Application of GmGRF5-1 and its encoded protein in increasing the protein content of soybean seeds

Abstract

The present invention relates to the field of genetic engineering, and specifically to the use of GmGRF5-1 and its encoded protein in increasing the protein content of soybean seeds. The amino acid sequence of the soybean photosynthesis-related protein GmGRF5-1 provided by the present invention is shown in SEQ ID NO.1. The invention also provides a gene GmGRF5-1 encoding the above protein, whose nucleotide sequence is shown in SEQ ID NO.2. Overexpression of the gene GmGRF5-1 can increase the protein content of soybean seeds, and has potential application value in agricultural production.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering, in particular to the application of soybean photosynthesis-related genes and proteins encoded therein in improving soybean quality. Background Art

[0002] Transgenic technology can break down species boundaries, enabling targeted modification, recombination, and transfer of genes. This has played a significant role in achieving the coordinated improvement of yield, quality, and resistance, traits that are difficult to overcome with conventional breeding techniques. Targeted manipulation of key genes controlling soybean quality using transgenic technology can significantly accelerate the selection and breeding of high-quality soybean varieties, fostering a diverse range of soybean varieties tailored to different consumer needs. This is crucial for increasing the nutritional value of soybeans, ensuring the safety of soy foods, and improving public health.

[0003] As the strongest plant-based protein provider, soybeans provide a major source of protein for humans and livestock. Using genetic engineering to introduce protein-enhancing genes into commercial varieties will significantly increase the protein supply for livestock and humans worldwide. Even a one-percentage-point increase in soybean protein yields millions of tons of additional protein.

[0004] Soybean seed proteins can be divided into three categories based on their biological functions: storage proteins, structural proteins, and defense-related proteins. Storage proteins predominate and are one of the most important plant proteins for human consumption. Storage proteins are primarily composed of glycinins and conglycinins. To date, seven homologous genes encoding glycinins have been cloned and sequenced: Gy1, Gy2, Gy3, Gy4, Gy5, Gy6, and Gy7 (Chen Jinling, 2020). Reports on other genes regulating the function of soybean seed protein content are less common.

[0005] Growth-regulating factors (GRFs) play a crucial role in regulating plant growth and development. Current research on GRF functions is primarily focused on plants such as Arabidopsis, rice, and corn. We have previously applied for and obtained a patent for the application of GmGRF5-1 in regulating soybean photosynthesis and yield. Summary of the Invention

[0006] The present invention further studies GmGRF5-1 and finds that overexpression of the GmGRF5-1 gene can also increase the protein content of soybean seeds. Therefore, the present invention seeks to protect the application of the GmGRF5-1 gene in improving soybean quality.

[0007] In a first aspect, the present invention provides the use of soybean photosynthesis-related protein GmGRF5-1 in increasing seed protein content.

[0008] In the application provided by the present invention, the amino acid sequence of the soybean photosynthesis-related protein GmGRF5-1 is shown as SEQ ID NO.1.

[0009] In the application provided by the present invention, the nucleotide sequence of the soybean photosynthesis-related protein GmGRF5-1 gene is shown as SEQ ID NO.2.

[0010] Soybean photosynthesis occurs during the vegetative growth phase. The demarcation between vegetative and reproductive growth is typically marked by flower bud differentiation, with the period before this being considered vegetative and the period after this being considered reproductive. However, there is no strict boundary between these two phases. For a considerable period, these two phases occur simultaneously, with significant competition for nutrients.

[0011] The physiological processes of oil and protein synthesis in plant seeds are highly complex. Proteins, in particular, contain a variety of amino acids, most of which have distinct synthesis pathways involving numerous genes and enzymes. Oil composition and accumulation are influenced by the activities of multiple enzymes in the fatty acid synthesis pathway. The expression of these genes is also regulated at pre-, transcriptional, and post-transcriptional levels, with numerous related genes involved in this process. Therefore, the role of the photosynthesis-related protein GmGRF5-1 in seed protein synthesis or accumulation during the late reproductive stages of plant growth remains uncertain.

[0012] In a second aspect, the present invention seeks to protect the use of a biological material containing the gene shown in SEQ ID NO. 2 in increasing the protein content of seeds.

[0013] The present invention provides an application of a biological material in increasing the protein content of seeds, wherein the biological material is an expression cassette, a vector, a host cell or a host bacterium.

[0014] In the third aspect, the present invention requests protection of the use of the soybean GmGRF5-1 protein having an amino acid sequence as shown in SEQ ID NO.1 or a gene having a nucleotide sequence as shown in SEQ ID NO.2 or a biological material containing the gene shown in SEQ ID NO.2 in the preparation of transgenic plants, wherein the transgenic plants are transgenic plants having an increased protein content relative to wild-type plant seeds.

[0015] In a fourth aspect, the present invention provides a method for preparing a transgenic plant with high protein content in seeds, wherein the gene of the soybean GmGRF5-1 protein with the amino acid sequence shown in SEQ ID NO. 1 is introduced into the plant genome or overexpressed.

[0016] In the method provided by the present invention, a gene having a nucleotide sequence as shown in SEQ ID NO. 2 is introduced into or overexpressed in the plant genome.

[0017] In the method provided by the present invention, when the gene with the nucleotide sequence shown in SEQ ID NO. 2 is introduced into or overexpressed in the plant genome, the promoter used is 35S promoter, GmGRF5-1 gene promoter or CAB3 mesophyll cell specific promoter.

[0018] In the method provided by the present invention, the transgenic plant is transgenic soybean.

[0019] The beneficial effect of the present invention is that the present invention is the first to discover the application of soybean photosynthesis-related gene GmGRF5-1 and its encoded protein in increasing the protein content of soybean seeds.

[0020] The present invention increases the protein content of seeds by overexpressing the GmGRF5-1 gene in plants, and the protein content in the seeds can be increased by 1.36%-1.89%.

[0021] Therefore, the GmGRF5-1 gene and the protein it encodes are used to improve plant quality. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] Figure 1 The present invention compares the amino acid sequence of the protein encoded by the soybean photosynthesis-related gene GmGRF5-1 with the amino acid sequence of the protein encoded by the Arabidopsis GRF5 gene.

[0023] Figure 2 Schematic diagram of the structure of the vector pGWCm in Example 2 of the present invention.

[0024] Figure 3 It is a structural schematic diagram of the intermediate carrier Fu76 in Example 2 of the present invention.

[0025] Figure 4 Schematic diagram of the structure of the plant expression vector pSoy2 of Example 3 of the present invention.

[0026] Figure 5 The protein content in soybean seeds measured by near-infrared spectrometer in Example 4 of the present invention; TL represents Tianlong No. 1; WS82 represents Williams 82.

[0027] Figure 6 This is the result diagram of soybean seed oil content in Example 5; TL represents: Tianlong No. 1; WS82 represents: Williams 82. DETAILED DESCRIPTION

[0028] The following examples are intended to illustrate the present invention but are not intended to limit the scope of the present invention. Without departing from the spirit and substance of the present invention, modifications or substitutions made to the methods, steps or conditions of the present invention are intended to fall within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental materials, reagents, instruments, etc. used in the examples of the present invention are all commercially available; unless otherwise specified, all technical means in the examples of the present invention are conventional means well known to those skilled in the art.

[0030] Example 1 Cloning of soybean photosynthesis-related gene GmGRF5-1

[0031] The GmGRF5-1 gene was cloned and sequenced from soybean Tianlong 1 (Glycine max L. Tianlong 1) using the forward primer 5'-ATGATGAGTGCAAGTGCAAGAA-3' (SEQ ID NO. 3) and the reverse primer 5'-TCATTCATCGGTTTGGATTCTG-3' (SEQ ID NO. 4), respectively. The cds sequence is shown in SEQ ID NO. 2; the amino acid sequence of the protein encoded by it is shown in SEQ ID NO. 1.

[0032] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, 25 cycles of 94°C for 30 s, 55°C for 35 s, and 72°C for 1 min 30 s, and extension at 72°C for 10 min.

[0033] The protein sequence of the soybean photosynthesis-related gene GmGRF5-1 has a similarity of 42% with the amino acid sequence of the Arabidopsis GRF5 protein. Figure 1 .

[0034] Example 2 Cloning vector of soybean photosynthesis-related gene GmGRF5-1

[0035] The PCR product obtained from Example 1 was directly cloned into Figure 2 The pGWCm vector was first hydrolyzed with Ahd I endonuclease and the digested product was recovered using a gel recovery kit to obtain a T vector. The PCR product and the T vector were then ligated at 16°C. The ligated product was transformed into E. coli DH5α and amplified therein. Positive clones were screened and sequenced. The 35S promoter / GmGRF5-1 gene promoter / CAB3 promoter were also ligated to the following vector using enzyme digestion and ligation: Figure 3 On the intermediate carrier Fu76 shown.

[0036] Example 3 Plant expression vector of soybean photosynthesis-related gene GmGRF5-1

[0037] The cloning vector of soybean photosynthesis-related gene GmGRF5-1 obtained in Example 2 and the intermediate vector connecting 35S promoter / GmGRF5-1 promoter / CAB3 promoter were combined with Figure 4 The plant expression vector pSoy2 shown below was mixed in equal proportions and then subjected to an LR reaction (50 ng of each plasmid, 1 μl of LR enzyme, and HO added to a final volume of 5 μl. Mix thoroughly and react at 25°C for at least 6 hours). GmGRF5-1 and three promoters were constructed on pSoy2 for overexpression of the soybean photosynthesis-related gene GmGRF5-1 in plants to study its function. Plant transformation was performed using Agrobacterium-mediated transformation. The selection marker in plants is Bar.

[0038] Example 4: Soybean photosynthesis-related gene GmGRF5-1 increases seed protein content

[0039] Referring to the soybean transformation method of Wang Kan et al. (Paz, M., Wang, K. Soybean transformation and regeneration using half-seed explants. US Patent #7,473,822 (Issued January 6, 2009)), one 35S overexpressing transgenic soybean line, two autologous promoter overexpressing transgenic soybean lines GmGRF5-1-OG, and two CAB3 overexpressing transgenic soybean lines GmGRF5-1-OC were obtained. The results of near-infrared spectroscopy are shown in Figure 2. Figure 5 It was shown that transformation of soybean with the photosynthesis-related gene GmGRF5-1 resulted in a significant increase in the protein content in soybean seeds.

[0040] Example 5 Correlation between soybean photosynthesis-related gene GmGRF5-1 and seed oil content

[0041] This example investigates the changes in oil content in the seeds of the aforementioned overexpressing transgenic material.

[0042] In this example, the oil content of the three transgenic soybean lines overexpressing transgenic soybeans, 35S:GmGRF5-1:GFP, GmGRF5-1-OG, and GmGRF5-1-OC, obtained in Example 4 were determined.

[0043] From the test results, it was found that: Figure 6 As shown in the figure, the oil content of the three transgenic materials was significantly reduced.

[0044] Although the present invention has been described in detail above using general descriptions and specific embodiments, it will be apparent to those skilled in the art that modifications and improvements may be made based on the present invention. Therefore, such modifications and improvements, which do not depart from the spirit of the present invention, are intended to be within the scope of protection claimed herein. Sequence Listing <110> Institute of Crop Sciences, Chinese Academy of Agricultural Sciences <120> Application of GmGRF5-1 and its encoded protein in increasing the protein content of soybean seeds <130> KHP221113374.4 <160> 4 <170> SIPOSequenceListing 1.0 <210> 1 <211> 345 <212> PRT <213> Artificial Sequence <400> 1 Met Met Ser Ala Ser Ala Arg Asn Arg Ser Pro Phe Thr Gln Thr Gln 1 5 10 15 Trp Gln Glu Leu Glu His Gln Ala Leu Val Phe Lys Tyr Met Val Thr 20 25 30 Gly Thr Pro Ile Pro Pro Asp Leu Ile Tyr Ser Ile Lys Arg Ser Leu 35 40 45 Asp Thr Ser Ile Ser Ser Arg Leu Phe Pro His His Pro Ile Gly Trp 50 55 60 Gly Cys Phe Glu Met Gly Phe Gly Arg Lys Val Asp Pro Glu Pro Gly 65 70 75 80 Arg Cys Arg Arg Thr Asp Gly Lys Lys Trp Arg Cys Ser Lys Glu Ala 85 90 95 Tyr Pro Asp Ser Lys Tyr Cys Glu Arg His Met His Arg Gly Arg Asn 100 105 110 Arg Ser Arg Lys Pro Val Glu Val Ser Ser Ala Ile Ser Thr Ala Thr 115 120 125 Asn Thr Ser Gln Thr Ile Pro Ser Ser Tyr Thr Arg Asn Leu Ser Leu 130 135 140 Thr Asn Pro Asn Met Thr Pro Pro Ser Ser Phe Pro Phe Ser Pro Leu 145 150 155 160 Pro Ser Ser Met Pro Ile Glu Ser Gln Pro Phe Ser Gln Ser Tyr Gln 165 170 175 Asn Ser Ser Leu Asn Pro Phe Phe Tyr Ser Gln Ser Thr Ser Ser Arg 180 185 190 Pro Pro Asp Ala Asp Phe Pro Pro Gln Asp Ala Thr Thr His Gln Leu 195 200 205 Phe Met Asp Ser Gly Ser Tyr Ser His Asp Glu Lys Asn Tyr Arg His 210 215 220 Val His Gly Ile Arg Glu Asp Val Asp Glu Arg Ala Phe Phe Pro Glu 225 230 235 240 Ala Ser Gly Ser Ala Arg Ser Tyr Thr Glu Ser Tyr Gln Gln Leu Ser 245 250 255 Met Ser Ser Tyr Lys Ser Tyr Ser Asn Ser Asn Phe Gln Asn Ile Asn 260 265 270 Asp Ala Thr Thr Asn Pro Arg Gln Gln Glu Gln Gln Gln Gln Gln His 275 280 285 Cys Phe Val Leu Gly Thr Asp Phe Lys Ser Thr Arg Pro Thr Lys Glu 290 295 300 Lys Glu Ala Glu Thr Ala Thr Gly Gln Arg Pro Leu His Arg Phe Phe 305 310 315 320 Gly Glu Trp Pro Pro Lys Asn Thr Thr Asp Ser Trp Leu Asp Leu Ala 325 330 335 Ser Asn Ser Arg Ile Gln Thr Asp Glu 340 345 <210> 2 <211> 1038 <212> DNA <213> Artificial Sequence <400> 2 atgatgagtg caagtgcaag aaataggtct cctttcacgc aaactcagtg gcaagagctt 60 gagcatcaag ctcttgtttt taagtacatg gttacaggaa cacccatccc accagatctc 120 atctactcta ttaaagaag tctagacact tcaatttctt caaggctctt cccacatcat ccaattgggt ggggatgttt tgaaatggga tttggcagaa aagtagaccc agagccaggg 240 aggtgcagaa gacagatgg caagaatgg agatgctcaa aggaggcata tccagactcc aagtactgtg aaagacacat gcacagaggc agaaaccgtt caagaaagcc tgtggaagtt 360 tcttcagca tagcaccgc cacaaacacc tcccaaaca tcccatcttc ttatacccga aacctttcct tgaccaaccc caacatgaca ccaccctctt ccttcccttt ctctcctttg ccctcttcta tgcctattga gtcccaaccc ttttcccaat cctaccaaaa ctcttctctc 540 aatcccttct tctactccca atcaacctcc tctagacccc cagatgctga ttttccaccc 600 caagatgcca ccacccacca gctattcatg gactctgggt cttattcgca tgatgaaaag aattataggc atgttcatgg aatagagaa gatgtggatg agagagcttt cttcccagaa gcatcaggat cagctaggag ctacactgaa tcataccagc aactatcaat gagctcctac aagtcctatt caaactccaa ctttcagaac atcaatgatg ccaccaccaa cccaagacag caagagcagc aacaacaaca acactgcttt gttttgggga cagacttcaa atcaacaaga 900 ccaactaaag agaaagaagc tgagacagct acgggtcaga gaccccttca ccgtttcttt 960 ggggagtggc caccaaagaa cacaacagat tcatggctag atcttgcttc caactccaga 1020 atccaaaccg atgaatga 1038 <210> 3 <211> 22 <212> DNA <213> Artificial Sequence <400> 3 atgatgagtg caagtgcaag aa 22 <210> 4 <211> 22 <212> DNA <213> Artificial Sequence <400> 4 tcattcatcg gtttggattc tg 22

Claims

1. Application of overexpression of soybean photosynthesis-related protein GmGRF5-1 in increasing soybean seed protein content; The amino acid sequence of the soybean photosynthesis-related protein GmGRF5-1 is shown in SEQ ID NO.

1.

2. The use according to claim 1, characterized in that The nucleotide sequence of the soybean photosynthesis-related protein GmGRF5-1 gene is shown in SEQ ID NO.

2.

3. Application of biological materials containing the gene shown in SEQ ID NO. 2 in increasing the protein content of soybean seeds.

4. The use according to claim 3, characterized in that The biological material is an expression cassette, a vector, a host cell or a host bacterium.

5. Use of the soybean GmGRF5-1 protein with an amino acid sequence as shown in SEQ ID NO.1 or the gene with a nucleotide sequence as shown in SEQ ID NO.2, or a biological material containing the gene shown in SEQ ID NO.2 in the preparation of transgenic soybeans, wherein the transgenic soybeans have an increased protein content compared to wild-type soybean seeds.

6. A method for preparing transgenic soybeans with high protein content in seeds, characterized in that: The soybean GmGRF5-1 protein gene having the amino acid sequence shown in SEQ ID NO. 1 is introduced into or overexpressed in the soybean genome.

7. The method according to claim 6, characterized in that A gene with a nucleotide sequence as shown in SEQ ID NO. 2 is introduced or overexpressed into the soybean genome.

8. The method according to claim 7, characterized in that When the gene with the nucleotide sequence shown in SEQ ID NO. 2 is introduced into or overexpressed in the soybean genome, the promoter used is the 35S promoter, the GmGRF5-1 gene promoter itself, or the CAB3 mesophyll cell-specific promoter.

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