Application of soybean GmLRR1 gene in regulation and control of soybean oil content

Abstract

The invention discloses application of a soybean GmLRR1 gene in regulation and control of soybean oil content, and belongs to the technical field of genetic engineering, the invention finds a GmLRR1 gene related to soybean oil synthesis, and the GmLRR1 gene can positively regulate and control the soybean oil content through comparison with a GmLRR1 gene mutant material. And a new thought and technical support are provided for molecular breeding and industrial application of the soybean oil content.

Description

Technical Field

[0001] The present invention belongs to the field of genetic engineering technology, and specifically relates to soybean GmLRR1 Application of genes in regulating soybean oil content. Background Art

[0002] Soybean (Glycine max (Linn.) Merr.) is one of the most important oil crops in the world and is widely used in the production of food, feed, industrial raw materials and bioenergy. Soybean is rich in protein and oil. Its oil is not only one of the main sources of vegetable oil for human consumption, but also has important applications in industry as biofuel and chemical raw materials. Therefore, research on increasing the oil content of soybean has important economic value and social significance.

[0003] In the soybean production process, oil content is a key factor affecting the benefits of soybean planting. Oil content not only determines the nutritional value and market competitiveness of soybeans, but is also an important economic indicator in soybean production. With the continuous increase in soybean demand, how to increase oil content while maintaining high soybean yield has become one of the important directions of soybean breeding research.

[0004] However, the molecular mechanism of soybean oil synthesis is complex and is subject to the interaction of multi-gene regulation and environmental factors. Although a large number of studies have explored the genetic factors affecting soybean oil content over the years, its specific molecular mechanism is still not fully understood. Soybean oil synthesis involves multiple biological pathways, including fatty acid synthesis, oil storage protein synthesis, and oil accumulation. Therefore, exploring and accurately locating key genes related to oil synthesis is of vital importance for optimizing the soybean oil synthesis pathway and increasing oil content.

[0005] In this context, GmLRR1 The gene is thought to play a role in soybean oil synthesis. GmLRR1 It is a typical LRR (Leucine-Rich Repeat) gene. The LRR gene family is usually involved in plant immune response, signal transduction and various metabolic regulation processes. Studies have shown that LRR genes play an important role in plant growth, development and stress resistance, but their specific function in oil synthesis has not been clearly verified. GmLRR1 The gene may affect oil accumulation by regulating the fatty acid synthesis pathway or oil storage process in soybean cells.

[0006] Although some studies have pointed out that the LRR gene family is related to lipid synthesis in other crops, the research on soybean is still relatively limited. GmLRR1 There is a lack of direct functional verification of the role of genes in soybean oil synthesis.GmLRR1 The functional verification of the gene provides a new experimental basis for a deeper understanding of the regulatory role of the gene in oil synthesis. This research can not only provide theoretical support for increasing the oil content of soybeans, but also promote the cultivation of high-oil soybean varieties through molecular breeding methods.

[0007] In addition, with the rapid development of genomics and molecular marker technology, traditional breeding methods have gradually failed to meet the demand for increasing soybean oil content. Therefore, the use of molecular breeding technology, gene function verification methods, and modern phenotypic evaluation technology has become an inevitable trend in the genetic research of soybean oil content. By identifying and verifying key genes related to oil content, combined with precise molecular marker-assisted breeding, it is possible to accelerate the improvement of soybean varieties, increase oil content, and thus promote the sustainable development of the soybean industry.

[0008] The present invention is proposed in this context, aiming to fill the gap in the functional verification of soybean oil synthesis genes and provide a new method for studying and applying GmLRR1 The function of the gene provides new ideas and technical support for the molecular breeding and industrial application of soybean oil content. Summary of the invention

[0009] The present invention aims to provide a soybean GmLRR1 Application of genes in regulating soybean oil content.

[0010] In order to achieve the above object, the technical solution of the present invention is as follows: Soybean GmLRR1 Gene, the sequence number of the gene in the Phytozome database is Glyma.16G192300 Its CDS sequence is shown in SEQ ID NO.1, and its nucleotide sequence consists of 3492 bases. Its encoded amino acid sequence is shown in SEQ ID NO.2, and consists of 1163 amino acids. GmLRR1 The gene is involved in regulating the synthesis of soybean oil. The regulation is manifested in: compared with the wild type, GmLRR1 The soybean oil content of the gene mutant was significantly reduced.

[0011] The above soybean GmLRR1 The gene may also include a nucleotide sequence formed by substitution, deletion or addition of one or more (e.g., 1-30; preferably 1-20; more preferably 1-10, such as 5, 3) nucleotide residues, and having GmLRR1 DNA derived from it that has the same or similar function as a gene; or GmLRR1 The defined nucleotide sequences have a homology of more than 80% (preferably more than 90%, such as 95%, 98%, 99% or higher) and haveGmLRR1 Functional DNA derived therefrom.

[0012] Those skilled in the art can easily mutate the nucleotide sequence encoding the LRR protein of the present invention by using known methods, such as directed evolution and point mutation. Those artificially modified nucleotides having 75% or higher identity with the nucleotide sequence of the LRR protein isolated from the present invention are derived from the nucleotide sequence of the present invention and are equivalent to the sequence of the present invention as long as they encode the LRR protein and have the function of the LRR protein.

[0013] The present invention obtains at least two strains through screening and mutagenesis. GmLRR1 The function of the gene is verified by using gene mutants. After multiple generations of backcrossing and background purification, the target trait of the mutant can be stably inherited in the offspring and can remain stable without additional selection.

[0014] The present invention has obtained two GmLRR1 Gene mutants, including MT-W82 GmLRR1-1 and MT-W82 GmLRR1-2 Both mutants can be stably inherited. GmLRR1-1 The 3277th base of the sequence shown in SEQ ID NO.1 undergoes a G→T mutation; the MT-W82 GmLRR1-2 The mutation of the base at position 1214 in the sequence shown in SEQ ID NO.1 changes from T to A.

[0015] Among them, the GmLRR1 The screening method for gene mutants is: (1) Treat soybean seeds with EMS (ethyl methyl sulfonate) to induce mutations; (2) Using the double-stranded DNA molecule shown in SEQ ID NO.1 as a template, MT-W82 GmLRR1-1 -F and MT-W82 GmLRR1-1 -R, MT-W82 GmLRR1-2 -F and MT-W82 GmLRR1-2 -R primer pair for PCR amplification; sequencing the amplified product; (3) According to the sequencing results, find the strains in which the single base of the sequence shown in SEQ ID NO.1 is mutated; by using known molecular markers or genotype analysis, ensure GmLRR1 Accuracy of genetic mutants; (4) After multiple generations of backcrossing and background purification, the strains with single base mutations were stably inherited. GmLRR1 Gene mutants; Wherein, the sequence of the primer pair is as follows: MT-W82 GmLRR1-1 -F: 5'-CTGGTGAAATCCCTCCAACCA-3'; MT-W82 GmLRR1-1 -R: 5'-GCGAAAAGATTGAAGTTTTGAACCA-3'; MT-W82 GmLRR1-2 -F:5'-GAACCTAAGGGCCAACTACT-3'; MT-W82 GmLRR1-2 -R:5'-TGCAGAGATTACCCAAAGAA-3'.

[0016] By testing the lipid content of the above mutants and the wild type, it was found that compared with the wild type, GmLRR1 The soybean oil content of the gene mutant was significantly reduced, and it was concluded that GmLRR1 Genes can regulate soybean oil content, providing new ideas and technical support for molecular breeding and industrial application of soybean oil content.

[0017] According to the oil content characteristics of wild type and mutant, GmLRR1 Genes can positively regulate the oil content of soybeans. Therefore, in practical applications, overexpression or overexpression can be used to perform transgenic operations on target plants to improve GmLRR1 The expression level of the gene is increased, thereby increasing the oil content of the target plant. Therefore, the present invention also provides a method for increasing the oil content of the plant, comprising the following method: (1) Improve the target plant GmLRR1 Gene expression to obtain target plants with increased oil content; (2) Increasing the content of LLR protein in the target plant, or increasing the activity of LLR protein in the target plant, to obtain the target plant with increased oil content.

[0018] Among them, increasing the GmLRR1 The expression of the gene is achieved by selecting from the following methods: Method (1) is to GmLRR1 Import target plants; Method (2) is to introduce a strong promoter and / or enhancer; Method (3) includes other common methods in the art such as small RNA regulation, methylation / demethylation, phosphorylation / dephosphorylation, and promoter binding site regulation.

[0019] More specifically, a method for increasing the oil content of soybeans comprises the following steps: A1) GmLRR1 Overexpression or over-expression of the gene in soybeans, orGmLRR1 Genes are introduced into target soybeans for genetic improvement; A2) Evaluate the agronomic traits of genetically improved soybeans, including crude oil content; A3) Through hybridization and backcrossing methods, soybean varieties with target traits (high oil content) are selected.

[0020] The present invention can be widely used in the fields of agricultural breeding, food processing and industrial production, including but not limited to: B1) Breed soybean varieties with high oil content to increase soybean oil production and reduce dependence on imported soybeans; B2) Produce high-protein and high-fat soybeans to improve the quality of soybean raw materials in the food processing industry; B3) Produce high-energy feed soybeans to improve the production efficiency of animal husbandry and optimize feed formula; B4) Promote the development of the biodiesel industry and increase the utilization rate of renewable energy; B5) Increase farmers’ economic benefits and promote agricultural economic development through agricultural extension and demonstration planting.

[0021] In the above application, the plant may be C1) or C2) or C3): C1) dicots or monocots; C2) Leguminosae; C3) Soybeans.

[0022] Advantages of the present invention: The present invention uses molecular breeding technology, gene function verification method, and modern phenotypic evaluation technology to find a gene related to soybean oil synthesis. GmLRR1 Genes, by identifying and verifying key genes related to oil content, combined with precise molecular markers to assist breeding, can accelerate the improvement of soybean varieties and increase oil content, thereby promoting the sustainable development of the soybean industry. GmLRR1 Genes can positively regulate soybean oil content, providing new ideas and technical support for molecular breeding and industrial application of soybean oil content. BRIEF DESCRIPTION OF THE DRAWINGS

[0023] Figure 1 Candidate genes GmLRR1 In mutant MT-W82 GmLRR1-1 and MT-W82 GmLRR1-2 Sequence analysis in; Figure 2 W82 and its mutant MT-W82 GmLRR1-1 and MT-W82 GmLRR1-2 Comparison of fat content in. DETAILED DESCRIPTION

[0024] The present invention will be further described below in conjunction with specific examples, and the advantages and features of the present invention will become clearer as the description proceeds. However, the specific experimental methods involved in the following examples are all conventional methods or implemented under the conditions recommended by the manufacturer's instructions unless otherwise specified.

[0025] Unless otherwise specified, the technical means used in the examples are conventional means known to those skilled in the art. The test methods in the following examples are conventional methods unless otherwise specified. Unless otherwise specified, the reagents and materials used can all be purchased from the market.

[0026] Unless otherwise defined, all professional and scientific terms used herein have the same meanings as those familiar to those skilled in the art. In addition, any methods and materials similar or equivalent to those described herein may be applied to the present invention. The preferred implementation methods and materials described herein are for demonstration purposes only.

[0027] Example 1 GmLRR1 Gene cloning DNA was extracted from wild soybean seeds, and specific primers were designed by PrimerPremier using the gene sequence obtained from the Phytozome database for PCR reaction. GmLRR1 The CDS sequence of the gene (as shown in SEQ ID NO.1) can also be obtained through the Phytozome database GmLRR1 The protein sequence corresponding to the CDS sequence of the gene (as shown in SEQ ID NO.2).

[0028] Example 2 Screening and identification of mutants Soybean variety W82 was selected for EMS mutagenesis treatment and obtained after screening. GmLRR1 Mutant; after multiple generations of backcrossing and background purification, the target trait of the mutant can be stably inherited in the offspring.

[0029] Using the double-stranded DNA molecule shown in SEQ ID NO.1 in the sequence list as a template, MT-W82 GmLRR1-1 -F, MT-W82 GmLRR1-1 -R, MT-W82 GmLRR1-2 -F and MT-W82 GmLRR1-2 PCR amplification was performed using a primer pair consisting of -R, and the PCR amplification product was recovered.

[0030] MT-W82 GmLRR1-1 -F: 5'-CTGGTGAAATCCCTCCAACCA-3'; MT-W82 GmLRR1-1-R: 5'-GCGAAAAGATTGAAGTTTTGAACCA-3'; MT-W82 GmLRR1-2 -F:5'-GAACCTAAGGGCCAACTACT-3'; MT-W82 GmLRR1-2 -R:5'-TGCAGAGATTACCCAAAGAA-3'.

[0031] PCR amplification and DNA sequencing were used to analyze the soybean samples. GmLRR1 The gene was subjected to genotyping analysis to confirm whether a mutation occurred. GmLRR1 The mutant had a single base substitution mutation (Table 1, Figure 1 ).

[0032] Specifically: the MT-W82 GmLRR1-1 The 3277th base of the sequence shown in SEQ ID NO.1 undergoes a G→T mutation; the MT-W82 GmLRR1-2 The mutation of the base at position 1214 in the sequence shown in SEQ ID NO.1 changes from T to A.

[0033] Table 1 GmLRR1 Gene mutant mutation information

[0034] From the above information, we can see that MT-W82 GmLRR1-1 and MT-W82 GmLRR1-2 It is indeed GmLRR1 Gene mutants. A gene with two or more mutant materials can be used to verify the function of the gene, so the phenotypic comparative analysis of oil content was continued.

[0035] Example 3 Phenotypic analysis of oil content Wild-type and GmLRR1 Gene mutant MT-W82 GmLRR1-1 and MT-W82 GmLRR1-2 .

[0036] Near infrared spectroscopy (NIR) was used to determine the seed oil content. Each sample was measured three times independently, and the average value was calculated and statistically analyzed.

[0037] Use statistical methods (such as t-test or analysis of variance (ANOVA)) to record the differences in lipid content between the wild type and each mutant to ensure the accuracy of the data.

[0038] As can be seen from Table 2, the lipid content of wild-type W82 is 21.64±0.4%, and that of MT-W82 GmLRR1-1 The lipid content of the mutant was 20.05±0.3%, MT-W82 GmLRR1-2 The lipid content of the mutant was 18.83±0.5%. GmLRR1 The lipid content of the mutant was significantly lower than that of the wild type ( Figure 2 ), it can be concluded that GmLRR1 Genes can regulate soybean oil content.

[0039] Table 2 Wild type and GmLRR1 Comparison of oil content in gene mutants

[0040] In summary, the present invention successfully constructed using EMS mutagenesis technology GmLRR1 The gene mutants were identified and their functions were verified through phenotypic analysis and molecular detection, which provides a new theoretical basis for the genetic research of soybean oil synthesis and can also be used for the molecular breeding and improvement of high-oil soybean varieties.

[0041] The embodiments described above are only preferred embodiments of the present invention and are only used to explain the present invention, not to limit the scope of implementation of the present invention. For those skilled in the art, other implementation methods can certainly be easily made by replacement or modification based on the technical contents disclosed in this specification. Therefore, all changes and improvements made on the principles of the present invention should be included in the scope of the patent application of the present invention.

Claims

1. Soybeans GmLRR1 The application of the gene in regulating the oil content of soybean is characterized in that: Said GmLRR1 The CDS sequence of the gene is shown in SEQ ID NO.

1. The regulation is manifested in that: compared with the wild type, GmLRR1 The soybean oil content of the gene mutant was significantly reduced.

2. The use according to claim 1, characterized in that: Said GmLRR1 Gene mutants include MT-W82 GmLRR1-1 and MT-W82 GmLRR1-2 Both mutants can be stably inherited. GmLRR1-1 The 3277th base of the sequence shown in SEQ ID NO.1 undergoes a G→T mutation; the MT-W82 GmLRR1-2 A mutation from T to A occurs at the 1214th base of the sequence shown in SEQ ID NO.

1.

3. The use according to claim 1 or 2, characterized in that: Said GmLRR1 The screening method for gene mutants is: (1) Treat soybean seeds with EMS to induce mutations; (2) Using the double-stranded DNA molecule shown in SEQ ID NO.1 in claim 1 as a template, MT-W82 GmLRR1-1 -F and MT-W82 GmLRR1-1 -R, MT-W82 GmLRR1-2 -F and MT-W82 GmLRR1-2 -R primer pair for PCR amplification; sequencing the amplified product; (3) According to the sequencing results, find the strain in which the single base mutation of the sequence shown in SEQ ID NO.1 occurs; (4) After multiple generations of backcrossing and background purification, the strains with single base mutations were stably inherited. GmLRR1 Gene mutants; Wherein, the sequence of the primer pair is as follows: MT-W82 GmLRR1-1 -F:5’-CTGGTGAAATCCCTCCAACCA-3’; MT-W82 GmLRR1-1 -R:5'-GCGAAAAGATTGAAGTTTGAACCA-3'; MT-W82 GmLRR1-2 -F:5’-GAACCTAAGGGCCAACTACT-3’; MT-W82 GmLRR1-2 -R:5’-TGCAGAGATTACCCAAAGAA-3’。

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