VIGS silencing system of walnut JrPDS gene as well as construction method and application of VIGS silencing system

Abstract

The invention discloses a VIGS silencing system of walnut JrPDS gene as well as a construction method and application of the VIGS silencing system, and relates to the technical field of biology. The walnut JrPDS gene interference fragment as shown in SEQ ID NO.1 provided by the invention can effectively interfere the expression of the walnut endogenous JrPDS gene, so that the walnut seedling endogenous JrPDS gene is silenced, the silencing rate can reach 48%, and the walnut JrPDS gene interference fragment is used for identifying the function of the walnut endogenous gene.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology, and in particular to a VIGS silencing system of a walnut JrPDS gene, a construction method thereof, and an application thereof. Background Art

[0002] Walnut, a genus of the Juglans genus in the Juglandaceae family, is an important timber, ecological, economic, and bioenergy species, widely cultivated in many countries and regions around the world. Over the past 30 years, extensive genetic research on walnut has been conducted, including surveys of diversity, identification of germplasm resources, phylogeny, construction of genetic maps, and investigations of biotic and abiotic stresses. However, research on walnut drought resistance remains incomplete, primarily focusing on analyses of biomass changes, morphological changes, and physiological and biochemical changes.

[0003] With the development of modern molecular biology and omics, these techniques are increasingly being used to study drought resistance in plants. While a genetic transformation system using somatic embryos has been established for walnuts, rooting and germination remain an unresolved issue. This significantly limits the large-scale propagation of superior walnut plants and lines, hindering the selection and breeding of high-quality walnut varieties and the development of improved walnut cultivation for the walnut industry. Furthermore, the incompleteness of the walnut genetic system limits the analysis of walnut gene function. Therefore, the development of a virus-induced gene silencing (VIGS) system for walnuts will facilitate the efficient and rapid identification of target gene functions.

[0004] The VIGS system is a rapid, efficient, and specific transient gene silencing system. It has become an indispensable method for analyzing gene function in many species, particularly those not amenable to stable genetic transformation. However, the efficiency and success rate of gene silencing vary significantly among different host plants. When silencing the walnut JrPDS gene, the insertion of the target gene fragment is crucial for the effectiveness of silencing, especially in walnut seedlings. Summary of the Invention

[0005] The main purpose of the present invention is to propose a VIGS silencing system for the walnut JrPDS gene and its construction method and application, aiming to improve the silencing effect of the walnut JrPDS gene.

[0006] To achieve the above object, the present invention provides a gene fragment for silencing the walnut JrPDS gene, wherein the gene fragment comprises the nucleotide sequence shown in SEQ ID NO.1.

[0007] The present invention also proposes a recombinant expression vector for silencing the walnut JrPDS gene, wherein the recombinant expression vector comprises a VIGS viral vector and the above-mentioned gene fragment, and the gene fragment is inserted into the VIGS viral vector.

[0008] Optionally, the VIGS viral vector is a tobacco rattle virus TRV2 vector.

[0009] The present invention also proposes a VIGS silencing system for the walnut JrPDS gene. The VIGS silencing system comprises an Agrobacterium bacterial solution containing an auxiliary vector and the Agrobacterium bacterial solution of the recombinant expression vector described above.

[0010] Optionally, the Agrobacterium strain is GV3101; and / or,

[0011] The auxiliary vector is a tobacco rattle virus TRV1 vector.

[0012] Optionally, the OD values of the Agrobacterium tumefaciens solution containing the recombinant expression vector and the Agrobacterium tumefaciens solution containing the auxiliary vector are 600 The value is 0.7~1.1.

[0013] Preferably, the OD values of the Agrobacterium tumefaciens solution containing the recombinant expression vector and the Agrobacterium tumefaciens solution containing the auxiliary vector are 600 The value is 1.1.

[0014] The present invention also proposes a method for constructing a VIGS silencing system for the walnut JrPDS gene, comprising the following steps:

[0015] Walnut leaf RNA was extracted and reverse transcribed to obtain walnut cDNA;

[0016] PCR amplification of the walnut cDNA was performed using the upstream primer JrPDS-Eco RI-F and the downstream primer JrPDS-Bam HI-R to obtain the walnut JrPDS gene interference fragment;

[0017] Inserting the walnut JrPDS gene interference fragment into the VIGS virus vector to obtain a recombinant plasmid containing the walnut JrPDS gene, and transforming Agrobacterium to obtain an Agrobacterium bacterial solution containing the recombinant expression vector;

[0018] Transforming the auxiliary vector into Agrobacterium to obtain an Agrobacterium bacterial solution containing the auxiliary vector;

[0019] The Agrobacterium solution containing the recombinant expression vector and the Agrobacterium solution containing the auxiliary vector are mixed and allowed to stand in the dark for 2 to 5 hours to prepare the VIGS silencing system;

[0020] Wherein, the nucleotide sequence of the upstream primer JrPDS-Eco RI-F is shown in SEQ ID NO.2;

[0021] The nucleotide sequence of the downstream primer JrPDS-Bam HI-R is shown in SEQ ID NO.3.

[0022] The present invention also provides the application of the VIGS silencing system in identifying walnut gene functions.

[0023] Optionally, the application comprises the following steps: injecting and infecting leaves of walnut seedlings at the true leaf stage using the VIGS silencing system, and continuing to culture the walnut seedlings after injection and infection.

[0024] Optionally, the Agrobacterium solution containing the auxiliary vector and the Agrobacterium solution containing the recombinant expression vector are mixed in an equal volume ratio, and the obtained infection solution is injected into the leaves of walnut seedlings for infection.

[0025] The technical solution of the present invention, by providing the nucleotide sequence shown in SEQ ID NO.1, can effectively interfere with the expression of the endogenous JrPDS gene in walnut seedlings, silencing the endogenous JrPDS gene. This invention, for the first time, constructs a VIGS silencing system for the JrPDS gene in walnut seedlings. The constructed silencing system has been validated and demonstrated to significantly reduce JrPDS expression in walnut seedling leaves, with a plant silencing rate of up to 48%. The gene silencing system of the present invention is simple, rapid, and highly effective, providing strong support for research related to walnut genetic improvement and molecular breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on the structures shown in these drawings without paying any creative work.

[0027] Figure 1 This is an agarose gel electrophoresis diagram of the JrPDS gene interference fragment amplified by PCR in Example 1 of the present invention;

[0028] Figure 2 This is an agar gel electrophoresis diagram of DH5α colony PCR in Example 1 of the present invention;

[0029] Figure 3 This is an agarose gel electrophoresis diagram of the GV3101 colony PCR in Example 1 of the present invention;

[0030] Figure 4This is the effect of the leaf injection inoculation method on the phenotype of walnut leaves in Example 1 of the present invention;

[0031] Figure 5 This is an agar gel electrophoresis diagram of the viral expression structure infecting plants in Example 1 of the present invention;

[0032] Figure 6 This is a diagram showing the relative expression structure of JrPDS in walnut leaves detected by real-time fluorescence quantitative detection in Example 1 of the present invention, wherein ** represents significant difference, P < 0.01.

[0033] The purpose, features and advantages of the present invention will be further described with reference to the accompanying drawings and in conjunction with the embodiments. DETAILED DESCRIPTION

[0034] The following will clearly and completely describe the technical solutions in the embodiments of the present invention in conjunction with the accompanying drawings. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making any creative efforts shall fall within the scope of protection of the present invention.

[0035] It should be noted that if the embodiments of the present invention involve directional indications (such as up, down, left, right, front, back, etc.), the directional indications are only used to explain the relative position relationship, movement status, etc. between the components under a certain specific posture. If the specific posture changes, the directional indications will also change accordingly.

[0036] In addition, if there are descriptions involving "first", "second", etc. in the embodiments of the present invention, the descriptions of "first", "second", etc. are only for descriptive purposes and cannot be understood as indicating or suggesting their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features limited to "first" and "second" may explicitly or implicitly include at least one of such features. In addition, if "and / or" or "and / or" appears in the full text, its meaning includes three parallel solutions. Taking "A and / or B" as an example, it includes solution A, solution B, or solutions that satisfy both A and B. In addition, the technical solutions between the various embodiments can be combined with each other, but it must be based on the ability of ordinary technicians in this field to implement. When the combination of technical solutions is mutually contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by the present invention.

[0037] The present invention provides a gene fragment for silencing the walnut JrPDS gene, wherein the gene fragment comprises a nucleotide sequence as shown in SEQ ID NO.1.

[0038]

[0039] The present invention also proposes a recombinant expression vector for silencing the walnut JrPDS gene, wherein the recombinant expression vector comprises a VIGS viral vector and the above-mentioned gene fragment, and the gene fragment is inserted into the VIGS viral vector.

[0040] In the technical solution of the present invention, the VIGS virus vector is preferably a tobacco rattle virus TRV2 vector.

[0041] The present invention also proposes a VIGS silencing system for the walnut JrPDS gene. The VIGS silencing system comprises an Agrobacterium bacterial solution containing an auxiliary vector and the Agrobacterium bacterial solution of the recombinant expression vector.

[0042] In the technical solution of the present invention, the Agrobacterium is preferably Agrobacterium GV3101; the auxiliary vector is preferably a tobacco rattle virus TRV1 vector.

[0043] In the technical solution of the present invention, the OD value of the Agrobacterium solution containing the recombinant expression vector is 600 The value is preferably 0.7 to 1.1, and further preferably 1.1; the OD of the Agrobacterium solution containing the auxiliary vector is 600 The value is preferably 0.7 to 1.1, more preferably 1.1.

[0044] In the technical solution of the present invention, the method for constructing the VIGS silencing system of the walnut JrPDS gene comprises the following steps:

[0045] Walnut leaf RNA was extracted and reverse transcribed to obtain walnut cDNA;

[0046] PCR amplification of the walnut cDNA was performed using the upstream primer JrPDS-Eco RI-F and the downstream primer JrPDS-Bam HI-R to obtain the walnut JrPDS gene interference fragment;

[0047] Inserting the walnut JrPDS gene interference fragment into the VIGS virus vector to obtain a recombinant plasmid containing the walnut JrPDS gene, and transforming Agrobacterium to obtain an Agrobacterium bacterial solution containing the recombinant expression vector;

[0048] Transforming the auxiliary vector into Agrobacterium to obtain an Agrobacterium bacterial solution containing the auxiliary vector;

[0049] The Agrobacterium solution containing the recombinant expression vector and the Agrobacterium solution containing the auxiliary vector are mixed and allowed to stand in the dark for 2 to 5 hours to prepare the VIGS silencing system;

[0050] The nucleotide sequence of the upstream primer JrPDS-Eco RI-F is shown in SEQ ID NO.2; the nucleotide sequence of the downstream primer JrPDS-Bam HI-R is shown in SEQ ID NO.3.

[0051] In the above technical solution, the extraction and reverse transcription in "extracting RNA from walnut leaves and reverse transcribing to obtain walnut cDNA" are performed using conventional methods in the art, for example, using a plant RNA extraction kit to extract walnut leaf RNA, and using a reverse transcription kit to reverse transcribe the RNA into cDNA. This application does not limit the extraction and reverse transcription methods.

[0052] The nucleotide sequence of the upstream primer JrPDS-Eco RI-F is shown in SEQ ID NO. 2, specifically: 5'-gtgagtaaggttaccgaattcATTGCTGGTGCAGGTTTGGC-3'; the nucleotide sequence of the downstream primer JrPDS-Bam HI-R is shown in SEQ ID NO. 3, specifically: 5'-cgtgagctcggtaccggatccTGGCATTGCAAATATCATAGAGTG-3'.

[0053] The PCR amplification procedure in "PCR amplifying the walnut cDNA" is preferably as follows: initial denaturation at 94°C for 2 minutes; 35 cycles of denaturation at 94°C for 30 seconds, annealing at 58°C for 30 seconds, and extension at 72°C for 30 seconds; and a final extension at 72°C for 2 minutes. The PCR amplification reaction system is preferably 50 μL and includes: 2 μL of cDNA, 2 μL of forward primer (10 μM), 2 μL of reverse primer (10 μM), 25 μL of high-fidelity enzyme, and ddH2O to 50 μL.

[0054] "Inserting the walnut JrPDS gene interference fragment into the VIGS virus vector" includes enzyme cutting and enzyme ligation, wherein the enzyme cutting linearizes the tobacco rattle virus TRV2 vector to obtain a linearized vector; the enzyme ligation connects the walnut JrPDS gene interference fragment to the linearized vector, and then transforms the competent Escherichia coli to obtain a recombinant plasmid, and then carries out Agrobacterium transformation.

[0055] When the Agrobacterium liquid containing the auxiliary vector and the Agrobacterium liquid containing the recombinant expression vector are mixed, the volume ratio is preferably 1:1.

[0056] The present invention also proposes the application of the VIGS silencing system in identifying walnut gene functions, which includes the following steps: injecting and infecting leaves of walnut seedlings in the true leaf stage using the VIGS silencing system, and continuing to culture the walnut seedlings after injection and infection.

[0057] In the above technical solution, the walnut seedlings are preferably 'Xiangling' or 'Qingxiang' walnut seedlings, more preferably 'Xiangling' walnut seedlings; the infection site is preferably the underside of the leaves. The method for cultivating walnut seedlings is to soak walnut seeds collected that year in water, cover them with soil to accelerate germination, sow the seeds in plug trays, and allow them to grow into plants after approximately 30 days.

[0058] The infected walnut seedlings were first cultured in the dark and then in the light. In the dark culture, the infected walnut seedlings were completely covered with black plastic bags and placed in an incubator. The plastic bags were opened after 1 day of treatment. The light culture was carried out at a temperature of 22°C, a relative humidity of 70%, and a light intensity of 300 μmol·m -2 ·s -1 , 16 h light / 8 h dark. Culture under light until silent albino phenotype appears.

[0059] The present invention uses a VIGS vector containing the JrPDS gene fragment and a tobacco rattle virus RNAi vector to infect true-leaf-stage walnut 'Xiangling' seedlings via foliar injection, rapidly and efficiently generating gene-silenced plants for studying functional walnut genes. Compared to traditional walnut genetic transformation systems, this system offers simplicity, low cost, and rapid efficiency, overcoming the problem of overexpression, but not gene silencing, of endogenous walnut genes in heterologous model plants.

[0060] The technical solutions of the present invention are further described in detail below in conjunction with specific embodiments and drawings. It should be understood that the following embodiments are only used to explain the present invention and are not used to limit the present invention.

[0061] Preparation Example 1

[0062] The construction of the VIGS recombinant expression vector pTRV2-JrPDS includes the following steps:

[0063] (1) The total RNA of the young leaves of 'Xiangling' walnut was extracted using the Novozyme kit. The extraction steps were carried out according to the kit instructions, and the RNA was reverse transcribed into cDNA according to the Novozyme reverse transcription kit instructions.

[0064] (2) Using online tools (NCBI and BLAST), specific primers for the walnut JrPDS gene were designed, and an Eco RI restriction site was added to the upstream primer and a Bam HI restriction site was added to the downstream primer to obtain the upstream primer JrPDS-EcoRI-F (as shown in SEQ ID NO. 2) and the downstream primer JrPDS-Bam HI-R (as shown in SEQ ID NO. 3).

[0065] (3) Using cDNA as a template, PCR amplification was performed using the upstream primer JrPDS-Eco RI-F and the downstream primer JrPDS-Bam HI-R. The PCR amplification reaction system is shown in Table 1 below; in the table, ddH2O, double-distilled water; high-fidelity enzyme, 2×Phanta Flash MasterMix (Dye Plus), purchased from Nanjing Novozymes Biotechnology Co., Ltd.

[0066] Table 1 PCR reaction system

[0067]

[0068]

[0069] The PCR amplification reaction program was as follows: pre-denaturation at 94°C for 2 min; denaturation at 94°C for 30 s, annealing at 58°C for 30 s, extension at 72°C for 30 s, 35 cycles; final extension at 72°C for 2 min; and storage at 4°C.

[0070] After PCR amplification, PCR products were detected by 1.2% agarose gel electrophoresis. Figure 1 As shown. Figure 1 It can be seen that the band size of the PCR amplification product is 255 bp, and the sequencing results show that the nucleotide sequence is shown in SEQ ID NO.1.

[0071] The target gene-specific bands obtained by agarose gel electrophoresis were recovered and purified using the Novozymes DNA gel recovery kit.

[0072] (4) Plasmid pTRV2 was double-digested with restriction endonucleases Eco RI and Bam HI. The enzyme digestion system and reaction procedures are shown in Tables 2 and 3 below. Restriction endonucleases Eco RI and Bam HI were purchased from New England Biolabs.

[0073] Table 2 Enzyme digestion system (50 μL)

[0074] Components Dosage pTRV2 3.5 μL EcoRI 1 μL BamHI 1 μL CutSmartBuffer 5μL <![CDATA[ddH2O]]> margin

[0075] Table 3 Reaction procedure

[0076] temperature time 37℃ 20min 65℃ 20min 4℃ ∞

[0077] The enzyme digestion product was detected by 1.2% agarose gel electrophoresis. If the band size was correct, the band obtained by agarose gel electrophoresis was recovered and purified using the Novozymes DNA gel recovery kit to obtain the linearized vector.

[0078] (5) Using the Novozyme recombinant kit ( The PCR amplification product from step (3) was ligated to the pTRV2 vector digested from step (4) using the II One Step Cloning Kit. The recombinase ligation system is shown in Table 4 below.

[0079] Table 4 Recombinase ligation system (20 μL)

[0080] Components Dosage Linearized vector 200ng Destination fragment 20ng 5×CEⅡBuffer 4μL ExnaseⅡ 2μL <![CDATA[ddH2O]]> margin

[0081] The recombination reaction conditions are: 37°C for 30 minutes, followed by 5 minutes on ice, and then transformation into competent E. coli. The recombinant solution can also be stored at -20°C for subsequent transformation.

[0082] (6) After the PCR amplification product was ligated to the pTRV2 vector (hereinafter referred to as the “ligation product”), it was transformed into Escherichia coli (DH5α, purchased from Shanghai ToloBio Biotechnology Co., Ltd.) in the following steps:

[0083] Take out the DH5α competent cells from -80℃, quickly put them in ice, and after they thaw, aliquot 50 μL into a 1.5 mL sterile centrifuge tube;

[0084] Add 5 μL of the ligation product to a centrifuge tube, flick the tube wall 5 times, and place on ice for 25 min;

[0085] Heat shock in a 42°C metal bath for 90 seconds, then quickly return to ice for 5 minutes;

[0086] 700 μL LB liquid medium (10 g / L Tryptone, 10 g / L NaCl, 5 g / L Yeast Extract) was added to the centrifuge tube and incubated at 37°C on a shaker at 200 rpm for 1 h. Tryptone, NaCl, and Yeast Extract were purchased from Beijing Coolbo Technology Co., Ltd.

[0087] The cells were collected by centrifugation at 5000 rpm for 1 min. 650 μL of culture medium was aspirated and discarded with a pipette. The cells were resuspended in the remaining 100 μL of culture medium and spread on LB solid plates (containing 50 mg / L kanamycin and 50 mg / L rifampicin). After drying, the plates were inverted and cultured in a 37°C incubator for 13 h.

[0088] Single colonies on the plate were picked and placed in LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin) and cultured at 37°C with shaking at 200 rpm for 16 h. Figure 2 As shown, the positive bacterial solution was sent to Wuhan Tianyi Biological Company for sequencing;

[0089] After the sequencing results were correct, an appropriate amount of bacterial solution was mixed with an equal volume of 50% glycerol (glycerol and ddH2O were mixed in equal volumes), and then stored at -80°C for a long term to obtain the recombinant expression vector pTRV2-JrPDS.

[0090] Preparation Example 2

[0091] The preparation of the infection solution comprises the following steps:

[0092] (1) The pTRV2-JrPDS plasmid was transformed into Agrobacterium using the freeze-thaw method. The specific steps are as follows:

[0093] Take out GV3101 (Agrobacterium, purchased from Shanghai ToloBio Biotechnology Co., Ltd.) competent cells from -80°C, partially thaw them in the palm of your hand or at room temperature for a while, and insert them into ice when they are in an ice-water mixture state;

[0094] Add 9 μL of the pTRV2-JrPDS plasmid obtained in Preparation Example 1 to every 100 μL of Agrobacterium competent cells, gently stir the bottom of the tube to mix, and place on ice for 1 min, in liquid nitrogen for 5 min, in a 37°C water bath for 5 min, and in an ice bath for 5 min.

[0095] 900 μL of LB liquid culture medium (10 g / L Tryptone, 10 g / L NaCl, 5 g / L Lyeast Extract) was added and cultured at 28° C. with shaking for 2.5 h.

[0096] The cells were collected by centrifugation at 6000 rpm for 1 min, and 100 μL of bacterial solution was left at the bottom. The cells were gently pipetted to resuspend the cells, spread on LB plates (containing 50 mg / L kanamycin and 50 mg / L rifampicin), and placed upside down in a 28°C incubator for 2 days.

[0097] After colonies grow, pick a single colony and add it to LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin). Place it in a 28°C shaker and shake at 200 rpm in the dark overnight (12-16 hours) for subsequent PCR identification.

[0098] Eight well-grown plaques were picked from the plate and inoculated into LB liquid medium (containing 50 mg / L kanamycin and 50 mg / L rifampicin) for 24 h of shaking culture. PCR amplification was performed using target gene-specific primers. The results are shown in Figure 3.

[0099] After the PCR amplification results are correct, an appropriate amount of bacterial solution is taken and thoroughly mixed with an equal volume of 50% glycerol and then stored at -80°C for a long term to obtain Agrobacterium containing the recombinant expression vector pTRV2-JrPDS.

[0100] (2) pTRV1 and pTRV2 were transformed into Agrobacterium GV3101 respectively by freeze-thaw method. The specific steps were as in step (1) to obtain Agrobacterium containing expression vector pTRV1 and Agrobacterium containing expression vector pTRV2.

[0101] (3) Agrobacterium containing the expression vector pTRV1, Agrobacterium containing the expression vector pTRV2, and Agrobacterium containing the recombinant expression vector pTRV2-JrPDS were inoculated into 5 mL of LB medium containing 50 mg / L kanamycin and 50 mg / L rifampicin for pre-culture, and shaken at 28°C overnight (220 rpm); 1 mL of the pre-culture was inoculated into 50 mL of LB medium (10 g / L Triptone, 10 g / L NaCl, 5 g / L Lyeast Extract), and cultured at 28°C for 30 h with shaking at 220 rpm. The bacterial cells were then collected and resuspended in infection solution (containing 10 mM MgCl2, 10 mM MES, and 200 μM AS), and the OD was adjusted. 600 The value is 1.1, and the OD 600 pTRV1 infection solution with an OD value of 1.1 600 pTRV2 infection solution with an OD of 1.1, and 600 The pTRV2-JrPDS infection solution had a value of 1.1. Among them, MES is 2-morpholinoethanesulfonic acid, AS is acetosyringone, and MES and AS were purchased from Beijing Coolbo Technology Co., Ltd.

[0102] (4) The pTRV1 infection fluid and the pTRV2-JrPDS infection fluid were mixed at a volume ratio of 1:1, and allowed to stand in the dark at 22°C for 3 h to obtain the pTRV2-JrPDS experimental group infection fluid for later use; the TRV1 infection fluid and the pTRV2 infection fluid were mixed at a volume ratio of 1:1, and allowed to stand in the dark at 22°C for 3 h to obtain the pTRV2-00 empty load treatment group infection fluid for later use.

[0103] Example 1

[0104] Infection of walnut seedling leaves includes the following steps:

[0105] (1) Soak the seeds of 'Xiangling' walnut in clean water for about 7 days, changing the clean water once a day. When the clean water is no longer turbid, cover the seeds with soil to accelerate germination. After the seeds turn white, transfer them to a round seedling pot (d = 10 cm) filled with a mixed culture soil of substrate soil, peat soil, vermiculite, and perlite (volume ratio 2:2:1:1), and then transfer them to a light incubator. The culture conditions are: 22℃, relative humidity 70%, and light intensity 300μmol·m -2 ·s -1Walnut 'Xiangling' seedlings with 5 to 10 true leaves were obtained under a 16-hour light / 8-hour dark cycle for 30 days. The seedlings were randomly divided into an untreated group (CK), a no-treatment group (pTRV2-00), and an experimental group (pTRV2-JrPDS), with 33 seedlings in each group.

[0106] (2) The three infection solutions prepared in Preparation Example 2 were injected into the back of leaves of walnut seedlings of the corresponding test groups (CK, pTRV2-00, and pTRV2-JrPDS) using a disposable 5 mL syringe. Three leaves were injected per plant, and the injection volume for each leaf was 100 μL. The infected walnut seedlings were cultured in a dark incubator for 24 h and then transferred to a growth chamber for normal culture. The culture conditions were: 22°C, 70% relative humidity, and a light intensity of 300 μmol·m -2 ·s -1 , 16h light / 8h dark.

[0107] Phenotypic observation, virus detection, and viral vector silencing effect detection were performed on the walnut plants infected in this example.

[0108] 1) During the normal culture period, observe the phenotype and growth of the plants every day. After 10 days, start recording phenotypic changes and count the number of albino plants. After 15 days of infection, if Figure 4 As shown, the leaves of the pTRV2-JrPDS experimental group showed obvious bleaching symptoms, while the untreated group (CK) and the empty-load treatment group (pTRV2-00) showed no changes. Statistics show that 16 of the 33 plants infected with the pTRV2-JrPDS experimental group were silent 15 days after infection, an infection rate of 48%.

[0109] 2) Take leaves from the CK untreated group, the pTRV2-00 empty vector treated group, and the pTRV2-JrPDS experimental group, with 3 replicates per group, and use the Novezum kit to extract total RNA from the leaves. The extraction steps are carried out according to the kit instructions, and the RNA is reverse transcribed into cDNA according to the Novezum reverse transcription kit instructions. PCR is used to detect whether the plants contain viruses. The test results are as follows: Figure 5 As shown, the primers used are as follows:

[0110] TRV-F: 5'-GCACGATGAGCTTTATTATTACGGA-3' (SEQ ID NO. 4);

[0111] TRV-R: 5'-CCGTAGTTTAATGTCTTCGGGAC-3' (SEQ ID NO. 5).

[0112] Depend on Figure 5It can be seen that no virus bands were detected in the CK untreated group, while virus bands were detected in the leaves of the pTRV2-00 empty-load treatment group and the pTRV2-JrPDS experimental group.

[0113] 3) Using GAPDH as the internal reference gene, the relative expression level of JrPDS gene was detected by real-time fluorescence quantitative PCR technology. The test results are as follows Figure 6 As shown, the primers used are as follows:

[0114] qpcrJrPDS-F: 5'-GAGGTTCTTCCTGCACCATTA-3' (SEQ ID NO. 6);

[0115] qpcrJrPDS-R: 5'-ACCAAGCATCGCAGGTAAA-3' (SEQ ID NO. 7);

[0116] qpcrJrGAPDH-F: 5'-ATTTGGAATCGTTGAGGGTCTTATG-3' (SEQ ID NO. 8);

[0117] qpcrJrGAPDH-R: 5'-AATGATGTTGAAGGAAGCAGCAC-3' (SEQ ID NO. 9).

[0118] Depend on Figure 6 It can be seen that the relative expression level of JrPDS gene in the pTRV2-JrPDS experimental group was significantly reduced, indicating that the infection solution of the pTRV2-JrPDS experimental group of the present invention can effectively silence the walnut JrPDS gene.

[0119] Example 2

[0120] This example is based on Example 1, and the difference is that in step (3) of Preparation Example 2, the OD values of the pTRV1 infection solution, the pTRV2 infection solution, and the pTRV2-JrPDS infection solution are 600 The values were all 0.7; 29 plants were infected. After testing, 7 plants were silent, and the infection rate was 24%.

[0121] Example 3

[0122] This example is based on Example 1, except that 'Qingxiang' walnut seeds were used instead of 'Xiangling'. Nineteen plants were infected. Testing revealed that nine plants were silent, for an infection rate of 47%. 'Qingxiang' was chosen as a representative late-maturing variety, while 'Xiangling' was chosen as an early-maturing variety.

[0123] Example 4

[0124] This example is based on Example 3, and the difference is that in step (3) of Preparation Example 2, the OD values of the pTRV1 infection solution, the pTRV2 infection solution, and the pTRV2-JrPDS infection solution are 600 The values were all 0.7; 28 plants were infected. After testing, 5 plants were silent, and the infection rate was 18%.

[0125] The above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Those skilled in the art will readily appreciate that the present invention is susceptible to various modifications and variations. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present invention shall be included within the scope of the present invention.

Claims

1. A gene fragment for silencing the walnut JrPDS gene, characterized in that: The gene fragment includes the nucleotide sequence shown in SEQ ID NO.

1.

2. A recombinant expression vector for silencing the walnut JrPDS gene, characterized in that: The recombinant expression vector comprises a VIGS viral vector and the gene fragment according to claim 1, and the gene fragment is inserted into the VIGS viral vector.

3. The recombinant expression vector for silencing the walnut JrPDS gene according to claim 2, wherein: The VIGS virus vector is a tobacco rattle virus TRV2 vector.

4. A VIGS silencing system for the JrPDS gene in walnut, characterized in that: The VIGS silencing system comprises an Agrobacterium bacterial solution containing an auxiliary vector and an Agrobacterium bacterial solution containing the recombinant expression vector according to claim 2 or 3.

5. The VIGS silencing system of the walnut JrPDS gene according to claim 4, characterized in that: The Agrobacterium strain is GV3101; and / or, The auxiliary vector is a tobacco rattle virus TRV1 vector.

6. The VIGS silencing system of the walnut JrPDS gene according to claim 4, characterized in that: The OD values of the Agrobacterium liquid containing the recombinant expression vector and the Agrobacterium liquid containing the auxiliary vector are 600 The value is 0.7~1.

1.

7. A method for constructing a VIGS silencing system for the walnut JrPDS gene, characterized in that: The following steps are involved: Walnut leaf RNA was extracted and reverse transcribed to obtain walnut cDNA; PCR amplification of the walnut cDNA was performed using the upstream primer JrPDS-Eco RI-F and the downstream primer JrPDS-Bam HI-R to obtain the walnut JrPDS gene interference fragment; Inserting the walnut JrPDS gene interference fragment into the VIGS virus vector to obtain a recombinant plasmid containing the walnut JrPDS gene, and transforming Agrobacterium to obtain an Agrobacterium bacterial solution containing the recombinant expression vector; Transforming the auxiliary vector into Agrobacterium to obtain an Agrobacterium bacterial solution containing the auxiliary vector; The Agrobacterium solution containing the recombinant expression vector and the Agrobacterium solution containing the auxiliary vector are mixed and allowed to stand in the dark for 2 to 5 hours to prepare the VIGS silencing system; Wherein, the nucleotide sequence of the upstream primer JrPDS-Eco RI-F is shown in SEQ ID NO.2; The nucleotide sequence of the downstream primer JrPDS-Bam HI-R is shown in SEQ ID NO.

3.

8. Use of the VIGS silencing system according to any one of claims 4 to 6 in identifying walnut gene function.

9. Use of the VIGS silencing system according to claim 8 in identifying walnut gene function, characterized in that: The application comprises the following steps: injecting and infecting leaves of walnut seedlings using the VIGS silencing system, and continuing to culture the injected and infected walnut seedlings.

10. Use of the VIGS silencing system according to claim 9 in identifying walnut gene function, characterized in that: The Agrobacterium liquid containing the auxiliary vector and the Agrobacterium liquid containing the recombinant expression vector are mixed in an equal volume ratio, and the obtained infection liquid is injected into the leaves of the walnut seedlings for infection.

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