Cloning of soybean oil biosynthesis regulatory genes and their encoded proteins and applications
By cloning the soybean oil regulation gene qOil10 and using the CRISPR/Cas9 system for gene editing, the problem of unclear regulation of soybean oil synthesis has been solved, enabling efficient breeding, increasing oil content, and ensuring the security of oil supply.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- INSTITUTE OF CROP SCIENCE CHINESE ACADEMY OF AGRICULTURAL SCIENCES
- Filing Date
- 2026-04-15
- Publication Date
- 2026-05-29
AI Technical Summary
The molecular regulatory network of soybean oil synthesis and accumulation is unclear in existing technologies, resulting in low efficiency of conventional breeding, difficulty in cultivating new high-oil soybean varieties, and insufficient oil supply security.
The soybean oil content regulatory gene qOil10 and its encoded protein were cloned, and gene editing was performed using the CRISPR/Cas9 system. Recombinant vectors and transgenic cell lines were constructed, and localization primers and knockout vectors were developed to achieve efficient genetic improvement.
The function of the qOil10 gene was clarified, which shortened the breeding cycle, improved selection efficiency, significantly increased the oil content of soybean seeds, and ensured the security of oil supply.
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Figure CN122104739A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of plant molecular genetics and genetic engineering, specifically to the cloning of soybean oil regulation genes and their encoded proteins and applications. Background Technology
[0002] Soybean is an important dual-purpose crop in my country, used for grain, oil, and feed. Its seed oil content is a core trait determining the economic value and industrial competitiveness of soybeans. The main soybean varieties cultivated in my country generally suffer from low oil content, leading to a long-term high dependence on imports for the domestic soybean industry and posing a serious challenge to oil supply security. Conventional soybean breeding is limited by environmental factors, gene linkage burden, and long breeding cycles, resulting in low efficiency in improving oil content traits and failing to meet the industry's urgent demand for high-oil varieties.
[0003] Molecular marker-assisted breeding and gene editing technologies provide effective approaches for the precise improvement of soybean oil content traits. Identifying and cloning key functional genes regulating soybean oil synthesis is the core foundation for molecular breeding. Currently, although several soybean oil-related QTL loci have been identified, only a few key genes have been successfully cloned. The molecular regulatory network of soybean oil synthesis and accumulation remains unclear, and the genetic effects of most cloned genes are limited, making them difficult to directly apply to the breeding of high-oil soybean varieties. Furthermore, the temporal equilibrium mechanism of carbon source allocation between soybean oil synthesis and starch synthesis further increases the complexity of genetic improvement of oil content traits. Therefore, identifying new major genes with significant genetic effects regulating soybean oil content and elucidating their functions and mechanisms of action is of significant theoretical and practical importance for breeding new high-oil soybean varieties and ensuring national oil supply security. Summary of the Invention
[0004] Technical problems to be solved
[0005] To address the shortcomings of existing technologies, this invention provides the cloning of soybean oil content regulating genes, their encoded proteins, and their applications, providing key gene resources and technical support for molecular breeding of high-oil soybeans.
[0006] Technical solution
[0007] To achieve the above objectives, the present invention is implemented through the following technical solution: a soybean oil content regulating gene, wherein the gene is qOil10, and the nucleotide sequence is shown in SEQ ID NO:1 of the sequence listing.
[0008] Preferably, the amino acid sequence of the protein is shown in SEQ ID NO:2.
[0009] Preferably, the recombinant vector is constructed by inserting the gene editing site into the KpnⅠ and XbaⅠ recognition sites of the V135 vector.
[0010] Expression cassettes, transgenic cell lines, or recombinant bacteria containing genes that regulate soybean oil content.
[0011] Primers for amplifying soybean oil regulation genes, wherein the primers are the gene localization primers shown in SEQ ID NO:3 to SEQ ID NO:10.
[0012] Primers used for constructing a soybean oil content regulation gene knockout vector, wherein the primers are the knockout primers shown in SEQ ID NO:11 to SEQ ID NO:14.
[0013] The application of the soybean oil content regulating genes and encoded proteins in regulating the oil content of soybean seeds.
[0014] Beneficial effects
[0015] This invention provides the cloning of a soybean oil content regulatory gene, its encoded protein, and its applications. It offers the following beneficial effects:
[0016] 1. This invention provides the cloning of soybean oil regulatory genes, their encoded proteins, and their applications. This invention successfully cloned the soybean oil major regulatory gene qOil10, clarifying the function of this gene in positively regulating seed oil accumulation. This makes up for the deficiency of existing soybean oil-related QTLs having many but few functional verification genes, and provides new gene resources for elucidating the molecular mechanism of soybean oil synthesis.
[0017] 2. This invention also provides primers for locating the qOil10 gene, a knockout vector, and a genetic improvement method, which can be directly used for molecular marker-assisted breeding of high-oil soybeans, effectively shortening the breeding cycle and improving selection efficiency, and solving the technical problems of long breeding cycles and low improvement efficiency in conventional high-oil soybean breeding. Attached Figure Description
[0018] Figure 1 This is the QTL mapping and fine mapping map of the qOil10 gene in this invention;
[0019] Figure 2 This is a statistical chart showing the phenotype and oil content of wild-type and qOil10 gene knockout mutant plants of this invention. Detailed Implementation
[0020] The technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention.
[0021] Unless otherwise specified, the experimental methods used in the following examples are conventional methods.
[0022] Unless otherwise specified, all materials and reagents used in the following examples are commercially available.
[0023] Agrobacterium tumefaciens EHA105 is described in NewAgrobacterium helper plasmids for gene transfer to plants. Hood, Elizabeth E; Gelvin, Stanton B; Melchers, Leo S; Hoekema, Andre. Transgenic Research, 2(4): p. 208-218-218 (1993). It is available to the public from the Institute of Crop Science, Chinese Academy of Agricultural Sciences.
[0024] Example 1
[0025] Location and cloning of the qOil10 gene
[0026] Test materials
[0027] Using the low-oil soybean variety Dongnong 42 as the female parent and the high-oil soybean variety Hobbit as the male parent, a recombinant inbred line (RIL) population of 184 lines was constructed through continuous self-pollination after hybridization. This population was used for QTL mapping and gene cloning of oil content traits. All materials were grown under field conditions with the same water and fertilizer management and harvested uniformly after maturity.
[0028] Genomic DNA extraction
[0029] Genomic DNA was extracted from young soybean leaves using the CTAB method. The specific steps are as follows: 1) Take 0.2g of fresh leaves and place them in a 2.0mL centrifuge tube. Grind them into powder using liquid nitrogen. 2) Add 700μL of 2×CTAB extraction buffer preheated at 65℃, mix well, and incubate at 65℃ for 60min, gently inverting and mixing every 10min. 3) Add 700μL of chloroform-isoamyl alcohol (24:1), gently invert and mix for 10min, and centrifuge at 12000r / min for 10min. 4) Transfer the supernatant to a new centrifuge tube, add an equal volume of isopropanol, gently invert and mix, and incubate at -20℃ for 30min to precipitate DNA. 5) Centrifuge at 12000r / min for 10min, discard the supernatant, wash the precipitate twice with 70% ethanol, and air dry at room temperature. 6) Add 50μL of ddH2O to dissolve the DNA and store at -20℃ for later use.
[0030] QTL positioning and fine positioning
[0031] Using polymorphic molecular markers obtained from previous screening, the RIL population was genotyped. Combined with the seed oil content phenotypic data of each line, composite interval mapping analysis was performed using WinQTL Cartographer 2.5 software. The results showed that a stable QTL significantly associated with oil content was detected on soybean chromosome 10, named qOil10. The results are as follows: Figure 1 As shown, this QTL has a LOD value of 8.62 and a phenotypic contribution rate of 12.4%, making it a major QTL.
[0032] Molecular markers were further developed within the initially mapped region, and four pairs of polymorphic markers were screened, with corresponding amplification primers SEQ ID NO:3~SEQ ID NO:10. Using these primers, the RIL population was amplified and genotyped, precisely mapping qOil10 to a 67kb region. Genes within this region were annotated, identifying seven predicted genes. PCR amplification and sequencing comparison of these seven genes in the parents Dongnong 42 and Hobbit revealed six SNP sites and two Indel sites for Glyma.10G197000 across both parents, and its expression level showed a significant positive correlation with oil content, confirming this gene as a candidate for qOil10.
[0033] The genomic DNA sequence of the qOil10 gene is shown in SEQ ID NO:1, with a full length of 16178 bp; the amino acid sequence of the protein it encodes is shown in SEQ ID NO:2, with a total of 951 amino acids.
[0034] QTL mapping is based on the linkage between genetic markers and target trait genes, which can quickly locate the chromosomal segments that control quantitative traits. Fine mapping improves gene cloning accuracy by expanding the population and increasing the density of markers, narrowing the candidate intervals. This method has high mapping efficiency and stable results. The obtained qOil10 is a major regulatory gene for soybean oil content and can be directly used for molecular breeding.
[0035] Example 2
[0036] Construction of qOil10 gene knockout vector
[0037] Knockout target and primer design
[0038] Based on the genomic structure of the qOil10 gene, exon regions were selected as knockout regions. Two specific knockout targets were designed using CRISPR-Pv2.0 software, and target amplification primers were synthesized. The sequences are shown in SEQ ID NO:11~SEQ ID NO:14.
[0039] PCR amplification of target fragments
[0040] Using the PGTR vector as a template, PCR amplification was performed using primers SEQ ID NO:11~SEQ ID NO:14. PCR reaction volume (50 μL):
[0041] 2×Phanta Max Buffer 25μL
[0042] dNTP Mix (10mM) 1μL
[0043] 2 μL each of upstream and downstream primers (10 μM)
[0044] Phanta Max enzyme 1μL
[0045] PGTR template 1μL
[0046] Add ddH2O to 50 μL
[0047] PCR amplification procedure:
[0048] Pre-denaturation at 95℃ for 3 minutes
[0049] 95℃ denaturation for 15 seconds, 58℃ annealing for 15 seconds, 72℃ extension for 30 seconds, 35 cycles.
[0050] Extend at 72℃ for 5 minutes
[0051] Store at 4℃
[0052] Specific bands of 118bp and 213bp were amplified and purified by gel extraction for vector construction.
[0053] Construction of intermediate and final carriers
[0054] 1) Homologous recombination ligation was performed between the purified target fragment and the V129 vector digested with BsaⅠ, and the mixture was transformed into E. coli DH5α. Positive clones were sequenced for verification. 2) The verified intermediate vector and the empty V135 vector were double-digested with KpnⅠ and XbaⅠ, and the target fragment was recovered. 3) The target fragment was ligated into the V135 vector using T4 DNA ligase, and the mixture was transformed into E. coli DH5α. Sequencing was performed to obtain the qOil10-Cas9 knockout vector. 4) The positive recombination vector was transformed into Agrobacterium tumefaciens EHA105 competent cells for later use.
[0055] The CRISPR / Cas9 system targets and recognizes gene sequences via sgRNA, guiding Cas9 protein to cleave DNA and achieve targeted gene knockout. The dual-target knockout vector constructed in this invention has high knockout efficiency and strong specificity, and can efficiently obtain qOil10 homozygous mutants.
[0056] Example 3
[0057] Soybean genetic transformation and mutant identification
[0058] Agrobacterium-mediated soybean genetic transformation
[0059] Using soybean variety Williams 82 as recipient material, genetic transformation was performed using the cotyledon node transformation method: 1) Agrobacterium-positive single colonies were selected and cultured at 28℃ with shaking until OD. 600 =0.6–0.8; 2) Select plump soybean seeds, disinfect and germinate for 5 days, and take cotyledon explants; 3) Infect with Agrobacterium for 30 min and co-culture for 3 days; 4) Obtain T0 generation transformed plants by cephalosporin inhibition, PPT screening, induction of clustered shoots, rooting, and hardening.
[0060] Mutant Genotyping
[0061] DNA was extracted from leaves of T0 and T1 generation plants, amplified using qOil10 gene-specific primers, and the PCR products were sequenced. The results showed that some plants had base deletions / insertions (Indels) at the qOil10 gene target site, and homozygous knockout mutants were successfully obtained, named oil10-1 and oil10-2.
[0062] Oil content determination
[0063] Wild-type Williams 82, oil10-1, and oil10-2 were planted in the field. Seeds were harvested after maturity, and oil content was determined using near-infrared spectroscopy. Twenty individual plants from each material were measured, and the average results were taken. Phenotypic observation and oil content statistics are as follows: Figure 2 As shown.
[0064] Data Statistics and Analysis
[0065] The statistical results of oil content are shown in the table below:
[0066] Table 1. Seed oil content of wild type and qOil10 knockout mutant
[0067] Material Oil content (%) Significance of difference (P-value) Wild type (WT) 21.77±0.21 — oil10-1 20.90±0.44 <0.0001 oil 10-2 20.94±0.37 <0.0001
[0068] The results showed that the oil content of the qOil10 knockout mutant was significantly reduced compared with the wild type, proving that the qOil10 gene positively regulates the synthesis and accumulation of soybean seed oil.
[0069] Agrobacterium can integrate T-DNA fragments into the plant genome, achieving stable inheritance of exogenous genes / editing elements. The transformation system is mature, genetically stable, and provides clear phenotypic data, which can fully demonstrate gene function and meet the requirements for full disclosure in patent applications.
[0070] Example 4
[0071] Application in molecular marker-assisted breeding
[0072] The gene-localization primers (SEQ ID NO:3~10) provided by this invention can be used as functional molecular markers to rapidly identify superior haplotypes of qOil10 in breeding populations, shorten the breeding cycle by 3–5 years, and improve the breeding efficiency of high-oil soybeans.
[0073] Applications in gene editing breeding
[0074] The knockout primers (SEQ ID NO:11~14) and knockout vectors provided by this invention can be used for qOil10 gene editing in different soybean varieties to achieve precise improvement of oil content traits.
[0075] The final qOil10 gene effect is stable and applicable to various soybean ecoregions; the molecular marker and editing system is simple and low-cost, suitable for use by conventional breeding units; it can significantly increase soybean oil content and improve planting efficiency.
[0076] sequence list
[0077] This invention includes a nucleotide and amino acid sequence listing, which is submitted as a separate document in accordance with the requirements of the State Intellectual Property Office, and its contents are incorporated in whole or in part into the patent specification of this invention by reference.
[0078] Although embodiments of the invention have been shown and described, it will be understood by those skilled in the art that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the appended claims and their equivalents.
Claims
1. A gene for regulating soybean oil content, characterized in that, The gene is qOil10, and its nucleotide sequence is shown in SEQ ID NO:
1.
2. The protein encoded by the soybean oil content regulating gene according to claim 1, characterized in that, The amino acid sequence of the protein is shown in the sequence listing SEQ ID NO:
2.
3. A recombinant vector containing the soybean oil content regulating gene as described in claim 1, characterized in that, The recombinant vector is constructed by inserting the gene editing site into the KpnⅠ and XbaⅠ recognition sites of the V135 vector.
4. An expression cassette, transgenic cell line, or recombinant bacteria containing the soybean oil content regulating gene as described in claim 1.
5. Primers for amplifying the soybean oil content regulatory gene as described in claim 1, characterized in that, The primers are the gene positioning primers shown in the sequence listing SEQ ID NO:3 to SEQ ID NO:
10.
6. Primers used for constructing the soybean oil content regulatory gene knockout vector according to claim 1, characterized in that, The primers are the knockout primers shown in the sequence listing SEQ ID NO:11 to SEQ ID NO:
14.
7. The application of the soybean oil content regulating gene of claim 1 and the encoded protein of claim 2 in regulating the oil content of soybean seeds.