Soybean high-protein gene cloning method, expression regulation mechanism and application

Abstract

According to the invention, systematic research is carried out on cloning and expression regulation of soybean high-protein genes and application of the soybean high-protein genes in cultivation of high-protein soybean varieties. The method comprises the following steps: firstly, accurately cloning a soybean high-protein gene through a specific primer pair and a high-fidelity PCR (Polymerase Chain Reaction) technology, and ensuring the accuracy of the gene through sequencing verification; secondly, the promoter region of the soybean high-protein gene is deeply analyzed, and the gene expression is precisely regulated and controlled by utilizing a gene editing technology and an RNAi technology, so that different breeding requirements are met. And finally, introducing the cloned and regulated high-protein gene into a soybean receptor variety, and successfully culturing a high-protein transgenic soybean strain with excellent other characters through molecular level detection and field trials. The invention provides a new thought and method for soybean breeding work, not only improves the protein content of soybean, but also maintains other excellent agronomic characters, makes an important contribution to sustainable development of soybean industry, and has wide application prospects and practical values.

Description

Technical Field

[0001] The invention relates to the technical field of soybean gene cloning, and in particular to a soybean high-protein gene cloning method, expression regulation mechanism and application. Background Art

[0002] Today, soybeans are an important oil crop and protein source in the world. The protein content of soybeans directly affects their nutritional value and economic benefits. However, there are large differences in the protein content of soybean varieties in their natural state, and high-protein varieties are often accompanied by the deterioration of other agronomic traits, such as reduced yield and weakened stress resistance. Therefore, breeding soybean varieties with high protein and other excellent traits through genetic engineering technology has become one of the hot topics in agricultural research.

[0003] At present, the research on soybean high-protein genes mainly focuses on gene cloning, functional verification and expression regulation. However, due to the complexity of the soybean genome and the multi-gene inheritance characteristics of high-protein traits, the cloning and precise expression regulation of high-protein genes still face many challenges. In addition, the introduction of cloned high-protein genes into soybean recipient varieties and the cultivation of high-protein soybean lines with stable inheritance and excellent performance are also issues that need to be solved urgently. Summary of the invention

[0004] The purpose of the present invention is to provide a soybean high-protein gene cloning method, expression regulation mechanism and application, so as to solve the prior art problems raised in the above background technology.

[0005] To achieve the above object, the present invention provides the following technical solution: a method for cloning a soybean high-protein gene, comprising the following specific steps:

[0006] (a) Total DNA was extracted from soybean germplasm resources with high protein characteristics. The high salt method was used for DNA extraction. Cells were lysed under high salt concentration to release DNA and ensure the purity and integrity of the DNA.

[0007] (b) designing and synthesizing specific primer pairs targeting target high-protein genes based on the soybean proteome database and gene sequence information;

[0008] (c) using high-fidelity PCR technology to amplify the target gene fragment using the extracted total DNA as a template, and optimizing the PCR reaction conditions to ensure amplification efficiency and specificity;

[0009] (d) cloning the amplified product into a cloning vector by TA cloning or restriction enzyme ligation reaction to construct a recombinant plasmid, and performing sequencing verification to ensure the correct insertion of the target gene fragment;

[0010] (e) The recombinant plasmid is introduced into competent E. coli cells, and positive clones containing the target high protein gene are obtained through screening and PCR verification.

[0011] Furthermore, the specific primer pair is configured to be able to specifically amplify the full coding sequence of the soybean high-protein gene.

[0012] Furthermore, the transformation and introduction method in step e is to mix a certain amount of competent cells (such as 50 μL) with a certain amount of recombinant plasmid DNA (such as 1-5 μL), and gently mix; place the mixture on ice for 20-30 minutes to allow the DNA to fully combine with the cells; transfer the mixture to a 42°C water bath for heat shock for 90 seconds to promote the entry of DNA into the cells; after heat shock, quickly transfer the mixture to ice for 2 minutes, then add 1 mL of preheated LB medium, and culture at 37°C with shaking for 45-60 minutes to allow the cells to resume growth and express resistance genes.

[0013] An expression regulation mechanism of a soybean high-protein gene comprises the following steps:

[0014] (a) Analyze the promoter region sequence of soybean high-protein gene and determine the location and function of key cis-acting elements;

[0015] (b) Using gene editing technologies, such as TALEN, ZFN or CRISPR / Cas9 genome editing technologies, key cis-acting elements in the promoter region can be site-directed mutated, deleted or inserted to regulate gene expression levels.

[0016] Furthermore, the application of the gene editing technology must ensure precise regulation of the expression of soybean high-protein genes without affecting other important agronomic traits of the plant.

[0017] Application of a soybean high-protein gene in breeding high-protein soybean varieties comprises the following steps:

[0018] (a) introducing the soybean high-protein gene into a soybean recipient variety by Agrobacterium transformation, pollen tube pathway or gene gun method;

[0019] (b) Conduct molecular level testing on transgenic plants and confirm the successful introduction, integration and expression of the target gene through the operation of PCR molecular biology technology;

[0020] (c) Conduct field trials on transgenic plants to evaluate their protein content, composition and other agronomic traits, and screen out transgenic soybean lines with high protein and other excellent traits.

[0021] Furthermore, the soybean recipient variety refers to a soybean variety to be improved that has good other traits (such as yield and resistance) except for its low protein content.

[0022] Furthermore, the method for breeding the high-protein soybean strain comprises:

[0023] Obtain homozygous transgenic loci through multiple generations of self-pollination to eliminate phenotypic segregation caused by flanking sequence recombination;

[0024] Using marker-assisted backcross breeding technology, the targeted gene can be introduced while maintaining ≥95% of the genetic background of the recipient variety;

[0025] Furthermore, the marker-assisted backcross breeding technology uses SNP markers that are closely linked to the target high-protein gene for foreground selection, and simultaneously uses 214 SSR markers covering 20 pairs of soybean chromosomes for background selection. After three generations of backcrossing, the recipient genetic background recovery rate is ≥95%, ultimately obtaining a stable variety with both high-protein characteristics and excellent agronomic traits.

[0026] Compared with the prior art, the present invention has the following beneficial effects:

[0027] 1. The present invention, a method for cloning a soybean high-protein gene

[0028] Extract total DNA from soybean germplasm resources with high protein characteristics, use high salt method or CTAB method for DNA extraction to ensure the purity and integrity of DNA;

[0029] Based on the soybean proteome database and gene sequence information, specific primer pairs targeting target high-protein genes were designed and synthesized;

[0030] High-fidelity PCR technology was used to amplify the target gene fragment using the extracted total DNA as a template;

[0031] The amplified product is cloned into a cloning vector to construct a recombinant plasmid, and sequencing verification is performed to ensure the correct insertion of the target gene fragment and the accuracy of the sequence;

[0032] The recombinant plasmid is introduced into competent cells such as Escherichia coli, and positive clones containing the target high-protein gene are obtained through screening and PCR verification.

[0033] 2. The present invention is a method for accurately regulating the expression of soybean high-protein genes

[0034] Analyze the promoter region sequence of soybean high-protein genes to determine the location and function of key cis-acting elements;

[0035] Gene editing technologies (such as TALEN, ZFN or CRISPR / Cas9) are used to perform site-directed mutation, deletion or insertion of key cis-acting elements in the promoter region to regulate the expression level of genes and achieve efficient or specific expression of high-protein genes.

[0036] 3. The present invention, application of soybean high protein gene in breeding high protein soybean varieties

[0037] The cloned soybean high-protein gene is introduced into the soybean recipient variety through Agrobacterium transformation, pollen tube channel method or gene gun method to achieve stable integration of the foreign gene;

[0038] Conduct molecular level tests on transgenic plants, such as PCR, Southern blot and Western blot, to confirm the successful introduction, integration and expression of the target gene;

[0039] Field trials are conducted on transgenic plants to evaluate their protein content, composition and other agronomic traits, such as yield, stress resistance, quality, etc., to screen out transgenic soybean lines with high protein and other excellent traits. DETAILED DESCRIPTION

[0041] In the description of the present invention, it should be noted that, unless otherwise clearly specified and limited, the terms "connected" and "connection" should be understood in a broad sense, for example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be a direct connection or an indirect connection through an intermediate medium. For ordinary technicians in this field, the specific meanings of the above terms in the present invention can be understood according to specific circumstances.

[0042] A method for cloning soybean high-protein gene;

[0043] Extract total DNA from soybean germplasm resources with high protein characteristics, use high salt method or CTAB method for DNA extraction to ensure the purity and integrity of DNA;

[0044] Based on the soybean proteome database and gene sequence information, specific primer pairs targeting target high-protein genes were designed and synthesized;

[0045] High-fidelity PCR technology was used to amplify the target gene fragment using the extracted total DNA as a template;

[0046] The amplified product is cloned into a cloning vector to construct a recombinant plasmid, and sequencing verification is performed to ensure the correct insertion of the target gene fragment and the accuracy of the sequence;

[0047] The recombinant plasmid is introduced into competent cells such as Escherichia coli, and positive clones containing the target high-protein gene are obtained through screening and PCR verification;

[0048] (1) The present invention uses specific primer pairs and high-fidelity PCR technology to ensure accurate amplification of the target high-protein gene and avoid false positives or sequence errors that may occur in traditional cloning methods.

[0049] (2) Through sequencing verification and positive clone screening, the accuracy and reliability of the cloned soybean high-protein gene are ensured, providing a solid foundation for subsequent gene function verification and expression regulation research.

[0050] A method for precise expression regulation of soybean high-protein genes;

[0051] Analyze the promoter region sequence of soybean high-protein genes to determine the location and function of key cis-acting elements;

[0052] Gene editing technology is used to perform site-directed mutation, deletion or insertion of key cis-acting elements in the promoter region to regulate the expression level of the gene and achieve efficient or specific expression of high-protein genes;

[0053] (1) The present invention determines the location and function of key cis-acting elements by sequence analysis of the promoter region of soybean high-protein gene, providing a theoretical basis for precise expression regulation.

[0054] (2) Gene editing technology is used to perform site-directed mutation, deletion or insertion in the promoter region to achieve precise regulation of soybean high-protein gene expression to meet different breeding needs.

[0055] (3) RNAi technology can be used to specifically inhibit the expression of target genes or negative regulatory factors, providing new means and methods for regulating the expression levels of high-protein genes.

[0056] Application of a soybean high-protein gene in breeding high-protein soybean varieties;

[0057] The cloned soybean high-protein gene is introduced into the soybean recipient variety through Agrobacterium transformation, pollen tube channel method or gene gun method to achieve stable integration of the foreign gene;

[0058] Conduct molecular level tests on transgenic plants, such as PCR, Southern blot and Western blot, to confirm the successful introduction, integration and expression of the target gene;

[0059] Conduct field trials on transgenic plants to evaluate their protein content, composition and other agronomic traits, such as yield, stress resistance, quality, etc., and select transgenic soybean lines with high protein and other excellent traits;

[0060] (1) The present invention introduces the cloned and regulated high-protein gene into a soybean recipient variety, and achieves stable integration of the exogenous gene through Agrobacterium transformation, pollen tube channel method or gene gun method.

[0061] (2) Through molecular level detection and field trials, the transgenic plants are comprehensively evaluated to ensure that the transgenic soybean lines cultivated have the characteristics of high protein and other excellent traits.

[0062] (3) The application scheme of the present invention provides new ideas and methods for soybean breeding, and is expected to make important contributions to the sustainable development of the soybean industry;

[0063] The backcross breeding operation steps

[0064] 1. First generation backcross (BC 1 )

[0065] Donor (transgenic) × recipient → F 1 Hybrids

[0066] F 1 Plants were heterozygous for foreground heterozygous (individuals that were confirmed to carry heterozygous genes) and background heterozygous (individuals in which the heterozygous recipient genome accounted for ≥50%).

[0067] 2. Second generation backcross (BC 2 ) and later miscellaneous

[0068] Choose BC 1 The plants with the highest proportion of heterotic receptor genes were backcrossed again with heterotic receptors, and the foreground and background were selected for heavy heterotic genes.

[0069] The background recovery rate formula for each generation is: (n is the number of backcross generations), combined with marker selection, it can be accelerated to 20-30% per generation.

[0070] Finally, the homozygous strains were obtained:

[0071] After 3 generations of backcrossing (BC 3 ) after hybridization, the plants retaining the target gene and with a recipient background ≥ 93.75% were self-pollinated for 1-2 generations to obtain genetically stable homozygous lines.

[0072] It is to be understood that the above embodiments are merely exemplary embodiments used to illustrate the principles of the present invention, but the present invention is not limited thereto. For those of ordinary skill in the art, various modifications and improvements can be made without departing from the spirit and essence of the present invention, and these modifications and improvements are also considered to be within the scope of protection of the present invention.

Claims

1. A method for cloning a soybean high-protein gene, characterized in that: The specific steps include: (a) Total DNA was extracted from soybean germplasm resources with high protein characteristics. The high salt method was used for DNA extraction. Cells were lysed under high salt concentration to release DNA and ensure the purity and integrity of the DNA. (b) designing and synthesizing specific primer pairs targeting target high-protein genes based on the soybean proteome database and gene sequence information; (c) using high-fidelity PCR technology to amplify the target gene fragment using the extracted total DNA as a template, and optimizing the PCR reaction conditions to ensure amplification efficiency and specificity; (d) cloning the amplified product into a cloning vector by TA cloning or restriction enzyme ligation reaction to construct a recombinant plasmid, and performing sequencing verification to ensure the correct insertion of the target gene fragment; (e) The recombinant plasmid is introduced into competent E. coli cells, and positive clones containing the target high protein gene are obtained through screening and PCR verification.

2. The method for cloning a soybean high-protein gene according to claim 1, characterized in that: The specific primer pair is configured to specifically amplify the full coding sequence of the soybean high-protein gene.

3. The method for cloning a soybean high-protein gene according to claim 1, characterized in that: The transformation and introduction method in step e is to mix a certain amount of competent cells (such as 50 μL) with a certain amount of recombinant plasmid DNA (such as 1-5 μL), and gently mix; place the mixture on ice for 20-30 minutes to allow the DNA to fully combine with the cells; transfer the mixture to a 42°C water bath for heat shock for 90 seconds to promote the entry of DNA into the cells; after heat shock, quickly transfer the mixture to ice for 2 minutes, then add 1 mL of preheated LB culture medium, and shake culture at 37°C for 45-60 minutes to allow the cells to resume growth and express resistance genes.

4. The expression regulation mechanism of a soybean high-protein gene according to claim 1, characterized in that: The following steps are involved: (a) Analyze the promoter region sequence of soybean high-protein gene and determine the location and function of key cis-acting elements; (b) Using gene editing technologies, such as TALEN, ZFN or CRISPR / Cas9 genome editing technologies, key cis-acting elements in the promoter region can be site-directed mutated, deleted or inserted to regulate gene expression levels.

5. A soybean high-protein gene expression regulation mechanism according to claim 4, characterized in that: The application of the gene editing technology must ensure precise regulation of the expression of soybean high-protein genes without affecting other important agronomic traits of the plant.

6. The use of a soybean high-protein gene in breeding high-protein soybean varieties according to claim 1, characterized in that: The following steps are involved: (a) introducing the soybean high-protein gene into a soybean recipient variety by Agrobacterium transformation, pollen tube pathway or gene gun method; (b) Conduct molecular level testing on transgenic plants and confirm the successful introduction, integration and expression of the target gene through the operation of PCR molecular biology technology; (c) Conduct field trials on transgenic plants to evaluate their protein content, composition and other agronomic traits, and screen out transgenic soybean lines with high protein and other excellent traits.

7. The use of a soybean high-protein gene according to claim 6 in breeding high-protein soybean varieties, characterized in that: The soybean recipient variety refers to a soybean variety to be improved that has good other traits (such as yield and resistance) except for its low protein content.

8. The use of a soybean high-protein gene in breeding high-protein soybean varieties according to claim 6, characterized in that: The method for cultivating the high-protein soybean strain comprises: Obtain homozygous transgenic loci through multiple generations of self-pollination to eliminate phenotypic segregation caused by flanking sequence recombination; Marker-assisted backcross breeding technology is used to achieve targeted introgression of the target gene while maintaining ≥95% of the genetic background of the recipient variety.

9. The use of a soybean high-protein gene in breeding high-protein soybean varieties according to claim 8, characterized in that: The marker-assisted backcross breeding technology uses SNP markers that are closely linked to the target high-protein gene for foreground selection, and simultaneously uses multiple SSR markers covering 20 pairs of soybean chromosomes for background selection. After three generations of backcrossing, the recipient genetic background recovery rate is ≥95%, and ultimately a stable strain with both high-protein characteristics and excellent agronomic traits is obtained.