Application of AsCFBP1 gene in regulating crude protein content in feed oats

Abstract

This invention discloses AsCFBP1 The application of genes in regulating the crude protein content of feed oats belongs to the field of genetic engineering technology. This invention clones [a specific gene] from oats through genome-wide association analysis. AsCFBP1 The gene, whose coding region nucleotide sequence is shown in SEQ ID NO.1, exhibits a significant positive correlation between its expression level and the crude protein content of feed oats. This invention constructs a stably inherited... AsCFBP1 Overexpression of transgenic lines, detection results showed that in transgenic lines AsCFBP1 Gene expression levels were significantly increased, and the crude protein content in the aboveground parts during the milk stage was significantly higher than that in wild-type recipient plants, without adversely affecting normal plant growth and development. This invention is the first to verify... AsCFBP1 This study provides a highly efficient and stable molecular improvement method for increasing the crude protein content of feed oats by identifying the major regulatory genes for crude protein in oats, thus providing key gene resources and technical support for molecular breeding of feed oats.

Description

Technical Field

[0001] This invention relates to the field of genetic engineering technology, and in particular to... AsCFBP1 Application of genes in regulating the crude protein content of feed oats. Background Technology

[0002] oat( Avena sativa Oat (L.) is a widely cultivated high-quality forage crop. With its comprehensive characteristics of high and stable yield, wide adaptability, outstanding regeneration ability, and excellent palatability, it has become a core forage resource in the integrated forage-livestock production system. The widespread cultivation of high-quality forage oats plays a vital supporting role in promoting the ecological protection of the agro-pastoral ecotone and the coordinated development of the livestock industry.

[0003] As a core indicator in the forage quality evaluation system, crude protein (CP) directly affects the nitrogen supply capacity of forage and the efficiency of ruminants in digesting and utilizing nutrients, thus determining animal growth performance and breeding efficiency. Existing studies have confirmed that increasing the crude protein content of forage can significantly promote daily weight gain in ruminants, increase milk protein synthesis levels, effectively improve feed utilization, reduce dependence on exogenous high-protein concentrates, thereby reducing breeding costs and improving the economic efficiency and sustainability of livestock production.

[0004] However, the main forage oat varieties currently have generally low crude protein content, which makes it difficult to meet the high-protein diet requirements of large-scale ruminant farming. This has become a key factor restricting the upgrading of forage oat quality and hindering the high-quality development of the integrated grass-livestock industry.

[0005] Molecular breeding is a highly efficient method for targeted improvement of forage quality, and the discovery and verification of key functional genes is its core prerequisite. Existing research has established highly efficient Agrobacterium-mediated genetic transformation technology for oats, providing technical support for oat molecular breeding. However, currently, genetic transformation technology has not been applied to the functional verification and genetic improvement of genes regulating crude protein content in forage oats, making it difficult to meet the practical needs of targeted quality improvement.

[0006] Chloroplast fructose-1,6-bisphosphatase (CFBP) is an important regulator of plant carbon metabolism in the FBPase protein family, participating in fructose-mediated signal transduction. Model plant studies have shown that... CFBP Abnormal gene function can lead to decreased photosynthetic rate, increased starch accumulation during the day, and decreased sucrose content. Since sucrose is a core component of carbon source allocation, its synthesis and transport directly affect nitrogen assimilation and protein synthesis, indicating... CFBP Genes can indirectly participate in protein synthesis by regulating carbon metabolism homeostasis, and thus possess the potential to be candidate genes for improving crude protein in oats. However, current understanding of...CFBP Research on genes in oats is still in its infancy, lacking verification of the function of their encoded proteins and their application in oat quality improvement, and their regulatory potential has not been explored. Summary of the Invention

[0007] The purpose of this invention is to provide AsCFBP1 The 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, filling the technical gap in the targeted improvement of crude protein in oats, and providing an efficient technical path and core gene support for the precise improvement of feed oat quality.

[0008] To achieve the above objectives, the present invention provides AsCFBP1 The application of genes in regulating the crude protein content of feed oats, the aforementioned AsCFBP1 The CDS sequence of the gene is shown in SEQ ID NO.1; the regulation is to enhance... AsCFBP1 Gene expression levels can increase the crude protein content in feed oats.

[0009] Preferably, the overexpression is achieved by constructing a structure containing AsCFBP1 Gene recombinant expression vectors were used to transform oats.

[0010] 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 shown in SEQ ID NO.1 AsCFBP1 Gene coding region; S2, constructing... AsCFBP1 Recombinant overexpression vectors for genes; S3. Transform the recombinant overexpression vector into oat receptor material via Agrobacterium-mediated transformation; S4, Filtering and obtaining AsCFBP1 The transgenic oat plants with overexpressed genes had significantly higher crude protein content than the wild type.

[0011] On the other hand, the present invention also provides AsCFBP1 The application of the gene in improving the quality of feed oats, through overexpression of the gene shown in SEQ ID NO.1 AsCFBP1 Genes can increase the crude protein content of whole oat plants and enhance their nutritional value for feed.

[0012] On the other hand, the present invention also provides AsCFBP1 The application of genes in oat variety breeding, as shown in SEQ ID NO.1 AsCFBP1 Genes are used as molecular markers or transgenic improvement targets to breed new feed oat varieties with high crude protein content.

[0013] Therefore, the present inventionAsCFBP1 The application of genes in regulating the crude protein content of feed oats has the following beneficial effects: (1) Through large-scale population phenotypic data and GWAS, the association with oat crude protein formation was accurately identified. AsCFBP1 The gene was identified, and its significant effect on increasing crude protein content was verified through transgenic technology, demonstrating a clear molecular basis and reliability. (2) Through Agrobacterium-mediated genetic transformation technology, significantly improve AsCFBP1 Gene expression levels in oats; (3) After sampling the aboveground tissues at the milk stage, the crude protein content was determined using the national standard method GB / T 6432-2018. The results showed that overexpression AsCFBP1 The crude protein content in the transgenic plants was significantly higher than that in the recipient plants, and the transgenic lines showed stable performance. (4) The present invention provides a method for enhancing AsCFBP1 The expression of feasible molecular improvement strategies to enhance the quality of oat forage has the advantages of clear technical path, significant effect and stable operation, and provides new breeding ideas, gene resources and effective means for breeding new high-protein oat varieties.

[0014] 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

[0015] 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.

[0016] Figure 1 For GWAS Manhattan plot; Figure 2 for pCAMBIA3300-AsCFBP1 Image of recombinant plasmid colony PCR identification results; Figure 3 for UBI:AsCFBP1 In transgenic lines AsCFBP1 Expression detection graph; Figure 4 For receptor materials and AsCFBP1 Phenotypic comparison of transgenic lines at different stages of growth and development; Figure 5 For receptor materials and AsCFBP1 Crude protein determination results of transgenic lines. Detailed Implementation

[0017] The technical solution of the present invention will be further described below with reference to the accompanying drawings and embodiments.

[0018] 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.

[0019] The instruments, equipment, reagents, and materials used in the examples were all obtained commercially.

[0020] The oats used in this example were Baiyan 10 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.

[0021] Example 1 Field trial data from two locations in Hebei Province were used: Baoding (2023, BD) and Zhangjiakou (2024, ZJK) in 2024. Crude protein levels were measured in 634 materials from Baoding in 2023 and in 628 materials from Zhangjiakou in 2024. All tested materials were 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. GWAS analysis was performed on the two independent datasets. Through reference genome annotation and further analysis, a significant association signal was identified in the 353-493 Mb region of oat chromosome 4A, revealing a key candidate gene significantly associated with oat crude protein content. AsCFBP1 .

[0022] GWAS Manhattan diagram Figure 1 As shown, the arrow points to a portion of the oat 4A chromosome, where, under the selection criteria, a stable major-effect locus significantly associated with crude protein content is located in the gene. AsCFBP1 The association signal of this site was higher than the significance threshold in both environments.

[0023] Obtained from the oat genome database website http: / / www.oatomics.com / home AsCFBP1 The gene number (AVESA.00022b.r1.4Ag00006836) was obtained, and the coding region sequence was obtained.

[0024] Example 2 AsCFBP1 Gene cloning and construction of overexpression vectors: (1) Amplification of the target gene: to extract Avena sativa 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′-ATGGATCACGCGGCG-3′ Reverse primer, SEQ ID NO.3: 5′-TCTGCGGGGTACAAGAATATA-3′ High-fidelity DNA polymerase 2× Phanta Flash Master was used for... AsCFBP1 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 employed a touch-down procedure: initial denaturation at 98°C for 30 seconds; followed by 35 cycles, each consisting of 5 seconds of denaturation at 98°C, 5 seconds of annealing, and 20 seconds of extension at 72°C; the annealing temperature was initially set at 64°C and decreased by 0.1°C per cycle until reaching 60.5°C. A final extension at 72°C for 1 minute followed by a final incubation at 4°C. The amplified products were purified and recovered after detection by 1% agarose gel electrophoresis.

[0027] (2) Linearization of the overexpression vector: The plant expression vector pCAMBIA3300 was linearized by double enzyme digestion in order to insert the foreign gene fragment. The digestion products were separated by agarose gel, and the target fragment was recovered and purified.

[0028] (3) AsCFBP1 Enzyme digestion of the insert fragment: Purification after PCR amplification AsCFBP1 The fragment was double-digested with the same restriction endonuclease as the vector at 37°C for 2 hours to expose sticky ends corresponding to those of the vector. After digestion, the target fragment was recovered using a DNA purification kit.

[0029] (4) Ligation reaction and acquisition of recombinant overexpression vector: T4 ligase was used to ligate the digested vector. AsCFBP1The fragment and the linearized support were ligated at a molar ratio of 8:1. The ligation product was used directly for subsequent transformations.

[0030] (5) Recombinant plasmid pCAMBIA3300-AsCFBP1 Construction and Identification: The ligation product was transformed into TOP10 competent cells using a heat shock method, and cultured at 37°C with shaking for 30 min. Single colonies were picked and plated onto LB agar containing the corresponding antibiotic. Individual colonies were selected for colony PCR and plasmid extraction, and sequencing was performed for verification to ensure... AsCFBP1 Genes are recombined into the expression vector in the correct direction.

[0031] pCAMBIA3300-AsCFBP1 Recombinant plasmid colony PCR identification, such as Figure 2 As shown in the figure, the red boxes indicate positive clones of the target gene and expression vector recombination.

[0032] (6) Agrobacterium strain transformed with recombinant plasmid: The correctly sequenced recombinant plasmid was transformed into [a specific gene] using a freeze-thaw method. Agrobacterium radiobacter GV3101. The transformed Agrobacterium was cultured on LB plates containing the corresponding antibiotics (rifampin + kanamycin), and 23 single colonies were picked for colony PCR identification using 2×Taq Master Mix as the reaction system.

[0033] The PCR amplification program was as follows: 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 using a touchdown program; after the cycles, a final extension at 72℃ for 10 min was performed, followed by incubation at 4℃. The PCR products were analyzed by 1% agarose gel electrophoresis to confirm the correct size of the target fragment, ensuring successful entry of the recombinant overexpression vector into Agrobacterium.

[0034] Example 3 Genetic transformation of oat embryo callus: (1) Explant preparation: Using the oat variety Baiyan 10 as the recipient material, plump and healthy mature oat seeds were selected, disinfected with 75% ethanol for 5 min, treated with 10% sodium hypochlorite solution for 20 min, and rinsed 5 times with sterile water. The embryo was cut off and inoculated into L3M callus induction medium, and cultured in the dark at 25℃ for about 30 days to obtain yellowish-white, loose and embryogenic callus tissue as the recipient material.

[0035] (2) Introduction of Agrobacterium overexpression vector: The constructed vector was introduced into Agrobacterium. AsCFBP1The overexpression recombinant plasmid was transformed into Agrobacterium GV3101 competent cells and plated on LB agar plates containing rifampicin and kanamycin (50 mg / L), and incubated at 28°C for 2-3 days. Single colonies were picked and identified as positive engineered strains by PCR for infection.

[0036] (3) Infection and co-culture: The positive engineered strain was shaken to OD 600 ≈0.5, centrifuge to collect bacterial cells, and resuspend in MS liquid medium containing 200 μmol / L acetylsyringone (AS) to the same OD value. Immerse the callus tissue in the bacterial solution for 10 min, aspirate excess bacterial solution, and spread it evenly on co-culture medium (containing AS), and incubate in the dark at 25℃ for 3 days.

[0037] (4) Resistance screening and plant regeneration: After co-culture, callus tissue was transferred to a selection medium containing hygromycin 25 mg / L and carbenicillin 350 mg / L, and cultured in the dark at 25°C for 3-4 weeks. Resistant callus was transferred to differentiation medium (MS + 6-BA 1.5 mg / L + NAA 0.4 mg / L + IAA 0.4 mg / L), and bud differentiation was induced by light. Then, it was transferred to rooting medium to obtain... AsCFBP1 Regenerated plants by overexpression of transgenes.

[0038] Example 4 Select transgenic oat plants and recipient materials with consistent growth status, and collect fresh young leaves. Quickly freeze the sampled tissues in liquid nitrogen and grind them into a fine powder. Extract total RNA according to the RNA extraction kit instructions.

[0039] Using 1 μg of RNA as a template, reverse transcription was performed according to the reverse transcription kit instructions to synthesize first-strand cDNA. The resulting cDNA was diluted under different conditions for subsequent qRT-PCR analysis.

[0040] based on AsCFBP1 Specific amplification primers SEQ ID NO.4 and SEQ ID NO.5 were designed based on the coding sequence of the gene to amplify the oat reference gene. AsActin Primers SEQ ID NO.6 and SEQ ID NO.7 were designed and synthesized by the company as a reference for expression level normalization.

[0041] AsCFBP1 Specific F-terminal primers for real-time quantitative PCR of genes, SEQ ID NO.4: 5'-ACTCGGTGAACGAGGGG-3' AsCFBP1 Specific R-terminal primers for real-time quantitative PCR of genes, SEQ ID NO.5: 5'-GGAATGACATGGGGAAGACT-3' Oat Reference Gene AsActinThe 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' UBI:AsCFBP1 In transgenic lines AsCFBP1 Expression detection graph as shown Figure 3 As shown, the results indicate that, compared to receptor materials, AsCFBP1 Overexpression lines AsCFBP1 Gene expression levels were significantly increased ( P <0.0001). This result demonstrates that the constructed overexpression vector can drive... AsCFBP1 The stable and high-level expression of the gene in oats lays the foundation for subsequent crude protein determination.

[0042] Receptor material (WT) and AsCFBP1 Phenotypic comparison of transgenic lines at different growth and development stages is shown in the figure below. Figure 4 As shown, the results indicate that AsCFBP1 Overexpression did not have a negative impact on the normal growth and development of oat plants.

[0043] Example 5 For a comprehensive assessment AsCFBP1 The effect of overexpression on the crude protein content of feed oats was determined uniformly after the plants reached the milk stage. Strict sampling strategies and standardized pretreatment procedures were employed for all tested materials to ensure the comparability and accuracy of the data.

[0044] (1) Sample collection and pretreatment: Select representative plants that are growing uniformly and free from pests and diseases, and cut their above-ground parts from the base. Remove withered and yellowed tissues and impurities from the harvested samples, and mix thoroughly to obtain a representative mixed sample. Immediately place the mixed sample in a 105℃ forced-air drying oven for 60 minutes (blanching), then adjust the temperature to 80℃ and continue drying until constant weight. After cooling to room temperature, pulverize the sample and sieve it uniformly to ensure the resulting powder is homogeneous. This powder is then used as the sample for crude protein content determination.

[0045] (2) The crude protein content of the samples was determined according to GB / T 6432-2018, using the Kjeldahl method. Based on the measured total nitrogen content of the samples, the crude protein content (CP) was calculated using the following formula: Crude protein content (CP, %) = Nitrogen content (%) × 6.25.

[0046] Receptor materials and AsCFBP1 The results of crude protein determination of transgenic lines at the milk stage are as follows: Figure 5As shown, the results indicate that, compared to the receptor material (WT), AsCFBP1 Transgenic overexpression lines ( AsCFBP1-OE1 , AsCFBP1-OE2 , AsCFBP1-OE3 , AsCFBP1-OE4 and AsCFBP1-OE5 The crude protein content of all samples was significantly increased, confirming that... AsCFBP1 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.

[0047] 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. AsCFBP1 The application of genes in regulating the crude protein content of feed oats is characterized by: The AsCFBP1 The CDS sequence of the gene is shown in SEQ ID NO.1; The regulation is overexpression. AsCFBP1 Genes increase the crude protein content in feed oats.

2. As described in claim 1 AsCFBP1 The application of genes in regulating the crude protein content of feed oats is characterized by: The overexpression is achieved by constructing a system containing... AsCFBP1 Gene recombinant expression vectors were used to transform oats.

3. A method for increasing the crude protein content of feed oats, characterized in that, Includes the following steps: S1. Cloning as shown in SEQ ID NO.1 AsCFBP1 Gene coding region; S2, constructing... AsCFBP1 Recombinant overexpression vectors for genes; S3. Transform the recombinant overexpression vector into oat receptor material via Agrobacterium-mediated transformation; S4, Filtering and obtaining AsCFBP1 The transgenic oat plants with overexpressed genes had significantly higher crude protein content than the wild type.

4. AsCFBP1 The application of genes in improving the quality of feed oats is characterized by, By overexpressing the expression shown in SEQ ID NO.1 AsCFBP1 Genes can increase the crude protein content of whole oat plants and enhance their nutritional value for feed.

5. AsCFBP1 The application of genes in oat variety breeding is characterized by: As shown in SEQ ID NO.1 AsCFBP1 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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