Application of sunflower HaMAPK6-1 gene in improving sunflower resistance to orobanche parasitism

Abstract

This invention relates to the field of genetic engineering technology, specifically to sunflowers. HaMAPK6‑1 Application of genes in improving sunflower resistance to broomrape parasites. This invention obtains complete genome sequences from sunflower transcriptome data. HaMAPK6‑1 Gene sequence, and HaMAPK6‑1 The gene was transferred into the native plant, sunflower, using transient conversion technology, and overexpressed. HaMAPK6‑1 In sunflower plants containing the gene, an experiment was conducted on transgenic plants with orobanche parasitism, and it was found that overexpression of the gene was observed. HaMAPK6‑1 The genetically modified sunflower showed significantly enhanced resistance to broomrape parasites compared to wild-type sunflowers, a finding of great value for molecular breeding and application of sunflowers.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, specifically to sunflowers. HaMAPK6-1 Application of genes in improving sunflower resistance to broomrape parasites. Background Technology

[0002] In modern agriculture and food processing, sunflower ( Helianthus annuus Sunflowers (L.) have gradually become a focus of research due to their significant economic and medicinal value. Sunflowers are one of the most widely cultivated oil crops globally, and due to their genetic diversity, they have diversified into three main cultivation types: edible, oil-producing, and ornamental. Sunflowers are not only a crop with important economic value in agriculture, but they also play an important role in food processing, industrial production, and environmental beautification; their medicinal value is equally undeniable.

[0003] However, large-scale sunflower cultivation still faces many challenges from biotic and abiotic stresses, among which the obligate root parasitic weed *Broomrape chinensis* (sunflower broomrape) is a major hazard. Orobanche cumana The most serious damage is caused by *Broomrape nigra* (also known as sunflower broomrape). This annual, holoparasitic herbaceous plant, belonging to the genus *Broomrape* in the family Orobanchaceae, ranges in height from 15 to 54 cm. As a holoparasitic plant, it specifically parasitizes the roots of sunflowers through its haustoria. Once successfully parasitized, *Broomrape nigra* absorbs nutrients and water from the sunflower through these haustoria connected to the roots. This competitive deprivation of water and nutrients from the host sunflower leads to physiological metabolic disorders, causing severe yield losses and becoming a key factor restricting sunflower production. After parasitism, *Broomrape nigra* significantly reduces the leaf area index of sunflowers during the grain-filling stage, decreases the photosynthetic rate of the sunflower population, and consequently affects the synthesis and accumulation of dry matter. It also damages the root morphology of sunflowers, affecting their normal growth and development.

[0004] Currently, the control of broomrape in sunflowers is mainly carried out through deep plowing, crop rotation, and pesticide application. Breeding broomrape-resistant varieties is also an important way to control broomrape. However, broomrape has the ability to evolve rapidly. Once a resistant sunflower variety that is effective against existing broomrape races is discovered, broomrape will evolve into more toxic physiological races after a period of time. Therefore, breeding broomrape-resistant varieties is a long-term and continuous task.

[0005] In plant pathogen-pathogen interactions, the mitogen-activated protein kinase (MAPK) cascade pathway is one of the core nodes in signal transduction. It can convert the sensory signals of potential pathogens into cellular responses, amplifying external stimuli at each stage to regulate plant responses to biotic stresses (such as pathogen infection) and abiotic stresses. However, whether the MAPK pathway participates in regulating the sunflower's defense response against broomrape parasites, and which specific MAPK family member plays a key role, remains unclear and unverified in current technology. Summary of the Invention

[0006] In view of this, there is an urgent need to discover sunflower genes resistant to broomrape and to breed sunflower varieties resistant to broomrape. This invention proposes a sunflower... HaMAPK6-1 Application of genes in improving sunflower resistance to broomrape parasites.

[0007] The specific technical solution is as follows: The present invention provides in a first aspect HaMAPK6-1 The use of genes or related biological materials in at least one of the following: A1) Regulating sunflower broomrape parasitism resistance or preparing products that regulate sunflower broomrape parasitism resistance; A2) Cultivate sunflowers with enhanced resistance to broomrape parasites or prepare products that enhance sunflower resistance to broomrape parasites; A3) Preparation of transgenic sunflowers; The HaMAPK6-1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0008] Furthermore, the biomaterial includes one or more of the following: The HaMAPK6-1 The gene-encoded protein and / or containing the above HaMAPK6-1 Gene recombinant vectors, recombinant microorganisms, or transgenic sunflower cell lines; The amino acid sequence of the HaMAPK6-1 protein is shown in SEQ ID NO.2.

[0009] Furthermore, the regulation of sunflower broomrape parasitism resistance includes: regulating the parasitism status of sunflower broomrape on sunflower roots.

[0010] Furthermore, the regulation of sunflower orobanche parasitism resistance includes: through positive regulation HaMAPK6-1 Gene expression is used to enhance sunflower broom parasitism resistance.

[0011] The present invention provides positive regulation in a second aspect. HaMAPK6-1 Application of gene-expressing biological materials in the cultivation of sunflowers resistant to broomrape parasites; HaMAPK6-1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0012] Further, the biomaterial includes: HaMAPK6-1 The gene encodes a protein and / or contains positive regulatory functions. HaMAPK6-1 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic sunflower cell lines.

[0013] Furthermore, the enhanced resistance to orobanche includes inhibiting the development of sunflower orobanche on sunflower roots.

[0014] In a third aspect, the present invention provides a method for improving sunflower broom parasitism resistance, comprising: Positive regulation HaMAPK6-1 Gene-expressing biological material was transferred into sunflowers.

[0015] The present invention provides a method for cultivating sunflowers with enhanced resistance to broomrape parasitism in a fourth aspect, comprising: Positive regulation HaMAPK6-1 Gene-expressing biological material was transferred into sunflowers.

[0016] In a fifth aspect, the present invention provides a sunflower with enhanced resistance to orobanche deserticola, the sunflower comprising positive regulation HaMAPK6-1 Biological materials for gene expression; The HaMAPK6-1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

[0017] Furthermore, the sunflower varieties mentioned are Tonghui 15 and TY0409.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention obtained complete transcriptome data from sunflowers. HaMAPK6-1 Gene sequence, and HaMAPK6-1 The gene was transferred into the native plant, sunflower, and overexpressed. HaMAPK6-1 Sunflower plants with the gene. Through experiments with transgenic plants and broomrape parasitism, it was found that overexpression... HaMAPK6-1 The genetically modified sunflower showed significantly enhanced resistance to broomrape parasites compared to wild-type sunflowers, a finding of great value for molecular breeding and application of sunflowers. Attached Figure Description

[0019] Picture 1 For sunflowers HaMAPK6-1 PCR electrophoresis image of a gene clone.

[0020] Picture 2 In the image, A represents a heatmap of transcriptome data; Picture 2 B in the text represents sunflower. HaMAPK6-1 A schematic diagram showing the relative expression levels of the gene before and after infection with *Orobanche deserticola* in sunflower. TH represents a normally growing Tonghui 15 plant, THO represents a Tonghui 15 plant after infection with *Orobanche deserticola*, TY represents a normally growing TY0409 plant, and TYO represents a TY0409 plant after infection with *Orobanche deserticola*.

[0021] Picture 3 For sunflowers HaMAPK6-1 Subcellular localization map of gene transfer into tobacco; scale bar: 20µm.

[0022] Picture 4 For the sunflower broomrape sensitive variety TY0409 HaMAPK6-1 Results of co-culturing sunflower broomrape with plants exhibiting transient gene overexpression and control groups; the circled areas indicate the parasitic sites of sunflower broomrape. Picture 4 Plant A represents the control group. Picture 4 B represents overexpression. HaMAPK6-1 Gene-based plants.

[0023] Picture 5 Transient overexpression of orobanche 15 in the sunflower cultivar Tonghui 15, which is resistant to orobanche, and TY0409, which is susceptible to orobanche. HaMAPK6-1 After gene modification, in sunflower plants HaMAPK6-1 A graph showing gene expression levels; among which, Picture 5 A represents resistant sunflower plants. HaMAPK6-1 Gene expression levels, Picture 5 B represents sensitive sunflower plants. HaMAPK6-1 Gene expression levels; WT represents the blank plant control group, and GFP represents the empty vector plant group. Detailed Implementation

[0024] To enable those skilled in the art to better understand the present invention, the technical solution of the present invention will be clearly and completely described below with reference to specific embodiments. It should be noted that the following detailed descriptions are exemplary and are only some embodiments of the present invention, not all embodiments.

[0025] Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort should fall within the scope of protection of the present invention.

[0026] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains. The experimental materials used in the embodiments of this invention are all conventional experimental materials in the art and are commercially available. Experimental methods not specifying detailed conditions were performed according to conventional experimental methods or the operating instructions recommended by the supplier.

[0027] In this invention, HaMAPK6-1 The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the HaMAPK6-1 protein is shown in SEQ ID NO.2.

[0028] SEQ ID NO.1:

[0029] SEQ ID NO.2: MDPSIHPSDTEMSDAAAPPPPPPQQQPVAGIDNIPATLSHGGRFIQYNIFGNIFEVTAKYKPPIMPIGKGAYGIVCSALNSETNEHVAIKKIANAFDNKIDAKRTLREIKLLRHMDHENVVAIRDIIPPPERNAFNDVYIAYELMDTDLHQIIRSNQSLSEEHCQYFLYQILRGLKYIHSANVLHRDLKPSNLLLNA NCDLKICDFGLARVTSETDFMTEYVVTRWYRAPELLLNSSDYTAAIDVWSVGCIFMELMDRKPLFPGRDHVHQLRLLMELIGTPSEAELGFLNENAKRYIRQLQHYPRQSFNEKFPQVHPAAIDLIEKMLTFDPRQRITVEDALAHPYLTSLHDISDEPVCMTPFNFDFEHHAPTEEQMKEMIYREALAFNPEYQHL.

[0030] In this invention, the sunflower varieties are: the resistant variety Tonghui 15 (TH15) and the sensitive variety TY0409; the sunflower broomrape species are: mixed seed samples of broomrape physiological races of grade E or above. In the early stage of sunflower broomrape parasitizing sunflower, root nodules are formed on the sunflower roots. The color is white in the early stage and gradually turns yellow in the later stage.

[0031] Example 1: Sunflower HaMAPK6-1 Gene amplification Take 0.2 g of fresh sunflower root tissue sample, add an appropriate amount of grinding beads to a 2 mL centrifuge tube, and quickly grind it into powder in a sample grinder pre-cooled with liquid nitrogen. Extract total RNA using the FastPure® Universal Plant Total RNA Isolation Kit. Detect its integrity and concentration using 1% agarose gel electrophoresis and a NanoDrop ultra-micro spectrophotometer. Then, reverse the extracted RNA into cDNA using the HiScript III 1st Strand cDNA Synthesis Kit (+gDNA wiper).

[0032] PCR amplification was performed using 2 × Phanta Max MasterMix (Dye Plus) with cDNA as a template (e.g.) Picture 1 (As shown), the amplification primers are: HaMAPK6-1-F: gagaacacgggggacgagctcATGGATCCATCAATTCACCCTTC (SEQ IDNO.3); HaMAPK6-1 -R: gctcaccatgtcgactctagaCAGATGCTGATATTCAGGATTAAAAGC (SEQ ID NO. 4).

[0033] 25 μL PCR amplification system: 12.5 μL 2 × Phanta Max MasterMix (Dye Plus), 1 μL forward primer, 1 μL reverse primer, 1000 ng template cDNA, and RNase-free ddH2O added to a final volume of 25 μL. PCR program settings: 95°C for 3 min, 95°C for 15 s, 60°C for 15 s, 72°C for 45 s, 35 cycles, 72°C for 5 min, and storage at 12°C.

[0034] 1% agarose gel electrophoresis was used to check if the target band size was correct. The gel was then cut and recovered using the EasyPure® Quick Gel Extraction Kit. The recovered product was sequenced to obtain the sunflower product. HaMAPK6-1 The nucleotide sequence of a gene.

[0035] Example 2: Sunflower HaMAPK6-1 Functional verification of genes The sunflower varieties used in this example are Tonghui 15 (TH15) and TY0409.

[0036] 2.0, Transcriptome Sequencing-Based HaMAPK6-1 Gene differential expression screening To investigate the molecular mechanism of sunflower response to broomrape parasitism, root tissues were collected from the resistant variety Tonghui 15 (TH15) and the susceptible variety TY0409 under normal growth conditions and after broomrape parasitism. Transcriptome sequencing analysis was performed. Through comparison of sequencing data and screening of differentially expressed genes, a gene encoding… MAPK6-1 The expression level of the gene was significantly upregulated in resistant varieties after parasitism, while the change was not significant in susceptible varieties. Picture 2 A).

[0037] 2.1 HaMAPK6-1 Differences in gene expression before and after orobanche parasitism Remove the husks from the sunflower seeds. Pour distilled water into a petri dish to soak the husks. Use tweezers to peel off the transparent seed coat. Gently scratch the peeled sunflower seeds a few times with the tweezers, being careful not to scratch the embryo or other important parts of the seed. Place the sunflower seeds in MS liquid medium. After germination, transfer them to MS solid medium. After 24-48 hours, transfer the sunflower seeds to square dishes and culture them in a tissue culture room for two weeks.

[0038] Sunflower seedlings that had grown normally for two weeks were co-cultured with sunflower broomrape seeds that had been pre-germinated with the germination stimulant GR24. After 10-14 days, it was observed that the sunflower seedlings were parasitized by sunflower broomrape. RNA was extracted from the sunflowers parasitized by sunflower broomrape and RNA was extracted from the normally growing sunflowers as a control. The RNA was reverse transcribed into cDNA and then subjected to real-time quantitative PCR to verify the gene expression.

[0039] like Picture 2 As shown in B, after sunflower seedlings were parasitized by *Broomrape nigra*, in the resistant variety Tonghui 15, HaMAPK6-1 Gene expression levels increased significantly, and in the sensitive variety TY0409, HaMAPK6-1 There was no significant change in gene expression levels.

[0040] 2.2 HaMAPK6-1 Subcellular localization of genes The pCAMBIA1300-eGFP vector was linearized using NEB restriction endonucleases Sac I and XbaI. The linearization system consisted of 5 µL rCutSmart buffer, 1 µg of vector, 1 µL each of Sac I and XbaI, with ddH2O added to a final volume of 50 µL. Enzyme digestion was performed at 37°C for 2 h, followed by purification and recovery via agarose gel electrophoresis. The gel-recovered product was compared with the cloned... HaMAPK6- 1 The gene product was subjected to homologous recombination using the pEASY®-Uni Seamless Cloning and Assembly Kit. The recombination system consisted of 5 µL of 2×Assembly Mix, 0.03 pmol of linearized vector, 0.06 pmol of gene fragment, and ddH2O added to a final volume of 10 µL. The reaction was carried out at 50°C for 15 min, and the product could be stored at -20°C.

[0041] The homologous recombination product was transformed into *E. coli* DH5α competent cells. One-tenth of the homologous recombination product was added to the competent cells, and the mixture was gently agitated by tapping the centrifuge tube. The cells were then placed on ice for 30 min, followed by heat shock at 42 °C for 45 s, and immediately placed on ice for 2 min. Antibiotic-free LB medium was added, and the cells were incubated on a shaker for 1 h. After centrifugation at 5000 rpm for 1 min, 30-50 µL of the resuspended cells were spread onto LB agar plates containing 50 ng / µL kanamycin and incubated overnight at 37 °C. Single colonies were selected for labeling, and culture PCR was performed by shaking. The correctly sequenced bands were sent for sequencing. The correctly sequenced bacterial cultures were shaken, plasmids were extracted, and stored at -20 °C for further experiments.

[0042] The extracted plasmid was transformed into Agrobacterium tumefaciens GV3101 (pSoup-p19) competent cells. The collected bacterial suspension was resuspended and plated on LB agar plates containing kanamycin, and incubated upside down at 28°C for 2-3 days. Single colonies were picked from LB liquid medium containing kanamycin and rifampin and shaken for PCR verification. Positive clones were screened, shaken, and injected into tobacco. Fluorescence results were observed using a laser confocal microscope.

[0043] The results show that HaMAPK6-1 Genes are present in various parts of the cell (e.g.) Picture 3 As shown), it exists in the cytoplasm, nucleus, and cell membrane of the cell. HaMAPK6-1 Gene expression.

[0044] 2.3 HaMAPK6-1 Obtaining gene overexpression plants The extracted plasmid was transformed into Agrobacterium tumefaciens GV3101 (pSoup-p19) competent cells. The resuspension was plated on LB agar plates containing kanamycin and incubated upside down at 28°C for 2-3 days. Single colonies were picked for colony PCR verification, and positive clones were selected for infecting sunflower seeds. The sunflower varieties TY0409 (sensitive to Orobanche deserticola) and TH15 (highly resistant) were selected. The seeds were dehulled, and distilled water was poured into a petri dish to soak the dehulled seeds. The transparent seed coat was peeled off with tweezers, and the seed coat was then gently scratched a few times with tweezers, taking care not to scratch the embryo or other important parts of the seed. The treated sunflower seeds were placed in a new petri dish, and the infection solution was poured into the dish. The dish was then incubated in the dark for 6 hours. Infected sunflower seeds were placed in MS liquid medium containing cephalosporin and termetidine and cultured in the dark for 48 h. Germinated seeds were then transferred to MS solid medium containing cephalosporin and termetidine. After 24-48 h, the sunflower seeds were transferred to MS solid medium containing cephalosporin and termetidine. Overexpression was obtained by growing the tissue culture medium in 13cm square petri dishes for two weeks in a tissue culture room. HaMAPK6-1Sunflower plants with the target gene overexpressed. After two weeks of normal growth, sunflower seedlings overexpressing the target gene were co-cultured with sunflower broomrape seeds pre-germinated by the germination stimulus GR24. Parasitism was observed for 10-14 days.

[0045] like Picture 4 As shown, during the same parasitism period, there were 4-6 orobanche parasites on the TY0409 blank plant group. Picture 4 A) Overexpression HaMAPK6-1 The TY0409 gene plant has no orobanche parasites on its roots or only 1-2 orobanche parasites. Picture 4 B).

[0046] Extracting overexpression HaMAPK6-1 RNA from the roots of sunflower plants containing TY0409 and TH15 genes, as well as from blank and empty vectors, was reverse transcribed into cDNA and then used for quantitative real-time PCR to verify gene expression. The results are as follows: Picture 5 A, Picture 5 As shown in B, it can be seen that... HaMAPK6-1 The gene was successfully overexpressed in sunflower Tonghui 15 and TY0409.

Claims

1. HaMAPK6-1 The use of genes or related biological materials in at least one of the following: A1) Regulating sunflower broomrape parasitism resistance or preparing products that regulate sunflower broomrape parasitism resistance; A2) Cultivate sunflowers with enhanced resistance to broomrape parasites or prepare products that enhance sunflower resistance to broomrape parasites; A3) Preparation of transgenic sunflowers; The HaMAPK6-1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. The application as described in claim 1, characterized in that, The biomaterials include one or more of the following: The HaMAPK6-1 The gene-encoded protein and / or containing the above HaMAPK6-1 Gene recombinant vectors, recombinant microorganisms, or transgenic sunflower cell lines; The amino acid sequence of the HaMAPK6-1 protein is shown in SEQ ID NO.

2.

3. The application as described in claim 1 or 2, characterized in that, The regulation of sunflower broomrape parasitism resistance includes: regulating the parasitism status of sunflower broomrape on sunflower roots.

4. The application as described in claim 3, characterized in that, The regulation of sunflower orobanche parasitism resistance includes: through positive regulation HaMAPK6-1 Gene expression is used to enhance sunflower broom parasitism resistance.

5. Positive regulation HaMAPK6-1 Application of gene-expressing biological materials in the cultivation of sunflowers resistant to broomrape parasites; HaMAPK6-1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

6. The application as described in claim 5, characterized in that, The biomaterial includes: HaMAPK6-1 The gene encodes a protein and / or contains positive regulatory functions. HaMAPK6-1 Recombinant vectors for gene expression, recombinant microorganisms, or transgenic sunflower cell lines.

7. The application as described in claim 5, characterized in that, The enhanced resistance to orobanche includes inhibiting the development of orobanche in the roots of sunflowers.

8. A method for improving the parasitic resistance of sunflower to broomrape, characterized in that, include: Positive regulation HaMAPK6-1 Gene-expressing biological material was transferred into sunflowers.

9. A method for cultivating sunflowers with enhanced resistance to orobanche deserticola, characterized in that, include: Positive regulation HaMAPK6-1 Gene-expressing biological material was transferred into sunflowers.

10. A sunflower with enhanced resistance to orobanche deserticola, characterized in that, The sunflower contains positive regulation. HaMAPK6-1 Biological materials for gene expression; The HaMAPK6-1 The nucleotide sequence of the gene is shown in SEQ ID NO.1.

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