Soybean auxin transporter GmPIN3 gene and its application

Abstract

The present invention discloses a soybean auxin transport carrier GmPIN3 gene and its application. The GmPIN3 gene is selected from GmPIN3a gene, GmPIN3b gene, GmPIN3d gene; the GmPIN3a gene has a nucleotide sequence as shown in SEQ ID NO.1; the GmPIN3b gene has a nucleotide sequence as shown in SEQ ID NO.3; the GmPIN3d gene has a nucleotide sequence as shown in SEQ ID NO.5. The present invention uses the CRISPR / Cas9 gene editing technology to obtain GmPIN3 gene knockout homozygous mutant lines, and proves that the mutant lines can effectively improve the chlorophyll content, nitrogen content and protein content of soybeans.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and more specifically, to the soybean auxin transporter GmPIN3 gene and its applications. Background Art

[0002] Nitrogen is an essential element for all life on earth. The utilization of nitrogen depends on the nitrogen cycle. The nitrogen cycle is a biochemical process by which nitrogen can be transformed into various forms, entering the soil from the atmosphere, reaching organisms, and then returning to the atmosphere. In this cycle, plants play a crucial role in nitrogen assimilation, and nitrogen is also a key element for plant growth. The impact of nitrogen on plant growth and development is very obvious. When nitrogen is sufficient, plants can synthesize more proteins, promoting cell division and growth. Under high-nitrogen conditions, the leaf area of plants grows rapidly, and more leaf area can be used for photosynthesis. Nitrogen deficiency in plants often manifests as slow growth, short plants, thin and small leaves, and yellowish leaves due to chlorophyll deficiency. In order to increase the yield of crops, the application of nitrogen fertilizers has been increasing year by year. Although the use of nitrogen fertilizers can meet the nitrogen requirements of crops, the utilization of industrial nitrogen fertilizers increases a large amount of agricultural costs, and the application of excessive nitrogen fertilizers will cause damage to the environment. Therefore, the rational use of nitrogen fertilizers is a necessary prerequisite for ensuring high yields and good quality of crops.

[0003] The nitrogen in soybeans mainly comes from biological nitrogen fixation in root nodules and nitrogen in fertilizers, of which 60%-70% of the nitrogen comes from fertilizers. Nitrogen is absorbed from the roots and transported through the transport system to the above-ground parts for utilization. Nitrate and ammonium salts are the two main forms of nitrogen that soybeans can utilize, and soybeans have a preference for nitrate. After plants absorb nitrate nitrogen, it is transported through the vascular tissue, and the members of the nitrate transporter (NRT) protein family work together to complete the absorption, transport, and distribution of nitrogen. Nitrogen is an important component of amino acids in plants and the main source of proteins. The accumulation of nitrogen in soybean plants determines the protein content of seeds. Therefore, on the basis of reasonably improving the biological nitrogen fixation function of soybeans, improving the absorption and utilization pathways of nitrogen, and effectively enhancing the nitrogen use efficiency of soybeans have become the key issues in soybean quality improvement. Summary of the Invention

[0004] The purpose of the present invention is to provide the soybean auxin transporter GmPIN3 gene and its applications, to provide necessary genetic materials for in-depth understanding of the mechanism of auxin regulating seed protein accumulation, and to provide good germplasm resource reserves and ideas for improving soybean seed quality.

[0005] The technical solution adopted by the present invention is as follows:

[0006] Application of soybean auxin transporter GmPIN3 gene in increasing soybean protein content. The GmPIN3 gene is selected from GmPIN3a gene, GmPIN3b gene, and GmPIN3d gene. The nucleotide sequence of the GmPIN3a gene is as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.2. The nucleotide sequence of the GmPIN3b gene is as shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.4. The nucleotide sequence of the GmPIN3d gene is as shown in SEQ ID NO.5, and the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.6.

[0007] Furthermore, the way of the application is: by knocking out GmPIN3a gene and GmPIN3b gene simultaneously, the protein content of soybean seeds is increased.

[0008] Furthermore, the way of the application can also be: by knocking out GmPIN3a gene, GmPIN3b gene and GmPIN3d gene simultaneously, the protein content of soybean seeds is increased.

[0009] Application of soybean auxin transporter GmPIN3 gene in increasing soybean chlorophyll content. The GmPIN3 gene is selected from GmPIN3a gene, GmPIN3b gene, and GmPIN3d gene. The nucleotide sequence of the GmPIN3a gene is as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.2. The nucleotide sequence of the GmPIN3b gene is as shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.4. The nucleotide sequence of the GmPIN3d gene is as shown in SEQ ID NO.5, and the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.6.

[0010] Furthermore, the way of the application is: by knocking out GmPIN3a gene and GmPIN3b gene simultaneously, the chlorophyll content of soybean leaves is increased.

[0011] Furthermore, the way of the application can also be: by knocking out GmPIN3a gene, GmPIN3b gene and GmPIN3d gene simultaneously, the chlorophyll content of soybean leaves is increased.

[0012] Application of soybean auxin transporter GmPIN3 gene in increasing nitrogen content of soybeans. The GmPIN3 gene is selected from GmPIN3a gene, GmPIN3b gene, and GmPIN3d gene. The nucleotide sequence of the GmPIN3a gene is as shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.2. The nucleotide sequence of the GmPIN3b gene is as shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.4. The nucleotide sequence of the GmPIN3d gene is as shown in SEQ ID NO.5, and the amino acid sequence of the protein encoded by it is as shown in SEQ ID NO.6.

[0013] Furthermore, the way of the application is: by simultaneously knocking out the GmPIN3a gene and the GmPIN3b gene, the nitrogen content in the roots, stems, leaves, and seeds of soybeans is increased.

[0014] Furthermore, the way of the application can also be: by simultaneously knocking out the GmPIN3a gene, the GmPIN3b gene, and the GmPIN3d gene, the nitrogen content in the roots, stems, leaves, and seeds of soybeans is increased.

[0015] The significant advantages of the present invention are as follows:

[0016] The present invention uses the CRISPR / Cas9 gene editing technology to obtain the GmPIN3a / GmPIN3b double gene knockout homozygous mutant line and the GmPIN3a / GmPIN3b / GmPIN3d triple gene knockout homozygous mutant line, and proves that the chlorophyll content in the leaves of the mutant line is significantly increased compared with that of the wild type, and the nitrogen content in the roots, stems, leaves, and seeds is significantly increased, and the seed protein content is significantly increased. The research results of the present invention have important significance for production and theoretical research. Description of the Drawings

[0017] Figure 1 : Gene editing nucleotide sequences of gene-edited soybean mutants of Gmpin3ab and Gmpin3abd. The sgRNA sequence of the gene editing site of CRISPR-Cas9 targets the third exon position of GmPIN3a, GmPIN3b, and GmPIN3d. The region framed in green is the target site, and the nucleotides marked in red are the deletion situations after gene editing of the gene-edited soybean double mutants and triple mutants of Gmpin3ab and Gmpin3abd. Gmpin3ab is the GmPIN3a / GmPIN3b double gene knockout homozygous mutant line, and Gmpin3abd is the GmPIN3a / GmPIN3b / GmPIN3d triple gene knockout homozygous mutant line.

[0018] Figure 2 : Phenotypes of the aboveground parts of wild-type and transgenic soybeans after 74 days of field growth. From left to right, columns 1-9 are wild-type Huachun 6, columns 10-15 are GmPIN3a / GmPIN3b / GmPIN3d triple gene knockout homozygous mutant lines, and columns 16-18 are GmPIN3a / GmPIN3b double gene knockout homozygous mutant lines.

[0019] Figure 3 :Determination of SPAD values ​​of wild-type and transgenic soybean leaves. WT is the wild-type Huachun 6, Gmpin3ab is the GmPIN3a / GmPIN3b double gene knockout homozygous mutant line, and Gmpin3abd is the GmPIN3a / GmPIN3b / GmPIN3d triple gene knockout homozygous mutant line.

[0020] Figure 4 :Determination of nitrogen content in wild-type and transgenic soybeans. WT is the wild-type Huachun 6, pin3ab is the GmPIN3a / GmPIN3b double gene knockout homozygous mutant line, and pin3abd is the GmPIN3a / GmPIN3b / GmPIN3d triple gene knockout homozygous mutant line.

[0021] Figure 5 :Determination of protein content in wild-type and transgenic soybean seeds. WT is the wild-type Huachun 6, pin3ab is the GmPIN3a / GmPIN3b double gene knockout homozygous mutant line, and pin3abd-L9 is the GmPIN3a / GmPIN3b / GmPIN3d triple gene knockout homozygous mutant line. DETAILED DESCRIPTION

[0022] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.

[0023] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0024] Unless otherwise specified, the test materials used in the following examples were purchased from conventional biochemical reagent stores.

[0025] The determination of vector sequencing in the following examples was determined by sequencing by a conventional sequencing company.

[0026] The soybean cultivar Huachun No. 6 is the background material of the present invention: Huachun No. 6 is a soybean variety bred by the College of Agriculture of South China Agricultural University, using the varieties Guizao No. 1×Brazil No. 8; it was approved at the third meeting of the Second National Crop Variety Approval Committee on July 28, 2009, and the approval number is Guoshendou 2009012.

[0027] The nucleotide sequence of the soybean GmPIN3a gene of the present invention is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded thereby is shown as SEQ ID NO.2; the nucleotide sequence of the soybean GmPIN3b gene is shown as SEQ ID NO.3, and the amino acid sequence of the protein encoded thereby is shown as SEQ ID NO.4; the nucleotide sequence of the soybean GmPIN3d gene is shown as SEQ ID NO.5, and the amino acid sequence of the protein encoded thereby is shown as SEQ ID NO.6.

[0028] Example 1: Obtaining of GmPIN3 gene knockout mutants

[0029] A DNA sequence was selected as the target sequence for CRISPR / Cas9 gene knockout in the third exon region of the soybean GmPIN3a gene, GmPIN3b, and GmPIN3d genes: 5’-GTCTGATGCTGGTCTTGGAA-3’

[0030] Primer pairs for constructing sgRNA were designed, and the specific sequences are as follows:

[0031] sgRNA Forward primer: 5’-ggattGATTGTCTGATGCTGGTCTTGGAA-3’

[0032] sgRNA Reverse primer: 5’-AAACAAACTTCCAAGACCAGCATCAGACCA-3’

[0033] The Forward primer and Reverse primer of sgRNA were annealed to form double-stranded DNA, which was constructed into the pGES201 vector by the GoldenGate method, transformed into Escherichia coli DH5α competent cells, and positive clones were screened to extract plasmids and sent to the company for sequencing. The correctly sequenced vector was transformed into the recipient soybean by the cotyledon node infection method mediated by Agrobacterium tumefaciens GV3101 to obtain GmPIN3 gene knockout mutant plants.

[0034] Among them, the GoldenGate reaction system was: 5 μL of pGES201 plasmid, 2 μL of T4 DNA Ligase Buffer (10X), 1 μL of T4 DNA ligase, 2 μL of BsaI-HFV, and 1 μL of DNA fragment; the GoldenGate reaction conditions were: 37°C for 5 min, 16°C for 5 min, for a total of 30 cycles; 37°C for 15 min; 85°C for 15 min.

[0035] Example 2: Verification of GmPIN3 gene knockout mutants

[0036] The primer sequences for verifying knockout mutants are as follows:

[0037] GmPIN3a - Forward primer: 5’-TACTCCAGTCTCATTGGTGTCATTT-3’

[0038] GmPIN3a - Reverse primer: 5’-CTATCACATTCAATCAAGTGCCGTC-3’

[0039] GmPIN3b - Forward primer: 5’-TGATTACTGTGTAGGGTGGTGTTTA-3’

[0040] GmPIN3b - Reverse primer: 5’-CTCATTGTGATGCCATTTTTCAACC-3’

[0041] GmPIN3d - Forward primer: 5’-TGCATTCAGGTAGGTTAATCAAAGT-3’

[0042] GmPIN3d - Reverse primer: 5’-GATCGATGTCTATGCGAAAGAGTGA-3’

[0043] Using the genomic DNA of the GmPIN3 gene knockout mutant soybean plants as a template, PCR amplification was carried out, and the PCR products were sequenced and compared. The results are as Figure 1 shown. On the third exon of GmPIN3a, there is an insertion of a single base G in Gmpin3ab; on the third exon of GmPIN3b, there is an insertion of a single base A in Gmpin3ab. On the third exon of GmPIN3a, there is a deletion of a single base G in Gmpin3abd - L9; on the third exon of GmPIN3b, there is a deletion of five bases AATGG in Gmpin3abd - L9; on the third exon of GmPIN3d, there is a deletion of two bases TG in Gmpin3abd - L9. On the third exon of GmPIN3a, there is an insertion of a single base G in Gmpin3abd - L85; on the third exon of GmPIN3b, there is an insertion of a single base A in Gmpin3abd - L85; on the third exon of GmPIN3d, there is a deletion of a single base G in Gmpin3abd - L85.

[0044] Example 3: Determination of Chlorophyll Content in GmPIN3 Gene Knockout Mutants

[0045] As Figure 2As shown, the leaves of Gmpin3ab and Gmpin3abd are significantly greener than those of the wild type.

[0046] Soybean transgenic plants and non-transgenic plants at 74 days of seedling age were selected, and a relative chlorophyll meter was used to measure the relative chlorophyll content of fully expanded functional leaves.

[0047] As Figure 3 shown, compared with the wild type, Gmpin3ab and Gmpin3abd showed a significant increase in chlorophyll content.

[0048] Example 4: Determination of nitrogen content in GmPIN3 gene knockout mutants

[0049] The determination of the nitrogen content of the samples was carried out by the Kjeldahl method. The samples of soybean pod-filling stage were dried, crushed and sieved for later use. Take 0.2 g of the sample and place it at the bottom of the digestion tube. Using copper sulfate and potassium nitrate as catalysts, add 2 g of the mixed catalyst to the digestion tube, then add 5 mL of concentrated sulfuric acid and shake well to digest the plant sample; when the liquid in the digestion tube shows a green and transparent color, remove the digestion tube and carry out the Kjeldahl method to determine the nitrogen content. At high temperature, the nitrogen-containing compounds in the plant leaves are converted into ammonium sulfate, then alkalized with sodium hydroxide, and ammonia is distilled out by heating. When the color of the liquid in the digestion tube is brown, it indicates that the reaction is complete. After absorption by boric acid, it is titrated with a standard sulfuric acid solution to calculate the nitrogen content.

[0050] As Figure 4 shown, compared with the wild type, Gmpin3ab and Gmpin3abd showed a significant increase in the nitrogen content of roots, stems, leaves and seeds.

[0051] Example 5: Determination of protein content in GmPIN3 gene knockout mutants

[0052] Wild type and pin3ab and pin3abd mutant seeds were selected and randomly divided into eight equal parts. The protein content was measured using a Matrix near-infrared light grain analyzer produced by Bruker.

[0053] As Figure 5 shown, compared with the wild type, Gmpin3ab and Gmpin3abd showed a significant increase in the seed protein content.

[0054] The above are only the preferred embodiments of the present invention. All equivalent changes and modifications made according to the scope of the patent application of the present invention shall fall within the scope covered by the present invention.

Claims

1. Knockout GmPIN3a Gene, GmPIN3b Genes and GmPIN3d The application of the gene in increasing the protein content of soybean seeds is characterized by: Said GmPIN3a The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmPIN3b The nucleotide sequence of the gene is shown in SEQ ID NO.

3. GmPIN3d The nucleotide sequence of the gene is shown in SEQ ID NO.

5.

2. Knockout GmPIN3a Genes and GmPIN3b The application of the gene in increasing the chlorophyll content of soybean leaves is characterized by: Said GmPIN3a The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmPIN3b The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

3. Knockout GmPIN3a Gene, GmPIN3b Genes and GmPIN3d The application of the gene in increasing the chlorophyll content of soybean leaves is characterized by: Said GmPIN3a The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmPIN3b The nucleotide sequence of the gene is shown in SEQ ID NO.

3. GmPIN3d The nucleotide sequence of the gene is shown in SEQ ID NO.

5.

4. Simultaneous knockout GmPIN3a Genes and GmPIN3b The application of the gene in increasing the nitrogen content of soybean roots, stems or leaves is characterized by: Said GmPIN3a The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmPIN3b The nucleotide sequence of the gene is shown in SEQ ID NO.

3.

5. Simultaneous knockout GmPIN3a Gene, GmPIN3b Genes and GmPIN3d The application of the gene in increasing the nitrogen content of soybean roots, stems, leaves or seeds is characterized by: Said GmPIN3a The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GmPIN3b The nucleotide sequence of the gene is shown in SEQ ID NO.

3. GmPIN3d The nucleotide sequence of the gene is shown as SEQ ID NO.5.

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