Application of the AsCINV2 gene in regulating the crude protein content of feed oats

Abstract

This invention discloses AsCINV2 The application of genes in regulating the crude protein content of feed oats belongs to the field of genetic engineering technology. This invention uses genome-wide association analysis to screen for key genes significantly associated with the crude protein content of feed oats. AsCINV2 Its CDS sequence is shown in SEQ ID NO.1. Experimental results indicate that in the transgenic lines... AsCINV2 Gene expression levels were significantly increased, but normal plant growth and development were not affected. Crude protein content at the milk stage was 17.5%–28.1% higher than that of the wild-type recipient. This invention is the first to verify… AsCINV2 Genes can significantly increase the crude protein content of feed oats, providing new gene resources and efficient improvement methods for molecular breeding of high crude protein oats, which has important theoretical value and industrial application prospects.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to... AsCINV2 Application of genes in regulating the crude protein content of feed oats. Background Technology

[0002] oat( Avena satava L. (L.) is a high-quality forage species in the northern forage industry. Due to its core advantages such as tender stems and leaves, rich nutrition, and excellent palatability, it has become the preferred raw material for the formulation of diets for ruminants such as cattle and sheep, and plays a key role in the integrated development of agriculture and animal husbandry.

[0003] Crude protein (CP) content is a core indicator for measuring the nutritional quality of forage, directly determining the nitrogen supply efficiency, rumen microbial activity, and feed digestibility in ruminants. Its level is closely related to livestock production efficiency. Studies have shown that increasing the crude protein content of forage can significantly promote the growth performance of ruminants, increase the yield of dairy and meat products, and effectively reduce the amount of concentrate feed input, achieving the dual goals of reducing feeding costs and optimizing production efficiency. However, the crude protein content of oat forage varies significantly among breeds and during the growth period, generally exhibiting a low level and large fluctuations. This makes it difficult to meet the large-scale production needs of high-protein diets for ruminants, becoming a key bottleneck restricting the upgrading of oat feed quality.

[0004] With the development of molecular breeding technology, molecular marker-assisted selection, transgenic technology and other technologies have provided new technical pathways for improving the quality of forage. However, the key genes and genetic regulation mechanisms that regulate the formation of crude protein in oats have not yet been clarified, which limits the application of molecular breeding technology in the improvement of the quality of feed oats. There is an urgent need to explore functional genes with the potential to regulate crude protein content.

[0005] CINV The (Cytosolic Invertase) gene encodes a cytosolic invertase, a crucial regulator of sucrose breakdown and carbon source allocation in plants, playing an irreplaceable role in plant energy metabolism, organ development, stress regulation, and the coordination of carbon and nitrogen metabolism. Existing research has confirmed that... CINV Genes can regulate the conversion efficiency of sucrose to bioactive monosaccharides in various crops, thereby indirectly regulating nitrogen absorption, assimilation, and protein synthesis. They have potential links to plant biomass formation and quality trait regulation, and thus possess application prospects as candidate genes for quality improvement. However, currently, there is a lack of research on this gene both domestically and internationally. CINV There is a lack of systematic research reports on genes in oats, especially regarding their functional verification in the process of crude protein accumulation in oats. Summary of the Invention

[0006] The purpose of this invention is to provide AsCINV2The application of genes in regulating the crude protein content of feed oats significantly increases the accumulation of crude protein in oat vegetative organs by improving the expression level of this gene in oats, effectively improving forage quality and providing a new gene resource and innovative technical path for the targeted improvement of crude protein content in feed oats.

[0007] To achieve the above objectives, the present invention provides AsCINV2 The application of genes in regulating the crude protein content of feed oats, the aforementioned AsCINV2 The CDS sequence of the gene is shown in SEQ ID NO.1; the regulation is to enhance... AsCINV2 Gene expression levels can increase the crude protein content in feed oats.

[0008] Preferably, the overexpression is achieved by... AsCINV2 The gene was constructed into a plant expression vector, and then transformed into feed oats via Agrobacterium-mediated transformation.

[0009] On the other hand, the present invention also provides a method for increasing the crude protein content of feed oats, comprising the following steps: S1, Cloning as described above AsCINV2 Gene; S2, constructing... AsCINV2 Recombinant overexpression vectors for genes; S3. The recombinant overexpression vector of S2 was transformed into Agrobacterium and then infected with callus tissue from feed oats. S4, obtained through resistance selection, differentiation, and rooting. AsCINV2 Transgenic oat plants with overexpressed genes; The crude protein content of the transgenic oat plants was significantly higher than that of the wild-type plants.

[0010] On the other hand, the present invention also provides the above-mentioned AsCINV2 The application of genes in improving the quality of feed oats, through overexpression AsCINV2 Genes can increase the crude protein content of whole oat plants and enhance their nutritional value for feed.

[0011] On the other hand, the present invention also provides the above-mentioned AsCINV2 The application of genes in oat variety breeding, AsCINV2 Genes are used as molecular markers or transgenic improvement targets to breed new feed oat varieties with high crude protein content.

[0012] Therefore, the present invention AsCINV2 The application of genes in regulating the crude protein content of feed oats has the following beneficial effects: (1) Through genome-wide association analysis, key candidate genes that are significantly associated with crude protein content in feed oats were precisely screened. AsCINV2 ; (2) Construction AsCINV2 After overexpression vector, genetic transformation of mature embryos mediated by Agrobacterium can yield transgenic materials with stable expression. (3) Overexpression AsCINV2 The gene can significantly enhance oat protein accumulation and increase the crude protein content of the whole plant, thereby improving the nitrogen supply of forage and reducing the dependence on concentrated feed, which has important economic and social value. (4) First time clearly defined AsCINV2 The role of genes in improving oat quality provides gene resources and molecular improvement strategies that can be directly used for breeding high crude protein varieties, and has good scalability and industrial application potential.

[0013] The technical solution of the present invention will be further described in detail below with reference to the accompanying drawings and embodiments. Attached Figure Description

[0014] To more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the description of the embodiments of the present invention 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.

[0015] Picture 1 For GWAS Manhattan plot; Picture 2 for pCAMBIA3300-AsCINV2 Recombinant plasmid map; Picture 3 for pCAMBIA3300-AsCINV2 PCR identification diagram of recombinant plasmid colonies; Picture 4 for UBI:AsCINV2 In transgenic lines AsCINV2 Gene expression detection graph; Picture 5 This is a comparison chart of the recipient material and the transgenic line during their growth and development stages. The left side shows the recipient material (WT), and the right side shows the transgenic line. AsCINV2 Overexpression lines ( AsCINV-OE ); Picture 6 The figure shows the results of crude protein content determination of recipient materials and transgenic lines at the milk stage. Detailed Implementation

[0016] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0017] To make the objectives, technical solutions, and advantages of this application clearer, more thorough, and more complete, the technical solutions of the present invention will be clearly and completely described below with reference to the accompanying drawings and embodiments. The following detailed descriptions are all illustrations of embodiments, intended to provide further detailed explanation of the present invention. Unless otherwise specified, all technical terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains.

[0018] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0019] The oats used in this example were Dingyan No. 2 and transgenic lines obtained using it as the recipient material. The oats were cultivated in an artificial greenhouse with a photoperiod of 16 hours of light / 8 hours of darkness, a daytime temperature of 22°C, a nighttime temperature of 16°C, and a relative humidity of 60%-70%. During the plant's growth period, watering and fertilization were carried out according to the water and fertilizer requirements at different stages to ensure sufficient water and nutrients.

[0020] Example 1 This invention utilizes field phenotypic data of crude protein content from two different ecological environments to conduct association analysis. The experimental data were obtained from 634 samples from Baoding, Hebei Province (2023 (BD)) in 2023 and 628 samples from Zhangjiakou, Hebei Province (2024 (ZJK)) in 2024. Crude protein content was measured in both batches of materials, and independent phenotypic datasets were generated. The tested materials were all cultivated oat germplasm resources, sourced from the Chinese Academy of Agricultural Sciences, Dingxi Academy of Agricultural Sciences, and the National Oat Germplasm Resource Mid-term Bank. Genome-wide association analysis (GWAS) was performed on all data from both locations. Combined with oat reference genome annotation information and functional predictions of candidate genes within the region, key candidate genes significantly associated with oat crude protein content were ultimately screened. AsCINV2 .

[0021] GWAS Manhattan diagram Picture 1 As shown, the arrow points to a region of approximately 505 Mb on oat chromosome 2D, where a stable major-effect site significantly associated with crude protein was located under the screening criteria. The association signal of this site was higher than the significance threshold in both environments.

[0022] Obtained from the oat genome database website http: / / www.oatomics.com / home AsCINV2 The gene number (AVESA.00022b.r1.2Dg00007491) was obtained, and the coding region sequence was acquired.

[0023] AsCINV2 The CDS sequence of the gene is shown in SEQ ID NO.1.

[0024] SEQ ID NO.1: Example 2 AsCINV2 Gene cloning and construction of overexpression vectors: (1) Amplification of the target gene: to extract Avena satava Using cDNA obtained by reverse transcription from fresh oat leaves of L. cv. Mavellous as a template, specific primers SEQ ID NO.2 and SEQ ID NO.3 containing restriction enzyme sites were designed to amplify the full-length CDS of the target gene by PCR.

[0025] Forward primer, SEQ ID NO.2: 5′-ATGGAGGCGGCGGG-3′ Reverse primer, SEQ ID NO.3: 5′-CACCGTCCACGAGGCG-3′ (1) High-fidelity DNA polymerase 2× Phanta Flash Master was used to... AsCINV2 The coding region of the gene was amplified by PCR. The PCR reaction system was 80 μL, containing: 40 μL 2× Phanta Flash Master, 1.6 μL DMSO, 0.5 μmol / L each of forward and reverse primers, approximately 100 ng cDNA template, and the remainder was made up with sterile deionized water.

[0026] The amplification reaction was performed using a touch-down program: first, pre-denaturation at 98℃ for 30 seconds; then 35 cycles, each cycle consisting of denaturation at 98℃ for 5 seconds, annealing at 64℃ for 5 seconds, and extension at 72℃ for 20 seconds; the annealing temperature was decreased by 0.1℃ per cycle until it reached 60.5℃. After the cycle, a final extension at 72℃ for 1 minute was performed, followed by a final hold at 4℃.

[0027] (2) After the reaction, 6 μL of the amplification product was taken for 1% agarose gel electrophoresis to confirm that the target fragment size was correct and the band was specific. The reaction solution was then purified using an agarose gel recovery kit for subsequent vector construction.

[0028] (3) The pCAMBIA3300 vector was double-digested with a predetermined restriction endonuclease, and the purified gene fragment was ligated with the linearized vector at a molar ratio of 8:1 to form a recombinant plasmid.

[0029] (4) The resulting ligation product was converted to [a specific substance] at 42°C. E. coli TOP10 competent cells were screened for positive monoclonal antibodies using LB plates containing selective antibiotics.

[0030] (5) Twenty-three single-clone colonies were picked with sterile toothpicks and identified by colony PCR using 2×Taq Master Mix. The amplification reaction was performed in a PCR instrument using a touch-down program: pre-denaturation at 95℃ for 5 min; followed by 34 cycles, each cycle consisting of denaturation at 95℃ for 30 s, annealing at 63℃ for 30 s, and extension at 72℃ for 1 min; the annealing temperature was decreased by 0.1℃ each cycle starting from the initial temperature of 63℃. After the cycle, the final extension was performed at 72℃ for 10 min, and finally, the temperature was maintained at 4℃. The PCR products were then subjected to 1% agarose gel electrophoresis to confirm the correct size of the target fragment.

[0031] (6) Select the successfully recombined positive clones and incubate them in LB liquid medium containing the corresponding antibiotics for 12-16 hours. Extract the plasmid using a plasmid mini-prep kit and perform sequencing verification to obtain the plasmids required for this invention. pCAMBIA3300-AsCINV2 The recombinant expression vector was used for subsequent Agrobacterium-mediated transformation and plant genetic transformation experiments.

[0032] pCAMBIA3300-AsCINV2 Recombinant plasmid map as follows Picture 2 As shown, pCAMBIA3300-AsCINV2 Recombinant plasmid colony PCR identification diagram as shown below Picture 3 As shown in the figure, the red boxes indicate positive clones of the target gene and expression vector recombination.

[0033] Example 3 Genetic transformation of oat mature embryo callus: (1) Explant preparation: Plump, healthy, and mold-free mature oat seeds of Dingyan No. 2 were selected as initial materials. The seeds were placed in 75% ethanol and gently shaken for 5 min for disinfection, then rinsed twice with sterile water, and then transferred to 10% sodium hypochlorite solution for 20 min for deep disinfection. Afterwards, the seeds were rinsed five times with sterile water to remove residual disinfectant. The treated seeds were inverted under sterile conditions for about 30 min to allow the surface to dry naturally, and then the embryo was cut off and inoculated into L3M callus induction medium with the cut surface facing down. The medium was cultured in complete darkness at 25℃ for about 30 days. After the formation of yellowish-white, loose callus with embryogenic characteristics, it was selected as the recipient material for genetic transformation.

[0034] (2) AsCINV2 Overexpression vector introduction into Agrobacterium: Take 100 μL of Agrobacterium GV3101 competent cells and add 1 μg of the constructed... AsCINV2Overexpressing the recombinant plasmid, the cells were gently mixed and incubated on ice for 30 min. The cells were then transformed using the heat shock method and added to 1 mL of antibiotic-free LB broth. The cells were incubated at 28°C and 200 rpm with shaking for 2-3 h. The incubated bacterial culture was then evenly spread onto LB agar plates containing rifampicin and kanamycin (50 mg / L) and incubated at 28°C for 2-3 days. Single colonies with normal growth were picked for colony PCR to screen for cells carrying the recombinant plasmid. AsCINV2 The positive Agrobacterium strain overexpressing the vector was used for subsequent infection steps.

[0035] (3) Agrobacterium infection and co-culture: The engineered strain that tested positive was inoculated into LB liquid medium containing the resistant strain and cultured until OD200. 600 After approximately 0.5, use 5000 g The bacterial cells were collected by centrifugation and resuspended in MS liquid induction medium containing 200 μmol / L acetylsuccinone (AS) to maintain the bacterial concentration at OD. 600 ≈0.5. Under aseptic conditions, the selected embryogenic callus tissue was immersed in the bacterial solution for about 10 minutes to allow Agrobacterium to fully adhere to the callus surface. After removing excess bacterial solution, the callus tissue was spread evenly on a co-culture medium containing the same concentration of AS and cultured at 25°C in complete darkness for 3 days to promote the completion of T-DNA transfer.

[0036] (4) Resistance screening and plant regeneration: After co-culture, the callus tissue was transferred to a selection medium containing 25 mg / L hygromycin and 350 mg / L carbenicillin, and cultured in the dark at 25°C for 3-4 weeks to screen for resistant callus tissue capable of growing under resistant conditions. These resistant callus tissues were then transferred to differentiation medium (MS + 1.5 mg / L 6-BA + 0.4 mg / L NAA + 0.4 mg / L IAA) and induced to differentiate into shoots under light conditions. Once green shoots formed, they were transferred to rooting medium to induce root development, ultimately yielding... AsCINV2 Transgenic oat regeneration plants overexpressing the gene.

[0037] Example 4 A suitable amount of healthy, young transgenic oat leaves from the same location were placed in 1.5m enzyme-free centrifuge tubes, rapidly immersed in liquid nitrogen for complete freezing, and then ground into a fine powder in a pre-cooled mortar. Total RNA was extracted from the leaves using Trizol extraction reagent according to the manufacturer's instructions, employing chloroform, isopropanol, and 75% anhydrous ethanol. The concentration and purity of the RNA were monitored to ensure it met the requirements for subsequent experiments. Subsequently, 2 μg of RNA was reverse transcribed into first-strand cDNA using a commercially available reverse transcription kit.

[0038] based on AsCINV2Specific amplification primers SEQ ID NO.4 and SEQ ID NO.5 were designed based on the CDS sequence of the gene to amplify the oat internal reference gene. AsActin As a reference for expression level normalization, primers SEQ ID NO.6 and SEQ ID NO.7 were both synthesized by the company.

[0039] AsCINV2 Specific F-terminal primers for real-time quantitative PCR of genes, SEQ ID NO.4: 5'-CGAGTTTCAAGGTGCTAAAGGA-3' AsCINV2 Specific R-terminal primers for real-time quantitative PCR of genes, SEQ ID NO.5: 5'-CGTCTCGGAGAGGGACAG-3' Oat Reference Gene AsActin The F-terminal primer, SEQ ID NO.6: 5'-TACTGGTATCGTTCTTGACTCT-3' Oat Reference Gene AsActin The R-terminal primer, SEQ ID NO.7: 5'-ATCAAGGGCTACATACGCAAG-3' The prepared qRT-PCR reaction system was added to the reaction tube of the real-time PCR instrument, sealed, and placed in the instrument for operation. The amplification program was set as follows: 95℃ pre-denaturation for 30 s; followed by 40 cycles, each cycle consisting of 95℃ denaturation for 10 s, 60℃ annealing for 10 s, and 72℃ extension for 30 s. The target gene was detected. AsCINV2 With internal reference genes AsActin The Ct value, using 2 -ΔΔCt The method was used to calculate the gene expression levels of transgenic lines.

[0040] Using the main oat variety Dingyan 2 as a control, if the test material contains AsCINV2 If the expression level of the protein was significantly higher than that of the control, the material was determined to be a transgenic overexpression plant. Each line was subjected to three replicate experiments, and the positive lines were used for subsequent phenotypic observation and crude protein content determination.

[0041] UBI:AsCINV2 In transgenic lines AsCINV2 Gene expression detection, such as Picture 4 As shown, the results indicate that, compared to the receptor material (WT), among the three overexpression materials... AsCINV2 Gene expression levels were significantly increased.

[0042] A comparison of the recipient material and the transgenic line during their growth and development stages is shown in the figure below. Picture 5 As shown, the results indicate that AsCINV2 Overexpression did not have a negative impact on the normal growth and development of oat plants.

[0043] Example 5 Crude protein content was determined after plants were cultured under identical conditions in an artificial climate chamber and entered the milk stage. Representative plants with uniform growth and free from pests and diseases were selected as experimental materials. The plant phenotypes were recorded by photographing the plants in the environment at the same time. The above-ground parts were completely cut from the base of the ground, and withered leaves and impurities were removed and thoroughly mixed to ensure that the samples were representative.

[0044] The resulting mixed sample was then placed in a 105℃ forced-air drying oven for 60 minutes for blanching, and then the drying temperature was adjusted to 80℃ until the sample reached constant weight. After drying, the material was cooled to room temperature, pulverized, and sieved uniformly. The resulting homogeneous powder was used as the sample for crude protein determination. The nitrogen content of the sample was determined using the Kjeldahl method as specified in the national standard GB / T 6432-2018.

[0045] Finally, the crude protein content difference between the transgenic material and the recipient plant control was compared to evaluate the results. AsCINV2 The effect of overexpression on crude protein content in oats. The crude protein content of the recipient material and the transgenic lines at the milk stage is shown in the figure below. Picture 6 As shown, the results indicate that, compared to the receptor material (WT), AsCINV2 Transgenic overexpression lines ( AsCINV2-OE1 , AsCINV2-OE2 and AsCINV2-OE3 The crude protein content of all samples was significantly increased, confirming that... AsCINV2 Overexpression of the gene in oats can significantly promote the increase of crude protein content, and it is a key functional gene for improving the quality of feed oats.

[0046] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention and not to limit them. 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 still be made to the technical solutions of the present invention, and these modifications or equivalent substitutions cannot cause the modified technical solutions to deviate from the spirit and scope of the technical solutions of the present invention.

Claims

1. AsCINV2 The application of genes in regulating the crude protein content of feed oats is characterized by: The AsCINV2 The CDS sequence of the gene is shown in SEQ ID NO.1; The regulation is overexpression. AsCINV2 Genes increase the crude protein content in feed oats.

2. As described in claim 1 AsCINV2 The application of genes in regulating the crude protein content of feed oats is characterized by: The overexpression will... AsCINV2 The gene was constructed into a plant expression vector, and then transformed into feed oats via Agrobacterium-mediated transformation.

3. A method for increasing the crude protein content of feed oats, characterized in that, Includes the following steps: S1, Cloning as described in claim 1 AsCINV2 Gene; S2, constructing... AsCINV2 Recombinant overexpression vectors for genes; S3. The recombinant overexpression vector of S2 was transformed into Agrobacterium and then infected with callus tissue from feed oats. S4, obtained through resistance selection, differentiation, and rooting. AsCINV2 Transgenic oat plants with overexpressed genes; The crude protein content of the transgenic oat plants was significantly higher than that of the wild-type plants.

4. As described in claim 1 AsCINV2 The application of genes in improving the quality of feed oats is characterized by, Through overexpression AsCINV2 Genes can increase the crude protein content of whole oat plants and enhance their nutritional value for feed.

5. As described in claim 1 AsCINV2 The application of genes in oat variety breeding is characterized by: by AsCINV2 Genes are used as molecular markers or transgenic improvement targets to breed new feed oat varieties with high crude protein content.

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