Soybean auxinamide synthase GH3 gene family and its application

Abstract

The present invention relates to the field of plant genetic engineering, and in particular to soybean auxin amide synthase GH3 Gene family and its application. The soybean auxin amide synthase GH3 Gene families include GH3.1 Gene, GH3.13 Gene, GH3.6 Genes and GH3.26 Gene, the GH3.1 Gene, GH3.13 Gene, GH3.6 Genes and GH3.26 The nucleotide sequences of the genes are shown in SEQ ID NO.1, SEQ ID NO.3, SEQ ID NO.5 and SEQ ID NO.7 respectively. GH3.1 Gene, GH3.13 Gene, GH3.6 Genes and GH3.26 When the genes were knocked out at the same time, the color of the nodule contents of the knockout plants was different from that of the recipient plants, the nodule nitrogenase activity was lower than that of the recipient plants, the seed size was larger than that of the recipient plants, the seed 100-grain weight was higher than that of the recipient plants, the seed protein content was higher than that of the recipient plants, and the seed fat content was lower than that of the recipient plants.

Description

Technical Field

[0001] The invention relates to the field of plant genetic engineering, and in particular to a soybean auxin amide synthase GH3 gene family and applications thereof. Background Art

[0002] The nitrogen in soybeans mainly comes from biological nitrogen fixation in nodules and nitrogen in fertilizers. Nitrogen is absorbed from the roots and reaches the aboveground part through the transport system for utilization. Nitrate and ammonium salts are the two main nitrogen sources that soybeans can utilize, and soybeans prefer nitrate. After absorbing nitrate nitrogen, plants transport it through vascular tissues, using the various members of the nitrogen transport protein family to collaboratively complete nitrogen absorption, transportation, and distribution. Nitrogen is an important component of amino acids in plants and is the main source of protein. The accumulation of nitrogen in soybean plants determines the protein content of the grains. Therefore, on the basis of reasonably improving the biological nitrogen fixation function of soybeans, improving the absorption and utilization of nitrogen and effectively improving the nitrogen fertilizer utilization efficiency of soybeans have become key issues in improving soybean quality. Summary of the invention

[0003] The purpose of the present invention is to provide a soybean auxin amide synthase GH3 gene family and application thereof.

[0004] The technical solution adopted by the present invention is as follows:

[0005] Application of soybean auxin amide synthase GH3 gene family in regulating the color of plant nodule contents and / or regulating plant nodule nitrogenase activity and / or regulating plant seed size and / or regulating plant seed 100-grain weight and / or regulating plant seed protein content and / or regulating plant seed fat content, wherein the soybean auxin amide synthase GH3 gene family includes GH3.1 gene, GH3.13 gene, GH3.6 gene and GH3.26 gene, the nucleotide sequence of the GH3.1 gene is shown in SEQ ID NO.1, the nucleotide sequence of the GH3.13 gene is shown in SEQ ID NO.3, the nucleotide sequence of the GH3.6 gene is shown in SEQ ID NO.5, and the nucleotide sequence of the GH3.26 gene is shown in SEQ ID NO.7;

[0006] Further, the amino acid sequence of the protein encoded by the GH3.1 gene is shown in SEQ ID NO.2, the amino acid sequence of the protein encoded by the GH3.13 gene is shown in SEQ ID NO.4, the amino acid sequence of the protein encoded by the GH3.6 gene is shown in SEQ ID NO.6, and the amino acid sequence of the protein encoded by the GH3.26 gene is shown in SEQ ID NO.8; further, a gene knockout method is used to cause base deletion in the GH3.1 gene, GH3.13 gene, GH3.6 gene and GH3.26 gene in the plant at the same time or to cause the protein to terminate prematurely, thereby being able to regulate the color of plant nodule contents and / or regulate the nitrogenase activity of plant nodules and / or regulate the size of plant seeds and / or regulate the 100-grain weight of plant seeds and / or regulate the protein content of plant seeds and / or regulate the fat content of plant seeds;

[0007] Furthermore, the plant is a dicotyledonous plant, the dicotyledonous plant is a leguminous plant, and the leguminous plant is soybean.

[0008] A method for changing the color of plant nodule contents and / or reducing plant nodule nitrogenase activity and / or increasing plant seed size and / or increasing plant seed 100-grain weight and / or increasing plant seed protein content and / or reducing plant seed fat content, comprising the step of simultaneously knocking out GH3.1 gene, GH3.13 gene, GH3.6 gene and GH3.26 gene in a recipient plant; the nucleotide sequence of the GH3.1 gene is shown in SEQ ID NO.1, the nucleotide sequence of the GH3.13 gene is shown in SEQ ID NO.3, the nucleotide sequence of the GH3.6 gene is shown in SEQ ID NO.5, and the nucleotide sequence of the GH3.26 gene is shown in SEQ ID NO.7;

[0009] Furthermore, the plant is a dicotyledonous plant, the dicotyledonous plant is a leguminous plant, and the leguminous plant is soybean.

[0010] A method for cultivating a gene knockout plant with changed color of nodule contents and / or reduced nodule nitrogenase activity and / or increased seed size and / or increased seed 100-grain weight and / or increased seed protein content and / or reduced seed fat content, comprising knocking out GH3.1 gene, GH3.13 gene, GH3.6 gene and GH3.26 gene in a recipient plant at the same time, so as to obtain a gene knockout plant with different color of nodule contents from that of the recipient plant and / or lower nodule nitrogenase activity and / or higher seed size and / or higher seed 100-grain weight and / or higher seed protein content and / or lower seed fat content than that of the recipient plant; the nucleotide sequence of the GH3.1 gene is shown in SEQ ID NO.1, the nucleotide sequence of the GH3.13 gene is shown in SEQ ID NO.3, the nucleotide sequence of the GH3.6 gene is shown in SEQ ID NO.5, and the nucleotide sequence of the GH3.26 gene is shown in SEQ ID NO.7;

[0011] Furthermore, the plant is a dicotyledonous plant, the dicotyledonous plant is a leguminous plant, and the leguminous plant is soybean.

[0012] The significant advantages of the present invention are:

[0013] The present invention created a soybean Gmgh3.1 / 13 / 6 / 26 quadruple mutant of the auxinamide synthase GH3 gene that lacks protein function, providing the necessary genetic material for in-depth understanding of the mechanism of auxin in regulating seed protein accumulation, and providing a good germplasm resource reserve and ideas for improving soybean seed quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 :Gene editing nucleotide sequence of the gene-edited soybean mutant of Gmgh3.1 / 13 / 6 / 26. The gene editing site sgRNA sequence of CRISPR-Cas9 targets the exon positions of GH3.1, GH3.13, GH3.6, and GH3.26. The nucleotides marked in blue are the editing status of the quadruple mutant Gmgh3.1 / 13 / 6 / 26.

[0015] Figure 2 :Partial phenotypes of wild-type soybean and transgenic soybean nodules. P-values ​​were tested by one-way ANOVA; ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0016] Figure 3:Partial phenotypes of wild-type soybean and transgenic soybean seeds. P-values ​​were tested by one-way ANOVA; ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001.

[0017] Figure 4 :Protein and fat content of wild-type soybean and transgenic soybean seeds. P-values ​​were tested by one-way ANOVA; ns, not significant, *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. DETAILED DESCRIPTION

[0018] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention. The experimental methods in the following examples are conventional methods unless otherwise specified. The experimental materials used in the following examples are purchased from conventional biochemical reagent stores unless otherwise specified. The determination of vector sequencing in the following examples is determined by sequencing by conventional sequencing companies.

[0019] The soybean cultivar Huachun 6 is the background material of the present invention: Huachun 6 is a soybean variety bred by the College of Agriculture of South China Agricultural University, using 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 Guoshendou 2009012.

[0020] The nucleotide sequence of the soybean GH3.1 gene of the present invention 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 soybean GH3.13 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 nucleotide sequence of the soybean GH3.6 gene is shown as SEQ ID NO.5, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.6; the nucleotide sequence of the soybean GH3.26 gene is shown as SEQ ID NO.7, and the amino acid sequence of the protein encoded by it is shown as SEQ ID NO.8.

[0021] Example 1: Method for obtaining a gene-edited soybean tetramer mutant of auxinamide synthase Gmgh3.1 / 13 / 6 / 26 with protein function loss

[0022] The sgRNA sequences targeting soybean GH3.1, GH3.13, GH3.6, and GH3.26 genes were designed using the CRISPR-GE (http: / / skl.scau.edu.cn / home / ) online tool. For soybean GH3.1 and GH3.13 genes, the sgRNA common to these two genes was selected: 5'-AAGGTTCCTTCAGCTCAATT-3'. The sgRNAs designed for soybean GH3.6 and GH3.26 genes were: 5'-ACACTGAAAGTGTCCAAGAG-3', 5'-TTTCATTGTGAAAGAATGCT-3'. The upstream and downstream primers of the sgRNA sequence were annealed to form double-stranded DNA, and the vector was constructed using the GoldenGate method. The purified double-stranded DNA fragment and vector pGES401 were added to the reaction system at the same time, and BsaI endonuclease and T4 DNA ligase were added to allow the enzyme to cut and ligate at the same time. Finally, DH5α Escherichia coli was transformed to screen positive clones, and plasmids were extracted to complete the construction of the knockout vector. The knockout vector was introduced into GV3101 Agrobacterium, and then Agrobacterium was used to infect the wild soybean cultivar Huachun No. 6, plant tissue culture was carried out, and the obtained tissue culture seedlings were screened for plant resistance, thereby obtaining a quadruple mutant Gmgh3.1 / 13 / 6 / 26 with simultaneous knockout of GH3.1, GH3.13, GH3.6 and GH3.26 genes. The gene-edited nucleotide sequence of the quadruple mutant Gmgh3.1 / 13 / 6 / 26 is shown in Figure 1 The gene editing site sgRNA sequence of CRISPR-Cas9 targets the exon positions of soybean GH3.1, GH3.13, GH3.6 and GH3.26 genes. The nucleotides marked in blue are the editing status of the quadruple mutant Gmgh3.1 / 13 / 6 / 26, and the nucleotides marked with ellipsis are the deletion status of the quadruple mutant Gmgh3.1 / 13 / 6 / 26 after editing.

[0023] Golden Gate reaction system (20 μL): pGES401 vector 5 μL, T4 DNA Ligase Buffer (10X) 2 μL, T4 DNA ligase 1 μL, BsaI-HFV2 2 μL, DNA fragment 1 μL. PCR reaction conditions: 37°C 5 min, 16°C 5 min, 30 cycles in total, 37°C 15 min, 85°C 15 min.

[0024] The primer pairs used to construct the above sgRNA guide sequences are as follows:

[0025] sgRNA1-F:

[0026] 5'-TGGTCTCGTGCAAAGGTTCCTTCAGCTCAATTGTTTTAGAGCTAGAAATAGC-3', sgRNA1-R: 5'-TGGTCTCGCACTTTCAGTGTTGCACCAGCCGGGAATCGAA-3';

[0027] sgRNA2-F: 5'-TGGTCTCGAGTGTCCAAGAGGTTTTAGAGCTAGAAATAGC-3',

[0028] sgRNA2-R:

[0029] 5'-TGGTCTCGAAACAGCATTCTTTCACAATGAAATGCACCAGCCGGGAATCGAA-3'; Example 2: Determination of nitrogenase activity in wild-type and soybean four-mutant homozygous plants

[0030] The nitrogenase activity was determined by the acetylene reduction method. The whole roots of the 14-day-old wild-type soybean cultivar Huachun No. 6 and the homozygous Gmgh3.1 / 13 / 6 / 26 quadruple mutant infected with the nodule nitrogen-fixing strain BXYD3 were cut with scissors (10-15 plants each) and placed in a 50mL measuring bottle. After the rubber stopper was plugged, 5ml of air in the bottle was extracted with a 10ml syringe, and the pinhole was immediately sealed with tape. Subsequently, 5ml of acetylene was injected into the bottle with a new syringe, and the pinhole was immediately sealed with tape. After reacting for 2 hours, the reaction was terminated with 5ml of 1M NaOH. Finally, 0.3ml of the gas in the bottle was extracted, and the nodule nitrogenase activity was measured with GC-2014.

[0031] like Figure 2 As shown, the contents of wild-type soybean nodules are red, while the contents of the quadruple mutant Gmgh3.1 / 13 / 6 / 26 nodules are white, and the nitrogenase activity in the nodules of the quadruple mutant Gmgh3.1 / 13 / 6 / 26 is significantly lower than that of wild-type soybean.

[0032] Example 3: Method for determining the protein and fat content in seeds of wild-type and homozygous soybean quadruple mutants. The sizes of all soybean seeds received from individual plants of the wild-type soybean cultivar Huachun No. 6 and the homozygous Gmgh3.1 / 13 / 6 / 26 quadruple mutant at maturity were compared, and their 100-grain weight was calculated. The seed protein (i.e., crude protein) and fat (i.e., crude fat) contents were determined using the Matrix near-infrared grain analyzer produced by Bruker.

[0033] like Figure 3 As shown in Figure 2, the seeds of the quadruple mutant Gmgh3.1 / 13 / 6 / 26 are larger than those of wild-type soybean, and the 100-grain weight is significantly increased. Figure 4As shown, compared with wild-type soybean, the seed protein content of the quadruple mutant Gmgh3.1 / 13 / 6 / 26 was significantly increased, and the fat content was significantly decreased.

Claims

1. Knockout GH3.1 Gene, GH3.13 Gene, GH3.6 Genes and GH3.26 The use of a gene for changing the color of plant nodule contents from red to white and / or reducing the nitrogenase activity of plant nodules and / or increasing the size of plant seeds and / or increasing the 100-grain weight of plant seeds and / or increasing the protein content of plant seeds and / or reducing the fat content of plant seeds, characterized in that: Said GH3.1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GH3.13 The nucleotide sequence of the gene is shown in SEQ ID NO.

3. GH3.6 The nucleotide sequence of the gene is shown in SEQ ID NO.

5. GH3.26 The nucleotide sequence of the gene is shown in SEQ ID NO.7; GH3.1 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

2. GH3.13 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

4. GH3.6 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.

6. GH3.26 The amino acid sequence of the protein encoded by the gene is shown in SEQ ID NO.8; The plant is a dicotyledonous plant, the dicotyledonous plant is a leguminous plant, and the leguminous plant is soybean.

2. A method for changing the color of plant nodule contents from red to white and / or reducing the nitrogenase activity of plant nodules and / or increasing the size of plant seeds and / or increasing the 100-grain weight of plant seeds and / or increasing the protein content of plant seeds and / or reducing the fat content of plant seeds, characterized in that: Including the recipient plant GH3.1 Gene, GH3.13 Gene, GH3.6 Genes and GH3.26 The step of simultaneously knocking out genes; GH3.1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GH3.13 The nucleotide sequence of the gene is shown in SEQ ID NO.

3. GH3.6 The nucleotide sequence of the gene is shown in SEQ ID NO.

5. GH3.26 The nucleotide sequence of the gene is shown in SEQ ID NO.7; The plant is a dicotyledonous plant, the dicotyledonous plant is a leguminous plant, and the leguminous plant is soybean.

3. A method for cultivating a gene knockout plant in which the color of nodule contents is changed from red to white and / or the nitrogenase activity of nodule is reduced and / or the seed size is increased and / or the 100-grain weight of seeds is increased and / or the seed protein content is increased and / or the seed fat content is reduced, characterized in that: In the recipient plant GH3.1 Gene, GH3.13 Gene, GH3.6 Genes and GH3.26 The genes are knocked out simultaneously, and a gene knockout plant is obtained whose root nodule contents are different in color from those of the recipient plant and / or whose root nodule nitrogenase activity is lower than that of the recipient plant and / or whose seed size is higher than that of the recipient plant and / or whose seed 100-grain weight is higher than that of the recipient plant and / or whose seed protein content is higher than that of the recipient plant and / or whose seed fat content is lower than that of the recipient plant; GH3.1 The nucleotide sequence of the gene is shown in SEQ ID NO.

1. GH3.13 The nucleotide sequence of the gene is shown in SEQ ID NO.

3. GH3.6 The nucleotide sequence of the gene is shown in SEQ ID NO.

5. GH3.26 The nucleotide sequence of the gene is shown in SEQ ID NO.7; The plant is a dicotyledonous plant, the dicotyledonous plant is a leguminous plant, and the leguminous plant is soybean.

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