Application of auxin transport carrier GmPIN3 gene family in legume variety improvement

Abstract

The present invention relates to the technical field of plant genetic engineering, and more specifically, to the application of the auxin transport carrier GmPIN3 gene family in the improvement of leguminous plant varieties. The auxin transport carrier GmPIN3 gene family includes GmPIN3a gene and GmPIN3b 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 improvement of leguminous plant varieties is to improve the photosynthetic capacity of leguminous plant leaves and / or increase the sucrose content of leguminous plant leaves and / or increase the oil content of leguminous plant seeds.

Description

Technical Field

[0001] The present invention relates to the technical field of plant genetic engineering, and more specifically, to the application of the auxin transport carrier GmPIN3 gene family in the variety improvement of leguminous plants. Background Art

[0002] To achieve the required yield increase and make agriculture more sustainable, it is crucial to obtain crops with high photosynthetic capacity and high nitrogen use efficiency (NUE). Nitrogen promotes leaf area growth, and plants have more leaf area for photosynthesis. The process of fixing carbon sources through photosynthesis in the above-ground part of plants and the process of the root system absorbing nitrogen from the soil are both mutually promoting and dependent, as well as mutually contradictory and restrictive, in order to achieve overall coordination and balance, and thus maintain the growth and development of plants. The nitrogen absorbed by the root system is an important component of a series of enzymes in the light and dark reactions of leaf photosynthesis, while the photosynthesis products of leaves provide the assimilation power ATP, NADPH, and carbon skeletons for nitrogen metabolism. Maintaining the carbon-nitrogen metabolic balance in the body is necessary for the normal growth and development of plants and is also a key factor affecting crop yields.

[0003] There are various factors affecting the carbon-nitrogen balance, including light signals, hormones, etc. The bZIP transcription factor HY5 (ELON-GATED HYPOCOTYL5) protein in the Arabidopsis thaliana light signal pathway can move over long distances and promote the long-distance carbon-nitrogen balance between the above-ground part and the root system by coordinately regulating the absorption of nitrate nitrogen in the root, photosynthetic carbon fixation in the above-ground part, and the transport of photosynthesis products to the root system, thereby ensuring the normal growth and development of plants. Research has found that the DELLA protein in the gibberellin signal pathway interacts with GRF4 (GROWTH-REGULATING FACTOR4) and inhibits the formation of the GRF4-GIF1 transcriptional activation protein complex. As a result, the photosynthetic carbon fixation ability of leaves and the nitrogen absorption ability of the root system are reduced. On the contrary, gibberellin promotes the degradation of the DELLA protein, enhances the transcriptional activation activity of GRF4, and achieves the coordinated regulation of the photosynthetic carbon fixation ability of plant leaves and the nitrogen absorption ability of the root system, thereby maintaining the balance between plant growth and carbon-nitrogen metabolism. In addition to gibberellin, carbon can promote the development of the hypocotyl and root of Arabidopsis thaliana by increasing the level of auxin. In rice, we can see that the ARF in the auxin signal pathway can directly bind to the nitrogen transporter in vivo, thereby promoting the absorption of nitrogen by the root system. It can be seen that the auxin concentration gradient is crucial for regulating carbon-nitrogen metabolism. Summary of the Invention

[0004] The purpose of the present invention is to provide the application of the auxin transport carrier GmPIN3 gene family in the variety improvement of leguminous plants.

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

[0006] Application of auxin transporter GmPIN3 gene family in legume variety improvement. The auxin transporter GmPIN3 gene family includes GmPIN3a gene and GmPIN3b gene. The nucleotide sequence of the GmPIN3a gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.2. The nucleotide sequence of the GmPIN3b gene is shown as SEQ ID NO.3, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.4. The legume variety improvement is to improve the photosynthetic capacity of legume leaves and / or increase the sucrose content of legume leaves and / or increase the oil content of legume seeds.

[0007] Furthermore, improving the photosynthetic capacity of legume leaves means increasing the net photosynthetic rate of legume leaves and / or increasing the stomatal conductance of legume leaves and / or increasing the transpiration ratio of legume leaves and / or decreasing the intercellular CO2 concentration of legume leaves.

[0008] Furthermore, by simultaneously knocking out the GmPIN3a gene and the GmPIN3b gene, the photosynthetic capacity of legume leaves and / or the sucrose content of legume leaves and / or the oil content of legume seeds can be increased.

[0009] Furthermore, the legume is soybean.

[0010] A method for increasing the photosynthetic capacity of legume leaves and / or increasing the sucrose content of legume leaves and / or increasing the oil content of legume seeds, which comprises the following steps: knocking out the GmPIN3a gene and the GmPIN3b gene in the recipient legume, or inhibiting the expression of the GmPIN3a gene and the GmPIN3b gene in the recipient legume, or inhibiting the activity of the proteins encoded by the GmPIN3a gene and the GmPIN3b gene in the recipient legume. The nucleotide sequence of the GmPIN3a gene is shown as SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.2. The nucleotide sequence of the GmPIN3b gene is shown as SEQ ID NO.3, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.4.

[0011] Furthermore, improving the photosynthetic capacity of legume leaves means increasing the net photosynthetic rate of legume leaves and / or increasing the stomatal conductance of legume leaves and / or increasing the transpiration ratio of legume leaves and / or decreasing the intercellular CO2 concentration of legume leaves.

[0012] Furthermore, the legume is soybean.

[0013] The remarkable advantages of the present invention are:

[0014] The present invention created a gene - edited soybean Gmpin3ab double mutant that does not contain the auxin transport carrier GmPIN3a and GmPIN3b proteins, providing necessary genetic materials for in - depth understanding of the mechanism of auxin in regulating seed oil accumulation, and providing good germplasm resource reserves and ideas for improving soybean seed quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0015] Figure 1 : Gene - edited nucleotide sequence of the gene - edited soybean mutant of Gmpin3ab. The sgRNA sequence of the gene - editing site of CRISPR - Cas9 targets the third exon position of GmPIN3a and GmPIN3b, and the red base sequence is the target site.

[0016] Figure 2 : Measurement of photosynthetic capacity of wild - type soybean and transgenic soybean leaves. P - values were tested by two - tailed Student’s t - test, ****p < 0.0001.

[0017] Figure 3 : Measurement of sucrose content in wild - type soybean and transgenic soybean leaves. P - values were tested by two - tailed Student’s t - test, **p < 0.01.

[0018] Figure 4 : Measurement of seed oil content in wild - type soybean and transgenic soybean. P - values were tested by two - tailed Student’s t - test, ****p < 0.0001. PT: Putian pilot; FZ: Fuzhou pilot; SY: Sanya pilot. DETAILED DESCRIPTION OF THE EMBODIMENTS

[0019] In order to make the content of the present invention easier to understand, the following further describes the technical solutions of the present invention in combination with specific embodiments, but the present invention is not limited thereto.

[0020] The following examples are convenient for better understanding of the present invention, but do not limit the present invention. The experimental methods in the following examples are all conventional methods unless otherwise specified. The test materials used in the following examples are all purchased from conventional biochemical reagent stores unless otherwise specified. The determination of vector sequencing in the following examples is all determined by conventional sequencing companies.

[0021] The soybean cultivar Huachun 6 is the background material (wild type) of the present invention: Huachun 6 is a soybean cultivar selected by the College of Agriculture, South China Agricultural University, and is bred from the varieties Guizao 1 × Brazil 8; it was approved by the third meeting of the Second National Crop Variety Approval Committee on July 28, 2009, and the approval number is GS Soya Bean 2009012.

[0022] In the present invention, the nucleotide sequence of the soybean auxin transport carrier GmPIN3a gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2; the nucleotide sequence of the soybean auxin transport carrier GmPIN3b gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.4.

[0023] Example 1: Obtaining a gene-edited soybean homozygous double mutant without the auxin transport carriers Gmpin3ab and GmPIN3abd. Use the CRISPR-GE (http: / / skl.scau.edu.cn / home / ) online tool to design an sgRNA that simultaneously targets the soybean auxin transport carrier GmPIN3a and GmPIN3b genes: 5’-GTCTGATGCTGGTCTTGGAA-3’. The primer pair designed for constructing this sgRNA is: Forward primer: 5’-ggattGATTGTCTGATGCTGGTCTTGGAA-3’, Reverse primer: 5’-AAACAAACTTCCAAGACCAGCATCAGACCA-3’. Anneal the primer pair of the sgRNA to form double-stranded DNA, construct it into the pGES201 vector by the GoldenGate method, transform the competent cells of Escherichia coli DH5α, screen the positive clones, extract the plasmids and send them to the company for sequencing. The correctly sequenced vector is transformed into the recipient soybean by the cotyledon node infection method mediated by Agrobacterium tumefaciens GV3101 to obtain the Gmpin3ab double mutant plants with the GmPIN3a and GmPIN3b genes simultaneously knocked out.

[0024] Golden Gate reaction system (20 μL): 5 μL of pGES201 plasmid, 2 μL of T4 DNA Ligase Buffer (10X), 1 μL of T4 DNA ligase, 2 μL of BsaI-HFV, 1 μL of DNA fragment. Golden Gate reaction conditions: 37°C for 5 min, 16°C for 5 min, a total of 30 cycles, 37°C for 15 min, 85°C for 15 min.

[0025] As Figure 1As shown, the sgRNA sequence of the gene editing site of CRISPR-Cas9 targets the third exon positions of GmPIN3a and GmPIN3b, and the red base sequence is the target site. On the third exon of GmPIN3a, there is a single-base G insertion in Gmpin3ab; on the third exon of GmPIN3b, there is a single-base A insertion in Gmpin3ab.

[0026] Example 2: Method for Measuring Photosynthetic Rate of Wild-Type and Homozygous Double-Mutant Soybeans

[0027] In the early flowering stage of soybeans, select a clear and cloudless morning from 9:00 to 12:00 to measure the leaves of wild-type and homozygous Gmpin3ab double-mutant soybeans using a Li-6800 portable photosynthetic fluorescence measurement system (LI-COR, Lincoln, USA). Select a 3 cm x 3 cm red-blue light source, adjust the detection light intensity to be consistent with the ambient light intensity, and use a small CO2 cylinder to control the CO2 concentration at 400 μmol mol -1 . The measured leaves are the middle leaves of the third trifoliate leaves that are fully expanded in the plant, and 15 samples are taken for each material for detection. The measured parameters include net photosynthetic rate (Pn), transpiration rate (Tr), intercellular CO2 concentration (Ci), and stomatal conductance (Gs).

[0028] As Figure 2 shown, compared with wild-type soybeans, the net photosynthetic rate, stomatal conductance, and transpiration ratio of Gmpin3ab double-mutant soybean leaves are all significantly increased, and the intercellular CO2 concentration is significantly decreased.

[0029] Example 3: Method for Measuring Sucrose Content of Wild-Type and Homozygous Double-Mutant Soybeans

[0030] According to the method provided by the sucrose content kit of Herui Biology. Weigh 0.1 g of leaf samples of wild-type and homozygous Gmpin3ab double-mutant soybeans at the early stage of pod filling, grind them at room temperature, add 1 mL of extraction solution, transfer them quickly to a 1.5 mL centrifuge tube after proper grinding, place them in a water bath at 80 °C for 10 min, shake 3 - 5 times, after cooling, centrifuge at 4000 g and 25 °C for 10 min, take the supernatant, add 2 mg of reagent five, decolorize at 80 °C for 30 min, then add 1 mL of extraction solution, centrifuge at 4000 g and 25 °C for 10 min, take 100 μl of the supernatant to a new centrifuge tube, add reagent two, mix well, and boil in a water bath for about 5 min (cover tightly to prevent water loss). After cooling to room temperature, add reagent three and reagent four in sequence, mix well, and boil in a water bath for 30 min. Zero the ultraviolet spectrophotometer with distilled water at 480 nm, and measure after the sample has cooled.

[0031] As Figure 3As shown, compared with wild-type soybeans, the sucrose content in the leaves of Gmpin3ab double mutant soybeans is significantly increased.

[0032] Example 4: Method for Determining Oil Content of Wild-Type and Homozygous Double Mutant Soybeans

[0033] Seeds received from individual plants of wild-type and homozygous Gmpin3ab double mutant soybeans at the mature stage were randomly divided into eight equal parts, and the oil (i.e., crude fat) content was measured using a Matrix near-infrared grain analyzer produced by Bruker.

[0034] As Figure 4 shown, compared with wild-type soybeans, the oil content in the seeds of Gmpin3ab double mutant soybeans is significantly increased.

[0035] 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 Genes and GmPIN3b The application of genes in improving leguminous plant varieties is characterized by: Said GmPIN3a The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2; GmPIN3b The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.4; the legume variety improvement is to improve the photosynthetic capacity of legume leaves and / or to improve the sucrose content of legume leaves and / or to improve the oil content of legume seeds; the improvement of the photosynthetic capacity of legume leaves is to improve the net photosynthetic rate of legume leaves and / or to improve the stomatal conductance of legume leaves and / or to improve the transpiration rate of legume leaves and / or to reduce the intercellular CO2 concentration of legume leaves; The leguminous plant is soybean.

2. A method for improving the photosynthetic capacity of leguminous plant leaves and / or improving the sucrose content of leguminous plant leaves and / or improving the oil content of leguminous plant seeds, characterized in that: The steps include: knocking out the receptor legume GmPIN3a Genes and GmPIN3b Gene, or inhibitory receptor in leguminous plants GmPIN3a Genes and GmPIN3b Gene expression, or inhibition of receptors in leguminous plants GmPIN3a Genes and GmPIN3b The activity of the protein encoded by the gene; GmPIN3a The nucleotide sequence of the gene is shown in SEQ ID NO.1, and the amino acid sequence of the protein encoded by it is shown in SEQ ID NO.2; GmPIN3b The nucleotide sequence of the gene is shown in SEQ ID NO.3, and the amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.4; the improving the photosynthetic capacity of the leaves of the legume plant is to improve the net photosynthetic rate of the leaves of the legume plant and / or to improve the stomatal conductance of the leaves of the legume plant and / or to improve the transpiration rate of the leaves of the legume plant and / or to reduce the intercellular CO2 concentration of the leaves of the legume plant; The leguminous plant is soybean.

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