Wild barley phytoene dehydrogenase gene HbPDS, specific fragments and applications

Abstract

This invention provides a wild barley phytoene dehydrogenase gene. HbPDS Specific fragments and applications. A [structure / system] was constructed. HbPDS Gene-specific fragments, recombinant viral vector pTRV2-HbPDS, and the first establishment of wild barley HbPDS The VIGS gene silencing system can silence the wild barley phytoene dehydrogenase gene. HbPDS This inhibits the synthesis of carotenoids in wild barley, leading to phenomena such as chlorophyll fading and whitening of leaves.

Description

Technical Field

[0001] This invention belongs to the field of plant gene function verification and plant genetic engineering technology, specifically relating to a wild barley phytoene dehydrogenase gene. HbPDS Specific fragments and their applications. Background Technology

[0002] Wild barley ( Hordeum brevisubulatium (Trin.) Link, also known as barley grass, wild rye, or short-awned barley grass, is a perennial herbaceous plant belonging to the genus *Hordeum* of the Poaceae family. It is widely distributed in Northeast my country, North China, Inner Mongolia, and Xinjiang. Its stems and leaves are soft, palatable, and nutritious, making it an excellent forage. Rich in high-quality resistance genes, it is highly adaptable, exhibiting tolerance to salinity, cold, and drought. It can safely overwinter at -40℃. Wild barley is also relatively tolerant of wet and waterlogging conditions and is not demanding in terms of soil requirements. It is suitable for planting in saline-alkaline soils with a pH of 8.3-9.5 and is a dominant species in saline and alkali meadow steppes. It has strong tillering and regeneration abilities, high yield, and good production performance, making it highly valuable for economic purposes.

[0003] The abundance of high-quality resistance genes in wild barley has made it a research hotspot. Currently, many foreign scholars have made some progress in the study of wild barley's stress resistance and shattering resistance. In addition to the aforementioned research directions, my country has mainly conducted research on tissue culture, endophytic fungi, and molecular marker development. However, an efficient gene function verification system adapted to wild barley has not yet been established, especially in the study of gene silencing at the seed stage, which is severely restricting the discovery of its resistance genes and the progress of molecular breeding.

[0004] Gene mining is fundamental to efficient plant genetic transformation, involving gene cloning, functional analysis, and research into regulatory mechanisms. Plant genetic transformation is a crucial technique for introducing exogenous genes into the plant genome and ensuring their stable expression. Agrobacterium-mediated transformation, as the most widely used genetic transformation method, has achieved breakthroughs in monocotyledonous crops such as rice and wheat. Constructing efficient genetic transformation systems is the core of plant transgenic technology, and many challenges remain that require further in-depth research.

[0005] Traditional breeding methods have played a crucial role in improving forage varieties, but they suffer from drawbacks such as long cycles, low efficiency, reliance on phenotypic selection, limited accuracy, high human and material input, and high costs, affecting breeding efficiency and precision. With the development of molecular biology techniques, gene function research has become a core direction for upgrading forage breeding. Technologies such as CRISPR / Cas9 and virus-induced gene silencing (VIGS) have provided strong support for gene function research in grasses, laying the foundation for molecular breeding, greatly accelerating the molecular breeding process, and promoting the rapid development of forage molecular breeding and animal husbandry.

[0006] VIGS (Vibration-Vibration Syndrome) is a post-transcriptional gene silencing technique that achieves silencing by specifically degrading the mRNA of target genes. It boasts advantages such as ease of operation, short cycle time, high efficiency, low cost, and wide applicability, making it an important method for rapid and efficient detection of plant gene function. Tobacco rattle virus (TRV) is a viral vector widely used in plant gene function research. It has advantages such as a wide host range, high silencing efficiency, and mild symptoms, making it the most widely used and effective VIGS viral vector in plants. It has shown good infection effects on model plants such as Nicotiana benthamiana, dicotyledonous plants such as Arabidopsis thaliana, tomato, and Populus euphratica, and monocotyledonous plants such as wheat and maize. However, research on gene silencing at the seed stage of wild barley has not yet been reported.

[0007] Phytoene desaturase (PDS) is a key enzyme in the biosynthesis of carotenoids, catalyzing the formation of colored carotenoids from colorless phytoene. The core of the VIGS technology system is the screening of reliable reporter genes and their silencing. PDS Genes can inhibit the synthesis of carotenoids in plants. When carotenoid levels decrease, chlorophyll degradation occurs, affecting the efficiency of photosynthesis and resulting in a photobleaching phenotype in plants. This visible phenotype gene is not only rapid but also highly effective. PDS Genes are often used as reporter genes to evaluate the success of establishing a VIGS technology system.

[0008] Because PDS gene sequences vary across species, heterologous PDS genes cannot be precisely adapted for validation in the VIGS system of wild barley. Furthermore, PDS gene-specific fragments are crucial for achieving gene silencing specificity, effectively preventing interference with other homologous genes within a plant during the silencing process. They are also key targets for subsequent design of specific primers, gene detection, and functional validation. Therefore, it is necessary to study the specific PDS gene fragments of wild barley itself. HbPDSThe gene was identified, and its specific fragments were determined, laying the foundation for the establishment of the wild barley VIGS technology system, subsequent gene function research, and molecular breeding research. Summary of the Invention

[0009] The purpose of this invention is to provide a wild barley HbPDS Gene, HbPDS Gene-specific fragments, recombinant viral vector pTRV2-HbPDS, and wild barley phytoene dehydrogenase gene HbPDS The construction method of the VIGS silencing system, and HbPDS Gene-specific fragments, recombinant viral vector pTRV2-HbPDS, and wild barley phytoene dehydrogenase gene HbPDS The VIGS Silent System in Silent Wild Barley HbPDS Applications in genes.

[0010] This study used wild barley seeds bred by Inner Mongolia Agricultural University as experimental material, utilizing the wild barley's... PDS Using the gene as a marker gene, a pTRV2-HbPDS recombinant vector was constructed to build a wild barley VIGS system, laying the foundation for gene function research in wild barley.

[0011] To achieve the above objectives, the present invention adopts the following technical solution.

[0012] A wild barley phytoene dehydrogenase gene HbPDS The nucleotide sequence of its full-length gene is shown in SEQ ID No. 1.

[0013] A wild barley phytoene dehydrogenase HbPDS Gene-specific fragments, the nucleotide sequences of which are shown in SEQ ID No. 6.

[0014] A recombinant viral vector pTRV2-HbPDS, wherein the recombinant viral vector pTRV2-HbPDS contains the above-mentioned... HbPDS Gene-specific fragments.

[0015] A method for preparing the recombinant viral vector pTRV2-HbPDS, the method comprising: using the wild barley phytoene dehydrogenase gene... HbPDS For the target gene, seamless DNA cloning technology was used to... HbPDS A gene-specific fragment was ligated into BamHI in the MCS region of the VIGS viral backbone vector pTRV2. The ligation product was transformed into DH5α competent E. coli cells, and bacterial culture PCR was performed for identification. Sequencing results were compared to obtain the constructed recombinant viral vector pTRV2-HbPDS. The wild barley phytoene dehydrogenase gene was also described. HbPDS The nucleotide sequence is shown in SEQ ID No. 1.

[0016] Furthermore, the primers used in the bacterial culture PCR identification are pTRV2-JC-F and pTRV2-JC-R; the sequence of pTRV2-JC-F is shown in SEQ ID No. 7, and the sequence of pTRV2-JC-R is shown in SEQ ID No. 8.

[0017] Wild barley phytoene dehydrogenase gene HbPDS The method for constructing the VIGS silencing system includes the following steps:

[0018] S1. The above recombinant viral vector pTRV2-HbPDS was transformed into Agrobacterium tumefaciens EHA105 competent cells by freeze-thaw method, and the PCR identification was positive.

[0019] S2. Mix the pTRV1 positive Agrobacterium tumefaciens bacterial solution with the transformed pTRV2-HbPDS positive Agrobacterium tumefaciens bacterial solution at a bacterial volume ratio of 1:1 to prepare a mixed bacterial solution; add cysteine, acetylsuccine and Tween-20 to the mixed bacterial solution and mix evenly to obtain an infection solution;

[0020] S3. After sterilizing the wild barley seeds with the seed coat completely removed, they are cultured aseptically. When more than half of the seeds have germinated to about 3 mm, the germinated seeds are infected with the above-mentioned infection solution and then dried.

[0021] S4. Detection: The dried seeds from S3 were spread on a sugar-containing solid culture medium and grown under conditions of 22℃ and a light / dark cycle of 14 h / 10 h. Phenotypic changes in wild barley seedlings were observed, and the number of albino wild barley seedlings was tested. HbPDS The expression level of the gene, i.e., the constructed wild barley phytoene dehydrogenase gene. HbPDS The VIGS silent system.

[0022] The above HbPDS Gene-specific fragments in the silent wild barley phytoene dehydrogenase gene HbPDS Applications in; the wild barley phytoene dehydrogenase gene HbPDS The nucleotide sequence is shown in SEQ ID No. 1.

[0023] The recombinant viral vector pTRV2-HbPDS was used to silence the wild barley phytoene dehydrogenase gene. HbPDS Applications in; the wild barley phytoene dehydrogenase gene HbPDS The nucleotide sequence is shown in SEQ ID No. 1.

[0024] The wild barley phytoene dehydrogenase gene obtained by the above construction method HbPDSThe VIGS silencing system silences the phytoene dehydrogenase gene in wild barley. HbPDS Applications in; the wild barley phytoene dehydrogenase gene HbPDS The nucleotide sequence is shown in SEQ ID No. 1.

[0025] Beneficial effects:

[0026] This invention provides a wild barley HbPDS Gene, HbPDS Gene-specific fragments, recombinant viral vector pTRV2-HbPDS, and wild barley phytoene dehydrogenase gene HbPDS The construction method of the VIGS silencing system, and HbPDS Gene-specific fragments, recombinant viral vector pTRV2-HbPDS, and wild barley phytoene dehydrogenase gene HbPDS The VIGS Silent System in Silent Wild Barley HbPDS The application of genes has the following advantages.

[0027] 1) This invention is the first to construct wild barley HbPDS The VIGS gene silencing system was developed, and silent wild barley was discovered. HbPDS Specific segments of a gene.

[0028] 2) This invention utilizes tobacco brittle virus technology to construct wild barley HbPDS Gene silencing systems can be systematically propagated in wild barley.

[0029] 3) The silencing system described in this invention can significantly reduce wild barley HbPDS When gene expression and carotenoid synthesis are inhibited, plants experience photobleaching, which results in chlorophyll fading and leaves turning white.

[0030] 4) The present invention first germinates the seeds after sterilization and complete removal of the seed coat, and ensures that they are not affected by other stresses or competition during the cultivation process. Then, pTRV1 and pTRV2 are mixed and infected by Agrobacterium tumefaciens-mediated method to introduce foreign genes and obtain positive plants.

[0031] 5) This invention establishes wild barley HbPDS Under the premise of gene silencing system, by constructing wild barley HbPDS Gene silencing system: Discover new functional genes from wild barley and integrate them with wild barley. HbPDS The gene was replaced in the MCS region of the recombinant viral vector pTRV2-HbPDS, and then its function was verified in wild barley, thus laying the foundation for discovering and creating superior genes in wild barley. Attached Figure Description

[0032] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0033] Figure 1 wild barley HbPDS Agarose gel electrophoresis image of gene-specific fragment CDS (307bp).

[0034] Figure 2 Agarose gel electrophoresis image of Escherichia coli DH5α culture for PCR detection of the recombinant viral vector pTRV2-HbPDS.

[0035] Figure 3 SnapGene Map of the recombinant viral vector pTRV2-HbPDS.

[0036] Figure 4 Agarose gel electrophoresis image of Agrobacterium EHA105 bacterial culture for PCR detection of the recombinant viral vector pTRV2-HbPDS.

[0037] Figure 5 Statistical graph showing the germination rate and contamination rate of wild barley seeds under different treatments.

[0038] Figure 6 The image shows the albino phenotype of wild barley seedlings cultured after infection with the pTRV2-HbPDS recombinant viral vector; in the figure, A represents wild-type wild barley seedlings, and B represents wild barley seedlings cultured after infection with the pTRV2-HbPDS recombinant viral vector.

[0039] Figure 7 Statistical chart of positive rate and conversion efficiency of wild barley.

[0040] Figure 8 This image shows the PCR detection results of wild barley seedlings infected with the recombinant viral vector pTRV2-HbPDS. In the image: M is the Trans2K DNA Marker; Positive Control is the positive recombinant vector pTRV2-HbPDS plasmid; Negative Control is the PCR product amplified in sterile water; WT is the PCR product amplified from the genomic DNA of non-transformed wild barley plants; and Transgenic Plant is the PCR product amplified from the genomic DNA of transformed wild barley plants.

[0041] Figure 9 Real-time quantitative RT-qPCR analysis of reverse transcription in transformed wild barley plants HbPDSGene expression level diagram; In the diagram: WT is a wild-type barley seedling, and HbPDS-1 and HbPDS-2 are two wild barley seedlings infected by the recombinant viral vector pTRV2-HbPDS. Detailed Implementation

[0042] The technical solutions in the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only a part of the embodiments, and not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0043] Technical approach: By constructing a system containing... HbPDS The pTRV2 viral silencing vector containing a gene-specific fragment was used to transform wild barley seeds via Agrobacterium tumefaciens-mediated transformation, inducing endogenous silencing in wild barley. HbPDS Gene silencing effectively reduced the risk of wild barley. HbPDS The gene expression level is adjusted to produce an albino phenotype. The specific steps are as follows: Construct the recombinant viral vector pTRV2. HbPDS; preparation of Agrobacterium infection solution; infection of seed materials that have reached germination conditions; statistical analysis of contamination rate of infected materials; and detection of gene silencing efficiency.

[0044] Example 1: HbPDS Obtaining the full-length gene sequence

[0045] In order to obtain wild barley PDS The gene sequence was first determined using the PDS sequence (GenBank: B8215DE3A) of its close relative, wheat, as the query sequence. A BLAST alignment was then performed on the published wild barley whole genome database (GWH-Project PRJCA019121). Through this alignment, the gene located in the wild barley genome was identified. PDS Homologous genes.

[0046] Based on the genomic sequence of this gene, specific primers were designed, and the wild barley phytoene dehydrogenase gene was amplified from wild barley DNA by PCR. HbPDS The full-length gene sequence was obtained, and the gene was named... HbPDS Its nucleotide sequence is shown in SEQ ID No. 1. The reaction system and reaction conditions for PCR identification are shown in Tables 1 and 2; among them, the HbPDS-DNA-F sequence is 5'-AATCAGGGGAGACAAGAATC-3' (SEQ ID No. 2), and the HbPDS-DNA-R sequence is 5'-CCAACCGCAGACAAGATG-3' (SEQ ID No. 3).

[0047] Table 1: PCR amplification system for full-length gene sequence

[0048]

[0049] Table 2: PCR amplification reaction procedure for the full-length gene sequence

[0050]

[0051] Example 2: HbPDS Cloning of gene-specific fragments

[0052] Provide a method for silencing the wild barley phytoene dehydrogenase gene ( HbPDS The preparation method for the gene-specific fragment is as follows.

[0053] 1. Total RNA extraction from samples

[0054] Wild barley seedlings were placed in a 1.5 mL centrifuge tube with small steel balls added. The mixture was then ground into powder using liquid nitrogen freezing. Total RNA was extracted using the Novizan RNA Extraction Kit and stored at -80°C for later use.

[0055] 2. Reverse transcription to synthesize cDNA

[0056] The RNA samples frozen at -80℃ were retrieved, and the first strand of cDNA was synthesized using the one-step method of the TIANGEN reverse transcription kit. The cDNA was then stored at -20℃. The reverse transcription PCR reaction system is shown in Table 3. The PCR reaction conditions were as follows: mix the reverse transcription PCR reaction system, remove the genome and reverse transcription reaction at 42℃ for 15 min, and inactivate the enzyme at 95℃ for 3 min.

[0057] Table 3: Reverse Transcription PCR Reaction System

[0058]

[0059] 3. HbPDS Gene-specific fragment amplification

[0060] wild barley phytoene dehydrogenase gene HbPDS The full-length sequence is used as a template and is designed for amplification. HbPDS Primers pTRV2-HbPDS-F and pTRV2-HbPDS-R were designed to target a 307 bp specific fragment on the CDS domain of the gene. Using the cDNA obtained from reverse transcription as a template, and adding the pTRV2 vector sequence adapter, primers pTRV2-HbPDS-F and pTRV2-HbPDS-R were finally designed. The mixtures were prepared according to the reaction system in Table 4, and PCR amplification was performed according to the reaction conditions in Table 5. HbPDSGene-specific fragments; among them, the pTRV2-HbPDS-F sequence is 5'-CTCTAGAAGGCCTCCATGGGGACACCGGCTGTCTATCATC-3' (SEQ ID No. 4), and the pTRV2-HbPDS-R sequence is 5'-AGACGCGTGAGCTCGGTACCGGATCGTTTACTGGGGCGTTCAC-3' (SEQ ID No. 5).

[0061] The amplification products were subjected to 1.0% agarose gel electrophoresis and photographed. The results are as follows: Figure 1 The product was recovered using a novizan product purification kit, and the resulting product was denoted as [product name missing]. HbPDS Gene-specific fragment (SEQ ID No. 6).

[0062] Table 4: PCR amplification system for specific fragments

[0063]

[0064] Table 5: PCR amplification reaction procedure for specific fragments

[0065]

[0066] Example 3: Construction of recombinant viral vector pTRV2-HbPDS

[0067] 1. Linearized pTRV2 vector

[0068] Using pTRV2 plasmid as a template, pTRV2 was digested with BamHI restriction endonuclease. The digestion reaction system is shown in Table 6. The digestion reaction program was 37℃ for 60 min. The product was recovered using a Novizan product purification kit, and the resulting linearized vector was designated as pTRV2-BamHI digested.

[0069] 2. Recombinant viral vector pTRV2-HbPDS

[0070] wild barley phytoene dehydrogenase gene HbPDS The target gene was the product recovered in Example 2. HbPDSUsing a gene-specific fragment as a template, seamless DNA cloning technology was employed. In this embodiment, ClonExpress® II recombinant cloning technology (a simple, rapid, and efficient seamless DNA cloning technology that can directionally clone insert fragments to any site on any vector) was used for ligation, specifically the ClonExpress® II OneStep Cloning Kit (i.e., In-Fusion ligation). The In-Fusion ligation system is shown in Table 7, and the In-Fusion ligation reaction program was 37°C for 30 min. HbPDS Gene-specific fragments were recombined into the BamHI restriction site in the multiple cloning site (MCS) of the VIGS viral backbone vector pTRV2 (i.e., pTRV2-BamHI restriction) which had been linearized with restriction endonuclease.

[0071] Table 6: Enzyme digestion reaction system

[0072]

[0073] Table 7: In-Fusion Connection System

[0074]

[0075] In Table 7, X and Y are calculated using the following formulas to determine the amount of carrier and insert fragment used:

[0076] The optimal amount of cloning vector used, X = [0.02 × number of base pairs in the cloning vector] ng (0.03 pmol);

[0077] The optimal amount of insert used is Y = [0.04 × number of base pairs of insert] ng (0.06 pmol).

[0078] Transform DH5α E. coli competent cells with the ligation product: Add all of the ligation product to DH5α E. coli competent cells, incubate on ice for 30 min, incubate at 42°C for 90 s, incubate on ice for 2 min, add 600 μL of LB liquid medium, incubate at 37°C on a shaker for 1 h, centrifuge at 2000 rpm for 2 min, resuspend the cells in 200 μL of supernatant, and plate the resuspended cells on solid LB medium containing Kan (Kan, 50 μg·mL⁻¹). -1After incubation at 37℃ for 12-16 h, single clones were picked and added to 600 μL of LB liquid medium. The culture was then incubated with shaking at 37℃ and 200 rpm for 3 h. Colony PCR was performed using pTRV2-JC-F and pTRV2-JC-R primers. The reaction system and conditions for PCR identification are shown in Tables 8 and 9. The pTRV2-JC-F sequence is 5'-GAACGTATTTGTTTTTATGTT-3' (SEQ ID No. 7), and the pTRV2-JC-R sequence is 5'-AAAAGACTTACCGATCAATCA-3' (SEQ ID No. 8). Agarose gel electrophoresis images of the PCR products are shown below. Figure 2 As shown; and sent to Qingke Biotechnology Co., Ltd. for sequencing, the sequencing results were compared to obtain the constructed recombinant viral vector pTRV2-HbPDS, whose SnapGene Map is shown below. Figure 3 As shown.

[0079] Table 8: Bacterial PCR Identification Reaction System

[0080]

[0081] Table 9: PCR identification reaction procedure for bacterial culture

[0082]

[0083] Example 4: Transformation of recombinant viral vector pTRV2-HbPDS into Agrobacterium tumefaciens EHA105 competent cells

[0084] 1. Freeze-thaw conversion

[0085] The recombinant viral vector pTRV2-HbPDS was transformed into Agrobacterium tumefaciens EHA105 competent cells using a freeze-thaw method: After thawing Agrobacterium tumefaciens EHA105 competent cells on ice, 5 μL of the correctly sequenced recombinant viral vector pTRV2-HbPDS plasmid from Example 3 was added. The cells were then incubated on ice for 30 min, flash-frozen in liquid nitrogen for 1 min, and then incubated in a 37°C water bath for 3 min. 600 μL of LB liquid medium was then added, and the cells were cultured at 28°C and 200 rpm with shaking for 3 h. The cells were then centrifuged at 4000 rpm for 4 min, and 200 μL of the supernatant was retained to resuspend the cells.

[0086] 2. Positive clones

[0087] The resuspended bacterial cells were spread onto solid LB medium containing Kan and Rif (Kan, 50 μg / mL). -1 Rif, 50 μg·mL -1After standing at 28℃ for 2-3 days to form single colonies, positive Agrobacterium tumefaciens colonies were preserved. The recombinant viral vector pTRV2-HbPDS was then identified by PCR. The reaction system and conditions for PCR identification are shown in Tables 8 and 9. The PCR amplification products were subjected to 1.0% agarose gel electrophoresis and photographed. The agarose gel electrophoresis image of the pTRV2-HbPDS Agrobacterium tumefaciens EHA105 bacterial culture is shown below. Figure 4 As shown. The correctly identified pTRV2-HbPDS positive Agrobacterium tumefaciens culture was mixed with 50% glycerol at a 1:1 ratio and stored at -80°C.

[0088] Example 5: Wild barley seed coat treatment and acquisition of sterile seedlings

[0089] 1. Seed coat treatment

[0090] During the growth and development of wild barley seeds, the radicle and plumule typically emerge from the basal embryo first to adapt to the external environment. Because seeds are easily contaminated by microorganisms in the natural environment, they may carry a certain amount of miscellaneous bacteria. These bacteria can affect seed germination and subsequent growth. Therefore, to achieve a balance between high germination rate and low contamination rate after disinfection, the entire seed coat is completely removed. The germination rate was observed and statistically analyzed, and the results are as follows: Figure 5 As shown, the germination rate of wild barley seeds after complete seed coat removal was 58.5%, and no contamination was observed. Therefore, wild barley seeds after complete seed coat removal were selected as the base material for aseptic culture.

[0091] 2. Obtaining sterile vaccines

[0092] In a clean bench, wild barley seeds treated with the above-mentioned seed coat were placed in sterile petri dishes and sequentially disinfected with 75% alcohol for 1 min, rinsed three times with sterile water, disinfected with 15% NaClO solution for 15 min, rinsed three times with sterile water, soaked in 0.1% H2O2 for 3.5 h, and rinsed three times with sterile water. The disinfected seeds were then spread on double-layered moistened sterile filter paper for aseptic culture for about 60 h. When more than half of the seeds germinated to about 3 mm, they could be used as inoculum. The environmental conditions for aseptic germination of the seeds were: temperature 27℃, photoperiod 24 h dark.

[0093] Example 6: TRV virus infection of wild barley seedlings

[0094] 1. Preparation of the infiltration solution

[0095] Following the experimental methods described in the master's thesis "Establishment of Transient Genetic Transformation System and Creation of New Germplasm of *Agrobacterium tumefaciens* _ Li Yuchen _ Inner Mongolia Agricultural University", pTRV1-positive *Agrobacterium tumefaciens* culture was obtained. 200 μL of pTRV2-HbPDS-positive *Agrobacterium tumefaciens* culture preserved in Example 4 and 200 μL of pTRV1-positive *Agrobacterium tumefaciens* culture were added to 50 mL of LB liquid medium containing Kan and Rif (Kan, 50 μg·mL⁻¹). -1 Rif, 50 μg·mL -1 Incubate at 28℃ and 200 rpm for 12-16 h; incubate until the bacterial concentration reaches OD500. 600 =0.4-0.6; then, the Agrobacterium tumefaciens culture carrying the pTRV1 vector and the Agrobacterium tumefaciens culture carrying the pTRV2-HbPDS recombinant virus vector were mixed at a volume ratio of 1:1 to obtain a mixed culture solution; cysteine, acetosyringone, and Tween-20 were added to the mixed culture solution, and the mixture was used as an infection solution to infect the germinating wild barley seeds in Example 5. The final concentrations of each reagent were: cysteine ​​400 mg / L, acetosyringone 19.62 mg / L, and Tween-20 5 mg / L.

[0096] 2. TRV virus infection

[0097] Germinated seeds that met the infection conditions in Example 5 were mixed with the infection solution, which was required to completely submerge the germinating seeds. The mixture underwent a 40-minute vacuum filtration followed by a 4-minute ultrasonic filtration followed by a 55-minute vacuum filtration. The vacuum filtration pressure was 0.1 MPa, and the ultrasonic temperature was 15°C with a power of 40 Hz. After the mixed infection treatment, the seeds and infection solution were co-cultured at 28°C and 200 rpm for 12 hours. The supernatant was discarded, and the seeds were then removed and placed on sterile filter paper to dry, removing excess Agrobacterium tumefaciens bacterial solution. The seeds were continuously turned over during the drying process until they were completely dry. The infected seeds were then placed on a sugar-containing solid culture medium for further cultivation.

[0098] The infected seeds were spread on a sugar-containing solid culture medium and cultured under the following conditions: 22℃, 2000 lux light intensity, and a light / dark cycle of 14 h / 10 h. Seed contamination status was statistically analyzed during this process, and the results are as follows: Figure 5 As shown, the phenotypes of wild barley seedlings cultured after infection with the pTRV2-HbPDS recombinant viral vector were photographed, and the results are as follows. Figure 6As shown, the pTRV2-HbPDS recombinant viral vector silenced the entire wild barley seedling phenotype after infection, resulting in obvious whitening of the entire plant phenotype, while the control phenotype remained green. After 3-4 weeks, the transformation efficiency of wild barley infected with the pTRV2-HbPDS recombinant viral vector was 50.7%, as shown in the figure. Figure 7 As shown.

[0099] The sugar-containing solid culture medium consisted of MS basal medium, 3 g / L sucrose, 50 mg / L gibberellin, 400 mg / L termethin, 4 g / L Phytagel, and pH 5.95.

[0100] Example 7: PCR detection of albino wild barley seedlings

[0101] Leaves from albino barley plants infected in Example 6 were placed in sterilized 2 mL centrifuge tubes. DNA was extracted using the CTAB method, and the DNA concentration was measured using a nanodrop micrometer. PCR amplification was then performed. The PCR identification reaction system is shown in Table 10, and the reaction conditions are shown in Table 9. The amplified products were subjected to 1.0% agarose gel electrophoresis and photographed. The results are as follows: Figure 8 As shown in the image, the PCR product size of the pTRV2-HbPDS recombinant viral vector in albino seedlings was 621 bp, as observed by agarose gel electrophoresis, which is consistent with expectations. (Wild barley) HbPDS The positive rate of the gene was 62.3%, as shown in the results. Figure 7 As shown.

[0102] Table 10: PCR identification reaction system for leaf DNA

[0103]

[0104] Example 8: Wild barley after infection HbPDS Determination of gene expression levels

[0105] RNA was extracted from albino wild barley seedlings, and RT-qPCR was used to analyze the RNA of individual albino wild barley seedlings. HbPDS Gene expression levels.

[0106] Wild barley albino seedlings were cultured for 3-4 weeks to observe phenotypic changes in the newly formed wild barley plants. The entire plant was albino. RNA was extracted from the whole plant samples using the Novizan RNA extraction kit. First-strand cDNA was synthesized using a one-step reverse transcription kit with the TIANGEN kit. The synthesized product was stored at -20℃ for later use. Using the reverse-transcribed cDNA as a template and Actin as an internal reference gene, the expression level of the silenced target gene was detected by qRT-PCR. The correspondence between each amplification target and primer in qRT-PCR is shown in Table 11 below.

[0107] Table 11: qRT-PCR primer design

[0108]

[0109] Wild barley HbPDS Gene expression level measurement results as follows Figure 9 As shown, the results indicate that, compared with the control WT, the infection rate of wild barley albino seedlings was significantly higher. HbPDS Gene expression levels were significantly reduced.

[0110] In summary, this invention successfully establishes a method for use with wild barley. HbPDS A rapid screening system for gene silencing using the VIGS technique. This system boasts advantages such as ease of operation, rapid and efficient processing, low cost, high transformation efficiency, short phenotypic identification cycle, and suitability for high-throughput processing, providing strong technical support for wild barley gene function research. Furthermore, this method can not only efficiently transform wild barley seeds but also obtain positive plants, potentially accelerating the genetic transformation and genetic engineering of wild barley, and providing an efficient tool for conducting functional genomics research on wild barley. The VIGS system established in this invention not only provides an efficient tool for wild barley gene function research but also provides a theoretical basis and practical reference for gene function research in other monocotyledonous plants using TRV-VIGS technology.

[0111] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. A method for preparing the recombinant viral vector pTRV2-HbPDS, characterized in that, The recombinant viral vector pTRV2-HbPDS contains HbPDS Gene-specific fragments; The preparation method includes: reverse transcription of wild barley RNA to synthesize cDNA, and using the cDNA obtained by reverse transcription as a template, performing PCR amplification using primers pTRV2-HbPDS-F and pTRV2-HbPDS-R to obtain... HbPDS Gene-specific fragments; using the wild barley phytoene dehydrogenase gene HbPDS For the target gene, seamless DNA cloning technology was used to... HbPDS A gene-specific fragment was ligated into BamHI in the MCS region of the VIGS viral backbone vector pTRV2. The ligation product was transformed into DH5α Escherichia coli competent cells, and bacterial culture PCR was performed for identification. Sequencing results were compared to obtain the constructed recombinant viral vector pTRV2-HbPDS. The pTRV2-HbPDS-F sequence is shown in SEQ ID No. 4, and the pTRV2-HbPDS-R sequence is shown in SEQ ID No.

5. The wild barley phytoene dehydrogenase gene... HbPDS The nucleotide sequence is shown in SEQ ID No.

1.

2. The method for preparing the recombinant viral vector pTRV2-HbPDS according to claim 1, characterized in that, The primers used in the bacterial culture PCR identification were pTRV2-JC-F and pTRV2-JC-R; the sequence of pTRV2-JC-F is shown in SEQ ID No. 7, and the sequence of pTRV2-JC-R is shown in SEQ ID No.

8.

3. A wild barley phytoene dehydrogenase gene HbPDS The method for constructing the VIGS silencing system includes the following steps: S1. The recombinant viral vector pTRV2-HbPDS described in claim 1 was transferred into Agrobacterium tumefaciens EHA105 competent cells by freeze-thaw method, and the PCR identification was positive. S2. Mix the pTRV1 positive Agrobacterium tumefaciens bacterial solution with the transformed pTRV2-HbPDS positive Agrobacterium tumefaciens bacterial solution at a bacterial volume ratio of 1:1 to prepare a mixed bacterial solution; add cysteine, acetylsuccine and Tween-20 to the mixed bacterial solution and mix evenly to obtain an infection solution; S3. After sterilizing the wild barley seeds with the seed coat completely removed, they are cultured aseptically. When more than half of the seeds have germinated to 3 mm, the germinated seeds are infected with the above-mentioned infection solution and then dried. S4. Detection: The dried seeds from S3 were spread on a sugar-containing solid culture medium and grown under conditions of 22℃ and a light / dark cycle of 14 h / 10 h. Phenotypic changes in wild barley seedlings were observed, and the number of albino wild barley seedlings was tested. HbPDS The expression level of the gene, i.e., the constructed wild barley phytoene dehydrogenase gene. HbPDS The VIGS silencing system; the wild barley phytoene dehydrogenase gene HbPDS The nucleotide sequence is shown in SEQ ID No. 1.

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