Cucumber aphid-regulated gene csago5a and application thereof

Abstract

The application discloses a cucumber aphid-susceptible regulation gene CsAGO5a and application thereof, including that a nucleotide sequence thereof is shown as SEQ ID NO. 1. An expression protein of the cucumber aphid-susceptible regulation gene CsAGO5a has an amino acid sequence shown as SEQ ID NO. 2. A plant virus carrier pV190 containing the cucumber aphid-susceptible regulation gene CsAGO5a. The carrier silences the CsAGO5a gene and has low expression in plants. The introduced carrier is pV190, and linearization treatment is carried out on the pV190 after enzyme cutting using BamH I to obtain a recombined carrier. The application takes a cucumber main cultivar "Superina" as material, silences the CsAGO5a gene through a VIGS technology, and carries out a non-selective colonization experiment and the like, proves that the application is related to plant aphid resistance, has important application value in the fields of cucumber breeding such as enhancing plant resistance, improving aphid resistance screening methods and creating germplasm resources and the like.

Description

Technical Field

[0001] This invention belongs to the field of plant genetic engineering technology, specifically relating to a cucumber aphid-susceptibility regulatory gene. CsAGO5a And its applications. Background Technology

[0002] Cucumber (Cucumis sativus L.) is an annual herbaceous crop belonging to the Cucurbitaceae family and the Cucurbita genus. It is one of the main cultivated vegetable crops in my country and plays an important role in food and medicine.

[0003] Aphids damage cucumber plants in multiple ways, including sucking sap, secreting honeydew, spreading viruses, and causing leaf damage. Plants infested with aphids typically exhibit yellowing and curling of leaves, stunted growth, significantly reduced yields, and possible premature wilting and death. In horticulture, the most common method for aphid control is the use of pesticides. However, pesticide use not only significantly increases production costs but also poses many potential hazards. Prolonged pesticide use can easily cultivate aphid resistance and cause environmental pollution and soil degradation. Furthermore, it can harm aphid predators, pollinators, and other insects. Therefore, developing high-quality aphid-resistant varieties is crucial, as it provides a fundamental solution to mitigating the threat and damage posed by aphids to crops.

[0004] AGOs are a class of relatively conserved proteins that are ubiquitous in organisms and play crucial roles in RNA silencing and related pathways across different species. They also regulate the growth and development of the model plant Arabidopsis thaliana. RNA silencing is a fundamental resistance mechanism against foreign nucleic acids, and AGOs, DCLs, and RDRs are components of this mechanism. AGOs participate in small RNA (sRNA) binding and act as part of the RNA-induced silencing complex in the RNA interference (RNAi) pathway, regulating gene expression at both the transcriptional and post-transcriptional levels, thereby implementing various fine-tuning functions in various biological processes. Studies have shown that... CsAGO5a It responds to cold and heat stress and is highly expressed in the stems and flowers of the plant. (Watermelon) AGO5 By upregulating and interacting with virus-derived siRNA during viral infection, and then promoting viral RNA degradation post-transcriptionally, it plays a role in resistance to cucumber green mottle mosaic virus. Currently, in cucumber... AGO5 The relationship between RNA silencing pathways and aphid stress has not yet been identified and elucidated. Summary of the Invention

[0005] The purpose of this section is to outline some aspects of embodiments of the present invention and to briefly describe some preferred embodiments. Simplifications or omissions may be made in this section, as well as in the abstract and title of this application, to avoid obscuring the purpose of these documents; however, such simplifications or omissions should not be construed as limiting the scope of the invention.

[0006] In view of the problems existing in the above and / or prior art, the present invention is proposed.

[0007] Therefore, the purpose of this invention is to overcome the shortcomings of the prior art and provide a method for cultivating transgenic plants.

[0008] To solve the above-mentioned technical problems, the present invention provides the following technical solution: a transgenic plant is obtained by containing the gene CsAGO5a of the target plant, thereby regulating the aphid resistance and / or the adaptability of the transgenic plant on cucumber leaves.

[0009] The nucleotide sequence of the CsAGO5a gene is shown in SEQ ID No. 1; the amino acid sequence is shown in SEQ ID No. 2.

[0010] The seeds containing the genetically modified plant.

[0011] The genetically modified plant is a cucumber.

[0012] The purpose of this invention is to overcome the shortcomings of the prior art and provide a recombinant carrier.

[0013] The purpose of this invention is to overcome the shortcomings of the prior art and provide a host cell.

[0014] The purpose of this invention is to overcome the shortcomings of the prior art and provide a method for regulating the aphid resistance of cucumbers.

[0015] To solve the above-mentioned technical problems, the present invention provides the following technical solution: after the recombinant vector is transformed into Agrobacterium, it is injected into the cotyledons of cucumber seedlings to silence the CsAGO5a gene.

[0016] In a preferred embodiment of the method described in this invention, the transfer vector is pV190, which is linearized by BamHI digestion and then recombined to obtain a recombinant vector.

[0017] In a preferred embodiment of the method described in this invention, the introduction of a nucleic acid molecule expressing the CsAGO5a protein into the recipient plant is achieved by introducing a recombinant expression vector containing a nucleic acid molecule capable of expressing the CsAGO5a protein into the recipient plant.

[0018] The nucleotide sequence of the recombinant vector is shown in SEQ ID No. 3.

[0019] Beneficial effects of this invention:

[0020] This invention uses the main cucumber variety "Superina" as material and clones it using RACE technology. CsAGO5a Genes, simultaneously through enzyme digestion, based on AGOs The recombinant reaction related to plant stress resistance, Agrobacterium-mediated transformation, injection into cucumber leaves, and subsequent aphid biomass statistics and non-selective colonization experiments demonstrated that it is a regulator of aphid susceptibility. This invention has significant application value in cucumber breeding, providing methods for enhancing plant resistance, improving aphid resistance screening, and creating germplasm resources. Attached Figure Description

[0021] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments 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. Wherein:

[0022] Figure 1 This is an evolutionary tree diagram of the AGOs gene family.

[0023] Figure 2 After inoculating aphids at 6h, 24h, and 72h in Example 2 of this invention CsAGO Family fluorescence quantitative data graph.

[0024] Figure 3 This is a plasmid map of the plant virus-mediated vector pV190 recombinant vector in Example 2 of the present invention.

[0025] Figure 4 The VIGS technology in Embodiment 1 of this invention was successfully silenced. CsAGO5a The cucumber plants afterward.

[0026] Figure 5 The silence in Embodiment 2 of the present invention CsAGO5a Plants with unsilenced genes CsAGO5a Differences in gene expression levels among plants.

[0027] Figure 6 The unsilenced gene plants and the silenced gene plants in Example 3 of this invention. CsAGO5a Identification of aphid resistance in plants. Detailed Implementation

[0028] To make the above-mentioned objects, features and advantages of the present invention more apparent and understandable, the specific embodiments of the present invention will be described in detail below with reference to the examples in the specification.

[0029] Many specific details are set forth in the following description in order to provide a full understanding of the invention. However, the invention may also be practiced in other ways different from those described herein, and those skilled in the art can make similar extensions without departing from the spirit of the invention. Therefore, the invention is not limited to the specific embodiments disclosed below.

[0030] Secondly, the term "one embodiment" or "embodiment" as used herein refers to a specific feature, structure, or characteristic that may be included in at least one implementation of the present invention. The phrase "in one embodiment" appearing in different places in this specification does not necessarily refer to the same embodiment, nor is it a single or selective embodiment that is mutually exclusive with other embodiments.

[0031] Unless otherwise specified, all raw materials used in the embodiments of this invention are commercially available.

[0032] This invention uses the main cucumber variety "Superina" as material, as detailed in the introduction of the literature "Inheritance and Quantitative Trait Locus Mapping of Fusarium Wilt Resistance in Cucumber".

[0033] Example 1

[0034] CsAGO5a Gene screening

[0035] like Figure 1 This is an evolutionary tree diagram of the AGOs gene family.

[0036] Aphid stress was applied to cucumber leaves by inoculating 20 aphids on the true leaves. Samples were taken at 6 h, 24 h, and 72 h after inoculation for transcriptome sequencing.

[0037] like Figure 2 As shown: 24h CsAGO5a Genes are significantly upregulated.

[0038] Example 2

[0039] CsAGO5a Acquisition of genes:

[0040] 1. Extraction of total RNA from cucumber leaves

[0041] Total RNA was extracted from cucumber using a rapid and universal plant RNA extraction kit. The experimental method was performed according to the kit's instructions, and the specific steps are as follows:

[0042] (1) Sample processing: The cucumber leaves were quickly ground into powder in liquid nitrogen. 30-150 mg of sample was added to 600 μl of lysis buffer SG and 10 μl of proteinase K. The mixture was immediately vortexed and shaken vigorously to mix. After mixing, it was placed at room temperature for 5 min.

[0043] (2) Centrifuge at 12,000 rpm (~13,400Xg) for 2 min, and take about 500 μl of supernatant for the following operations.

[0044] (3) Add the obtained supernatant to the genomic DNA removal column, centrifuge at 12,000 rpm (~13,400Xg) for 30 sec, and retain the filtrate.

[0045] (4) Slowly add 0.5 times the volume of supernatant of anhydrous ethanol (about 250 μl) to the above filtrate, mix well (precipitation may occur at this time), and transfer the resulting solution and precipitate into an RNase-Free adsorption column CR4 (the adsorption column is placed in the collection tube), centrifuge at 12,000 rpm (~13,400Xg) for 30 seconds, discard the waste liquid in the collection tube, and put the adsorption column back into the collection tube.

[0046] (5) Add 700 μl of protein removal solution RW3 to the RNase-Free adsorption column CR4, centrifuge at 12,000 rpm (~13,400 x g) for 30 seconds, discard the waste liquid, and put the adsorption column back into the collection tube.

[0047] (6) Add 500 μl of washing solution RW to the RNase-Free adsorption column CR4, let it stand at room temperature for 2 min, centrifuge at 12,000 rpm (~13,400xg) for 30-60 sec, discard the waste liquid, and put the adsorption column back into the collection tube.

[0048] (7) Repeat step 6.

[0049] (8) Centrifuge at 12,000 rpm (~13,400 x g) for 2 min and discard the waste liquid. Place the RNase-Free adsorption column CR4 at room temperature for 2 min to thoroughly dry any residual washing liquid in the adsorption material.

[0050] (9) Transfer the RNase-Free adsorption column CR4 into a new RNase-Free centrifuge tube, add 30-100 μl of RNase-Free ddHzO dropwise to the middle of the adsorption membrane, incubate at room temperature for 2 min, and centrifuge at 12,000 rpm (~13,400 x g) for 2 min to obtain the RNA solution.

[0051] 2. Synthesis of the first strand of cDNA

[0052] Residual DNA was removed using a one-step gDNA removal and cDNA synthesis kit, and the first strand of cDNA was synthesized. The specific method is as follows:

[0053] (1) Add the ingredients listed in Table 1 below to a DNase / RNase-free centrifuge tube:

[0054] Table 1

[0055]

[0056] Mix thoroughly by pipetting. Incubate at 42°C for 2 minutes.

[0057] (2) Add 5×HiScriptⅡqRTSuperMixⅡ to the first step reaction tube

[0058] Perform reverse transcription at 50℃ for 15 min, then at 85℃ for 5 sec, and store at -20℃.

[0059] CsAGO5a gene cloning:

[0060] Gene-specific primers CsAGO5a-F (5'-TGCCAAGCCGGTTTATTTGC-3') and CsAGO5a-R (5'-CCTTGATGATACAACATTAATGCCA-3') were designed based on the CsAGO5a sequence. Cucumber leaf cDNA was used as a template for PCR amplification. The PCR amplification reaction system is shown in Table 2 below, and the PCR amplification reaction program is shown in Table 3 below.

[0061] Table 2 PCR amplification reaction system

[0062]

[0063] Table 3 PCR amplification reaction procedure

[0064]

[0065] Example 3

[0066] VIGS technology

[0067] 1. Select CsAGO5a Silent genes

[0068] Based on the important role of AGOs in plant stress resistance, selection CsAGO5a The target gene is silenced.

[0069] 2. Constructing a VIGS carrier

[0070] I. Cloning the target fragment (PCR)

[0071] PCR refers to the process of generating a large number of specific DNA sequences by replicating DNA fragments. The DNA template to be amplified, primers, DNA polymerase, four nucleotides (dNTPs), and buffer are mixed together to prepare the PCR reaction mixture. A large number of specific DNA sequences are obtained through repeated denaturation, annealing, and extension processes. Gene-specific primers CsAGO5a-F were designed based on the CsAGO5a sequence: 5'-AGGACTTTACTTAATGGATCCACTATACAAGTTCTTGATGTTGTTTTGAGA-3'; CsAGO5a-R: 5'-CCTAGACCTATAACTGGATCCCTTTTTGATCTTGCGACAATCCT-3'. Cucumber leaf cDNA was used as a template for PCR amplification. The PCR amplification reaction system is shown in Table 4 below, and the PCR amplification reaction procedure is shown in Table 5 below.

[0072] Table 4 PCR amplification reaction system

[0073]

[0074] Table 5 PCR amplification reaction procedure

[0075]

[0076] II. Constructing a Recombination Vehicle

[0077] The reaction system consisted of: (0.04 × 416 bp) target fragment, [(0.02 × 10000) / carrier concentration] carrier, and 2 μL LExnase II;

[0078] Reaction procedure: 37 ℃ for 30 min.

[0079] like Figure 3 Image shown: Plasmid map of plant virus-mediated vector pV190;

[0080] 3. Transformation

[0081] I. Escherichia coli transformation

[0082] Escherichia coli transformation is the process of introducing exogenous DNA into E. coli cells to introduce plasmids or other exogenous DNA into bacteria for expression or research.

[0083] II. Screening

[0084] Transformed E. coli cells were distributed on agar plates containing antibiotics. Only bacteria carrying the target plasmid were able to grow in the presence of antibiotics. Colony PCR-positive cultures were sent to Qingke Biotechnology Co., Ltd. for sequencing.

[0085] III. Preservation of bacteria and plasmid extraction

[0086] Once bacteria have grown on the streak plate, select a single colony and incubate it overnight in a 1.5 mL centrifuge tube containing 1 mL of 1 / 1000 Kan LB liquid medium. Then add 70% glycerol, mix well, and store at -80°C. Use the TIANGEN kit to extract plasmids from the bacterial culture and perform Agrobacterium transformation.

[0087] IV. Transformation with Agrobacterium tumefaciens (GV3101)

[0088] All Agrobacterium transformation operations were performed on ice to maintain a low-temperature environment: Frozen Agrobacterium GV3101 competent cells were thawed on ice. 1000 ng of the purified fragment-vector ligation product was added to 100 μL of competent cells, shaken to mix, and then placed on ice, in liquid nitrogen, a 37°C water bath, and an ice bath for 5 min each. 700 μL of LB liquid medium was added, and the mixture was incubated at 28°C, 200 rpm for 2-3 h. After centrifugation at 6000 rpm for 1 min, most of the LB liquid medium was removed. The remaining 100 μL was gently pipetted and mixed, then spread onto LB solid medium containing the antibiotic Kan+Rif using a spreader. The mixture was then incubated upside down at 28°C for 2-3 days.

[0089] VI. Colony PCR Identification

[0090] The selected positive clones were subjected to shaking and preservation.

[0091] The obtained Agrobacterium was subjected to two shaking experiments. The first shaking system consisted of 5 mL LB liquid medium, 1 / 1000 antibiotic (Kan + Rif), and 150 μL of bacterial culture, incubated overnight at 28°C for 200 rpm. The second shaking system consisted of 150 mL LB liquid medium, 1 / 1000 antibiotic (Kan + Rif), and 150 μL of the bacterial culture from the first shaking, incubated overnight at 28°C for 200 rpm.

[0092] V. Injection:

[0093] (1) After preparing the resuspended solution, inject it in the dark at 28°C for 2-3 hours;

[0094] (2) Injection:

[0095] The suspension was drawn up with a 5 mL syringe and injected into the cotyledons of cucumber. After 17-21 days, the whitening of the cucumber plants injected with PDS was observed, indicating that the operation was successful.

[0096] Phytoene desaturase (PDS) is a key enzyme in the carotenoid synthesis pathway. When PDS expression is inhibited, plants lose the photoprotective effect of carotenoids, thus exhibiting an albinism effect. It is often used as a reference for the success or failure of experimental procedures.

[0097] like Figure 4 VIGS technology successfully silenced CsAGO5a The cucumber plants were injected with the VIGS solution; three weeks after injection, the plant leaves began to turn white, indicating that the VIGS experiment was successful. CsAGO5a The plant exhibits gene silencing function.

[0098] Example 4

[0099] Quantitative fluorescence technique

[0100] 1. RNA extraction from plant leaves

[0101] After plants with the PDS gene silenced became albino, the silenced gene was removed. CsAGO5a Plant leaves with unsilenced genes CsAGO5a The plant leaves containing the gene were rapidly ground into powder in liquid nitrogen, and the resulting RNA solution was obtained using the RNA Easy Fast Plant Tissue Kit and stored at -80°C.

[0102] 2. Reverse transcription

[0103] The RNA solution obtained from the above operations was reverse transcribed into cDNA using the Vazyme HiScript III 1st Strand cDNA Synthesis Kit (+gDNAwiper).

[0104] 3. Quantitative fluorescence analysis

[0105] Using a SYBR Premix Ex Taq (Takara) and a Q225 quantitative PCR instrument (Coolbot), and following the manufacturer's instructions for the PCR system and procedure, quantitative PCR was performed to obtain the number of cycles required to reach the fluorescence threshold, and the silencing factor was calculated. CsAGO5a The relative expression level after gene expression.

[0106] like Figure 5 The silence shown CsAGO5a Plants with unsilenced genes CsAGO5a Differences in gene expression levels among plants.

[0107] Example 5

[0108] To those who did not remain silent CsAGO5a Cucumber plants with silent genes CsAGO5aAphid resistance was assessed by counting aphid populations in cucumber plants treated with the gene over 10 days. Using GraphPad software, four cucumber plants of similar growth were selected from each of the two treatments for biomass analysis. CsAGO5a Differences in resistance after gene silencing. Ten days later, the aphid biomass on plants with silenced CsAGO5a was 84, compared to those without silencing. CsAGO5a The total aphid biomass on the plants was 228. The results indicate... CsAGO5a The number of aphids after silencing was less than the number before silencing. CsAGO5a Cucumber plants. (The control group consisted of cucumber plants with unsilenced CsAGO5a.)

[0109] Table 6 shows the number of aphids after CsAGO5a silencing and the number of aphids without CsAGO5a silencing.

[0110]

[0111] like Figure 6 As shown: Not silent CsAGO5a Genes and Silence CsAGO5a Identification of aphid resistance in plants based on gene expression.

[0112] Example 6

[0113] To those who have remained silent CsAGO5a Plants with unsilenced genes CsAGO5a Genetic life tables for aphids were constructed. Five plants with similar growth were selected, and a layer of aluminum foil was placed on the soil surface. Two wingless, healthy adult aphids were then grafted onto the plantlets and covered with a net. After the adult aphids laid nymphs, the adults and excess newly laid nymphs were removed, leaving only one newly laid nymph. The nymph's molting and survival were observed every 24 hours. When the nymph grew into an adult, observations were made every 24 hours, and aphid production was recorded. After recording, the laid nymphs were removed, and observations continued until the aphids died. This yielded various parameters of population variation, including net adult aphid proliferation rate (R0), cyclical growth rate (λ), intrinsic growth rate (rm), and average generation period (T).

[0114] Table 7 lists those who have been silenced. CsAGO5a Aphid life table on plants with genetic information

[0115]

[0116] Table 8 lists those who have been silenced. CsAGO5a Parameters of the life table of aphids on plants with genes

[0117]

[0118] Table 9 shows those who are not silent. CsAGO5a Aphid life table on plants with genetic information

[0119]

[0120] Table 10 shows those who were not silent. CsAGO5a Parameters of the life table of aphids on plants with genes

[0121]

[0122] It should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and are not intended to limit it. Although the present invention has been described in detail with reference to preferred embodiments, those skilled in the art should understand that modifications or equivalent substitutions can be made to the technical solutions of the present invention without departing from the spirit and scope of the technical solutions of the present invention, and all such modifications or substitutions should be covered within the scope of the present invention.

Claims

1. The application of the CsAGO5a gene in regulating cucumber aphid susceptibility, characterized by... The invention includes enhancing the aphid resistance of cucumber plants by silencing the CsAGO5a gene and regulating the adaptation of aphids on cucumber leaves; the nucleotide sequence of the CsAGO5a gene is shown in SEQ ID No. 1; the amino acid sequence is shown in SEQ ID No. 2.

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