDS gene of Capparis spinosa, the sequence of which is shown in SEQ ID NO.1.

[0008] The method for constructing a VIGS silencing system of a PDS gene of Capparis spinosa comprises: connecting a specific nucleotide fragment for silencing the PDS gene of Capparis spinosa as shown in SEQ ID NO.1 to a pTRV2 vector to construct a vector pTRV2:CsPDS, i.e., a VIGS silencing system of the PDS gene of Capparis spinosa.

[0009] The method for constructing the VIGS silencing system of the PDS gene of Capparis spinosa L. specifically comprises the following steps:

[0010] 1) Obtaining the mRNA sequence information of the PDS gene of Capparis spinosa based on the gene annotation information of the transcriptome database, and designing a specific nucleotide fragment for silencing the PDS gene of Capparis spinosa;

[0011] 2) Designing amplification primers to amplify specific nucleotide fragments for silencing the PDS gene of Capparis spinosa;

[0012] 3) The amplified product was connected to the pTRV2 vector to construct the vector pTRV2:CsPDS, i.e., the VIGS silencing system of the PDS gene of Capparis spinosa.

[0013] The amplification primer sequences are as follows:

[0014] CsPDS-F:

[0015] 5′-CCTCCATGGGGGATCCGGTACCCAGGTTTACATATATTCTTTGGTGCC-3′,

[0016] CsPDS-R:

[0017] 5′-GAGACGCGTGAGCTCGGTACCGAAGAAATCGATTCAAGGCAATC-3′.

[0018] The construction method constructs the VIGS silencing system of the PDS gene of Capparis spinosa L.

[0019] The use of the VIGS silencing system of the PDS gene of Capparis spinosa L. in inhibiting the expression of the PDS gene of Capparis spinosa L. comprises the following steps:

[0020] 1) Construction of the VIGS silencing system of the PDS gene of Capparis spinosa, i.e., the vector pTRV2:CsPDS;

[0021] 2) The vectors pTRV2:CsPDS, pTRV2:00, and pTRV1 plasmids were transformed into Agrobacterium competent cells respectively, and the positive bacterial liquid was collected after cultivation;

[0022] 3) The pTRV2:CsPDS and pTRV2:00 positive bacterial solutions were mixed with the pTRV1 bacterial solution in proportion and injected into Nicotiana benthamiana leaves;

[0023] 4) Cultivate, screen and obtain Nicotiana benthamiana plants with significantly reduced expression of the PDS gene of Capparis spinosa.

[0024] The mixing ratio is 1:1.

[0025] Application of the VIGS silencing system of Capparis spinosa PDS gene in identifying the function of Capparis spinosa PDS gene.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] The specific nucleotide fragment for silencing the PDS gene of Caper is disclosed in the present invention, and the sequence is shown in SEQ ID NO.1. The present invention obtains the mRNA sequence information of the PDS gene of Caper according to the gene annotation information of the transcriptome database, designs the specific nucleotide fragment for silencing the PDS gene of Caper; designs amplification primers to amplify the specific nucleotide fragment for silencing the PDS gene of Caper; connects the amplified product to the pTRV2 vector to construct the vector pTRV2:CsPDS, i.e. the VIGS silencing system of the PDS gene of Caper. The results of the embodiment show that the expression level of the PDS gene in the leaves of Nicotiana benthamiana after silencing by the VIGS silencing system is significantly reduced, resulting in the phenomenon of white spots and bleaching on the leaves. The present invention establishes a low-cost, short-cycle, easy-to-operate, and high-efficiency VIGS virus silencing system for Caper, which can be used for the preliminary screening and identification of functional genes of Caper. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 The electrophoresis diagram of PCR amplification of the specific fragment of CsPDS gene (M is DL1500; lane 1 is the negative control; lanes 2 to 3 are the amplification products of CsPDS gene);

[0029] Figure 2 This is the PCR identification diagram of the positive single colony of Agrobacterium tumefaciens GV3101 transformed with pTRV2:CsPDS (M is DL1500; lanes 1 to 7 are the fragments amplified with the upstream primer of gene CsPDS and the downstream primer of NOS terminator, with a size of 1283 bp);

[0030] Figure 3 The VIGS silencing diagram of CsPDS gene in tobacco (left is pTRV2:00 silenced plant; right is pTRV2:CsPDS silenced plant);

[0031] Figure 4 The expression analysis diagram of PDS gene in silenced plants of pTRV2:00 and pTRV2:CsPDS (A is the semi-quantitative PCR detection of PDS silencing effect in tobacco; B is the fluorescence quantitative PCR detection of PDS silencing effect in tobacco; "*" is p<0.05, significant difference; "**" is p<0.01, extremely significant difference). DETAILED DESCRIPTION

[0032] In order to make the purpose, technical scheme and advantages of the present invention clearer, the present invention is further described below in conjunction with specific embodiments. Unless otherwise specified in the following embodiments, the technical means used are conventional means well known to those skilled in the art. Molecular biology experimental methods not specifically described can be carried out with reference to the methods listed in the book "Molecular Cloning Experiment Guide" (3rd Edition) by J. Sambrook or conventional methods in the art, or according to the kit and product instructions.

[0033] The materials used in this application are Capparis spinosa seeds, which were collected from Turpan, Xinjiang.

[0034] Example 1

[0035] 1. Cloning of Capparis spinosa CsPDS silencing fragment

[0036] First, the mRNA sequence information of phytoene desaturase (PDS) was obtained based on the gene annotation information in the transcriptome database, and a specific nucleotide fragment for silencing the PDS gene was designed. The sequence is shown in SEQ ID NO.1. The restriction site KpnI on the plant gene silencing vector pTRV2 was selected, and the primers were designed:

[0037] CsPDS-F: 5'-CCTCCATGGGGGATCCGGTACCCAGGTTTACATATATTCTTT GGTGCC-3',

[0038] CsPDS-R: 5'-GAGACGCGTGAGCTCGGTACCGAAGAAATCGATTCAAGG CAATC-3'.

[0039] The RNA of Capparis spinulosa leaves was extracted using a column-type plant total RNA extraction and purification kit (Shanghai Bioengineering Co., Ltd.) and converted into cDNA. Using Capparis spinulosa cDNA as a template, a CsPDS gene-specific fragment with a total length of 483 bp ( Figure 1 The amplification conditions were as follows: pre-denaturation at 95°C for 5 min; denaturation at 95°C for 15 s, annealing at 58°C for 20 s, extension at 72°C for 60 s, for 35 cycles; and final extension at 72°C for 10 min.

[0040] 2. Vector Construction

[0041] The PCR amplification product was recovered and connected to the pTRV2 vector. The ligation product was transformed into E. coli DH5α competent cells. After resistance screening, positive single colonies were picked for colony PCR ( Figure 2 ), and the plasmid was sequenced and verified, and the vector successfully connected to CsPDS was named pTRV2:CsPDS.

[0042] 3. VIGS infection of Nicotiana benthamiana leaves

[0043] The pTRV2:00, pTRV 2:CsPDS, and pTRV1 plasmids were respectively transferred into GV3101 competent cells by heat shock method. The positive Agrobacterium colonies after transformation were picked and inoculated into 2 mL YEB resistance medium (containing 50 mg·L -1 Rifampicin and 50 mg L -1 Kanana antibiotics), cultured overnight at 28°C and 200 rpm; the bacterial solution was inoculated into 15 mL YEB resistance medium at a ratio of 1:100 and cultured until the bacterial solution OD 600 The cells were collected by centrifugation at 5000 rpm for 5 min and the cells were gently resuspended with an equal volume of Agrobacterium infection solution (final concentration of AS: 100 μM) to a concentration of 0.6. 600 Keep it at 0.6, mix the resuspended pTRV2:00 and pTRV2:CsPDS with the pTRV1 bacterial solution in a ratio of 1:1, and let it stand in the dark for 2 hours; use a sterile syringe needle to poke a small hole on the back of the mature leaves of the Nicotiana benthamiana seedlings, and then slowly inject the resuspended solution, and place the infected plants in the dark for 1 day, and then switch to a normal light cycle for continued cultivation. After about 3 weeks, the leaves showed obvious whitening and white spots, and the leaves were taken for subsequent corresponding experiments.

[0044] 4. Identification of the expression level of Nicotiana benthamiana PDS after VIGS silencing

[0045] When the leaves of the infected plants have a bleaching or white spot phenotype ( Figure 3 ), fresh leaves were taken, and total RNA of the leaves was extracted using an RNA kit. NbActin (accession number: AY179605) of Nicotiana benthamiana was used as the internal reference gene, and NbPDS (accession number: DQ469932.1) was used as the detection PDS gene. Semi-quantitative PCR and fluorescence quantitative PCR were used to detect the expression level of the target gene after silencing.

[0046] The primers used were:

[0047] NbActin-qRT-F: 5'-CCGTGGAGAAGAGCTACGAG-3',

[0048] NbActin-qRT-R: 5'-CAGCTTCCATTCCGATCATT-3';

[0049] NbPDS-qRT-F: 5'-CCCGCAGAAGAGTGGATAAA-3',

[0050] NbPDS-qRT-F: 5'-TTGGGGTTTTGACAACATGA-3'.

[0051] The results are as follows Figure 4 As shown, compared with the negative control pTRV2:00, the expression level of the PDS gene in Nicotiana benthamiana leaves after VIGS silencing was significantly reduced.

[0052] The above description is only illustrative rather than restrictive of the present invention. Those skilled in the art will understand that many modifications, changes or equivalents may be made without departing from the spirit and scope defined by the appended claims, but all will fall within the scope of protection of the present invention.

Claims

1. A specific nucleotide fragment for silencing the PDS gene of Capparis spinosa, the sequence of which is shown in SEQ ID NO.

1.

2. A method for constructing a VIGS silencing system for the PDS gene of Capparis spinosa, characterized in that: include: The specific nucleotide fragment for silencing the PDS gene of Capparis spinosa shown in SEQ ID NO.1 was connected to the pTRV2 vector to construct the vector pTRV2:CsPDS, i.e. the VIGS silencing system of the PDS gene of Capparis spinosa.

3. The construction method according to claim 2, characterized in that: The specific steps include: 1) Obtaining the mRNA sequence information of the PDS gene of Capparis spinosa based on the gene annotation information of the transcriptome database, and designing a specific nucleotide fragment for silencing the PDS gene of Capparis spinosa; 2) Designing amplification primers to amplify specific nucleotide fragments for silencing the PDS gene of Capparis spinosa; 3) The amplified product was connected to the pTRV2 vector to construct the vector pTRV2:CsPDS, i.e., the VIGS silencing system of the PDS gene of Capparis spinosa.

4. The construction method according to claim 3, characterized in that: The amplification primer sequences are shown below: CsPDS-F: 5′-CCTCCATGGGGGATCCGGTACCCAGGTTTACATATATTCTTTGGTGCC-3′, CsPDS-R: 5′-GAGACGCGTGAGCTCGGTACCGAAGAAATCGATTCAAGGCAATC-3′.

5. The VIGS silencing system of the PDS gene of Capparis spinosa obtained by the construction method according to any one of claims 1 to 4.

6. Use of the VIGS silencing system of the Capparis spinosa PDS gene according to claim 5 in inhibiting the expression of the Capparis spinosa PDS gene.

7. The use according to claim 6, characterized in that: The steps include: 1) Construction of the VIGS silencing system of the PDS gene of Capparis spinosa, i.e., the vector pTRV2:CsPDS; 2) The vectors pTRV2:CsPDS, pTRV2:00, and pTRV1 plasmids were transformed into Agrobacterium competent cells respectively, and the positive bacterial liquid was collected after cultivation; 3) The pTRV2:CsPDS and pTRV2:00 positive bacterial solutions were mixed with the pTRV1 bacterial solution in proportion and injected into Nicotiana benthamiana leaves; 4) Cultivate, screen and obtain Nicotiana benthamiana plants with significantly reduced expression of the PDS gene of Capparis spinosa.

8. The use according to claim 7, characterized in that: The mixing ratio is 1:

1.

9. Use of the VIGS silencing system of the Capparis spinosa PDS gene according to claim 5 in identifying the function of the Capparis spinosa PDS gene.

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