Soybean gmft4 gene, protein encoded by the gene and application thereof

Abstract

The application discloses a soybean GmFT4 gene, a coding protein and application thereof, and belongs to the field of plant biotechnology.The application solves the problem that relatively few genes capable of regulating soybean seed size are disclosed at present.The nucleotide sequence of the GmFT4 gene is shown in SEQ ID NO:1 in the sequence listing, and the amino acid sequence of the GmFT4 gene coding protein is shown in SEQ ID NO:2 in the sequence listing.Through overexpression of GmFT4 in soybean, it is found that the grain of soybean is larger and the hundred-grain weight is increased after overexpression of GmFT4.The GmFT4 gene can be used for biological breeding of soybean and its closely related species, and the grain size of soybean can be increased through the gene, so that the yield of crops is increased.

Description

Technical Field

[0001] This invention belongs to the field of plant biotechnology, specifically relating to a soybean gene, its encoded protein, and its applications. Background Technology

[0002] Soybean is an important food and economic crop, providing humans with essential plant protein and oil. Seed size is one of the main traits affecting soybean yield. The main factors influencing soybean yield include the number of pods per plant, the number of seeds per pod, and the weight of 100 seeds. Seed size determines seed weight, and thus soybean yield. Controlling soybean seed weight through molecular breeding and bio-breeding to cultivate high-yielding soybean varieties is an effective means to increase soybean yield. However, to date, the molecular mechanisms regulating soybean seed size have not been fully elucidated, the regulatory networks between various genes are still unclear, and relatively few genes reported that can regulate soybean seed size have been identified. Therefore, further exploration of genes related to soybean seed size is of great significance for a deeper understanding of the regulatory mechanisms of seed size and for improving soybean yield. This invention uses Dongnong 50, a small-seeded soybean with a 100-seed weight of 6-7g, as the research object, providing an effective means to regulate soybean seed size. Summary of the Invention

[0003] This invention provides a soybean GmFT4 gene, its encoded protein, and its applications, which solves the problem that there are relatively few publicly available genes that can regulate soybean seed size. At the same time, by overexpressing the GmFT4 gene in soybean plants, soybean seed size and grain weight are increased, thereby achieving the goal of increasing soybean yield.

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

[0005] The soybean GmFT4 gene, the nucleotide sequence of which is shown in SEQ ID NO.1 of the sequence listing.

[0006] The protein encoded by the soybean GmFT4 gene, the amino acid sequence of which is shown in SEQ ID NO.2 in the sequence listing.

[0007] Application of the soybean GmFT4 gene in regulating soybean seed size.

[0008] Application of the soybean GmFT4 gene in breeding improved soybean varieties.

[0009] A method for preparing genetically modified soybeans, the method comprising the following steps:

[0010] Step 1: Construct the GmFT4 gene overexpression vector pTF101.1:GmFT4;

[0011] Step 2: The constructed vector pTF101.1:GmFT4 was transferred into the recipient soybean genome to obtain transgenic soybean lines overexpressing the GmFT4 gene;

[0012] Step 3: Molecular identification of transgenic soybean lines overexpressing the GmFT4 gene was performed to obtain transgenic soybeans in which the GmFT4 gene was overexpressed.

[0013] Furthermore, the method for transferring the constructed vector pTF101.1:GmFT4 into the recipient soybean genome in step 2 is the Agrobacterium tumefaciens-mediated soybean cotyledon node method.

[0014] Furthermore, the recipient soybean variety in step 2 is Dongnong 50.

[0015] This invention discloses the soybean GmFT4 gene, its encoded protein, and its applications. By overexpressing the soybean GmFT4 gene in soybean, this invention has determined that the soybean GmFT4 gene regulates soybean seed size, increasing both seed size and grain weight, thus addressing the problem of relatively few currently disclosed genes capable of regulating soybean seed size. Therefore, the soybean GmFT4 gene can be applied in processes involving molecular or genetic engineering breeding to improve the yield of soybeans and their closely related species. Attached Figure Description

[0016] Figure 1 Semi-quantitative RT-PCR analysis of the GmFT4 gene in overexpressing transgenic soybean and non-transgenic soybean;

[0017] Figure 2 qPCR analysis of the GmFT4 gene in transgenic and non-transgenic soybeans overexpressing the gene;

[0018] Figure 3 Statistical analysis of seed size phenotypic changes and 100-seed weight data for GmFT4 overexpressing transgenic soybeans and non-transgenic soybeans. Detailed Implementation

[0019] The present invention will be further described in detail below with reference to the accompanying drawings. The embodiments described are only used to explain the present invention and are not intended to limit the scope of the present invention.

[0020] The nucleotide sequence of the soybean GmFT4 gene in this embodiment is shown in SEQ ID NO.1 of the sequence listing.

[0021] The amino acid sequence of the protein encoded by the soybean GmFT4 gene of this invention is shown in SEQ ID NO.2.

[0022] Unless otherwise specified, the following examples are all conventional methods.

[0023] Example 1: Functional verification of the soybean GmFT4 gene.

[0024] (I) Construction of GmFT4 gene overexpression vector.

[0025] Using cDNA from soybean leaves overexpressing E1 (in a Kariyutaka background) as a template, and GmFT4-F1 (Seq ID No. 3) and GmFT4-R1 (Seq ID No. 4) as primers, PCR amplification was performed using the high-fidelity enzyme FastPfu Fly DNA Polymeras purchased from Beijing TransGen Biotech Co., Ltd. PCR reaction conditions: 94℃ pre-denaturation for 5 min, (94℃ denaturation for 30 s, 58℃ annealing for 30 s, 72℃ extension for 60 s) × 6 cycles, 94℃ denaturation for 30 s, 68℃ annealing for 30 s, 72℃ extension for 60 s, 26 cycles, followed by a final extension at 72℃ for 5 min. The enzyme used was Promega. Gel recovery was performed using an SV Gel and PCR CleanUp System. The GmFT4 gene amplification band was obtained via PCR amplification, and an XbaI restriction enzyme site was introduced upstream of the GmFT4 gene, followed by a SacI restriction enzyme site downstream. The resulting PCR product and vector pTF101.1 were simultaneously digested with XbaI and SacI, ligated overnight with T4 DNA ligase, and transformed into competent *E. coli* cells. pTF101.1:GmFT4 vector contains the Bar selection marker gene (glufosinate resistance), and the GmFT4 gene is driven by a 35S promoter.

[0026] The constructed vector pTF101.1:GmFT4 was transferred into the genome of soybean variety Dongnong 50 via Agrobacterium tumefaciens-mediated transfer of cotyledonary nodes, resulting in transgenic soybean lines overexpressing the GmFT4 gene.

[0027] (II) Molecular identification of transgenic soybeans overexpressing the GmFT4 gene.

[0028] 1. Through genetic transformation of soybean, using a 160 mg / L glufosinate application experiment, two transformation events (35S:GmFT4#L1 and 35S:GmFT4#L2) were obtained in the T1 generation to develop glufosinate resistance. RNA was extracted from mature soybean leaves of these two transformation events. The RNA extraction method is described in the Invitrogen TRIzol kit manual.

[0029] 2. The total RNA extracted in step 1 was treated with DNase. The RNA content and quality of the treated total RNA were determined using NanoDrop, which was purchased from Toyobo.

[0030] 3. After obtaining 2 μg of total RNA from step 2 (NanoDrop assay) to meet the standard, it was collected using RNA purchased from Beijing TransGen Biotech Co., Ltd. Use the One-Step gDNA Removal and cDNA Synthesis SuperMix kit and the dT18 primers provided with the kit to synthesize cDNA according to the kit's instruction manual.

[0031] 4. Take 2 μl of cDNA diluted 5-fold in step 3 as an RT-PCR template. Use qGmFT4-F1 (Seq ID No. 5) and qGmFT4-R1 (Seq ID No. 6) as primers and perform PCR amplification using EasyTaq (purchased from Beijing TransGen Biotech Co., Ltd.). PCR reaction conditions: 94℃ pre-denaturation for 5 min, 94℃ denaturation for 30 s, 60℃ annealing for 30 s, 72℃ extension for 30 s, 27 cycles for the internal reference gene TUA5, 29 cycles for the GmFT4 gene, and a final extension at 72℃ for 5 min. The primer sequence for the soybean internal reference gene TUA5 is qTUA5 (Seq ID No. 7 and Seq ID No. 8), and the primer sequence for the internal reference gene TUA5 is the publicly available sequence from a published paper (Ruibo Hu, et al. 2009). Take 5 μl of the PCR product for agarose gel electrophoresis. The results are as follows. Figure 1 As shown, compared with the non-GMO soybean Dongnong 50, the expression level of the GmFT4 gene was significantly higher in both independent transformation events. This indicates that the GmFT4 gene was successfully overexpressed in soybean.

[0032] 5. Take 1 μl of cDNA diluted 30-fold in step 3 as a qPCR template. Use qGmFT4-F1 (Seq ID No. 5) and qGmFT4-R1 (Seq ID No. 6) as primers, and the primer sequence for the soybean internal reference gene TUA5 is qTUA5 (Seq ID No. 7 and Seq ID No. 8). Follow the TransStart Top Green qPCR Super Mix Kit instructions, using SYBR Green I as dye, and employ a three-step PCR reaction. qPCR reaction conditions: 95℃ pre-denaturation for 30 s, 95℃ denaturation for 5 s, 60℃ annealing for 15 s, 72℃ extension for 20 s, 40 cycles. Use the 2-ΔΔCT algorithm to calculate the relative expression level of the GmFT4 gene. The results are shown below. Figure 2 As shown, the expression level of the GmFT4 gene was significantly higher in soybeans than in non-GMO soybeans in both independent transformation events. This indicates that the GmFT4 gene was successfully overexpressed in soybeans.

[0033] (III) Phenotypic analysis of transgenic soybeans overexpressing the GmFT4 gene.

[0034] Transgenic soybeans overexpressing the GmFT4 gene (generation T2) and the non-transgenic control variety Dongnong 50 were sown in pots, with 10 plants per line, and placed under natural light (long day). The plant phenotypes were then investigated in detail. Results showed that the GmFT4-overexpressing transgenic soybeans had significantly larger seeds and a significantly increased 100-seed weight. The 100-seed weight of the transgenic soybeans was approximately 8.7 g, while that of the control variety Dongnong 50 was approximately 6.8 g. The 100-seed weight of the transgenic soybeans was approximately 1.9 g higher than that of the non-transgenic control variety Dongnong 50. Figure 3 ).

[0035] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the protection scope of the present invention.

Claims

1. Overexpression of soybean GmFT4 The application of genes in increasing soybean seed size is characterized by, The soybean GmFT4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

2. Overexpression of soybean GmFT4 The application of genes in breeding soybean varieties with larger seeds is characterized by, The soybean GmFT4 The nucleotide sequence of the gene is shown in SEQ ID NO.

1.

3. A method for preparing transgenic soybeans with increased seed size, characterized in that, The preparation method includes the following steps: Step 1: Constructing soybeans GmFT4 Gene overexpression vector pTF101.1: GmFT4 The soybeans GmFT4 The nucleotide sequence of the gene is shown in SEQ ID NO.1; Step 2: The constructed vector pTF101.1: GmFT4 Transplanted into the recipient soybean genome, obtaining GmFT4 Gene overexpression transgenic soybean lines; Step 3: Put GmFT4 Molecular identification of transgenic soybean lines with overexpressed genes was performed to obtain... GmFT4 Genetically modified soybeans in which the gene is overexpressed.

4. The preparation method according to claim 3, characterized in that, The vector pTF101.1 constructed in step 2: GmFT4 The method for transferring the soybean genome into the recipient soybean genome was Agrobacterium tumefaciens-mediated soybean cotyledonary nodes.

5. The preparation method according to claim 3, characterized in that, The recipient soybean variety in step 2 is Dongnong 50.

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