Soybean GmGA20ox4 Gene, Encoded Protein Thereof and Application

Abstract

The soybean GmGA20ox4 gene, its encoded protein and applications relate to the field of soybean genetic engineering, and particularly to a soybean GmGA20ox4 gene, its encoded protein and applications. The present invention provides a GmGA20ox4 gene encoding gibberellin 20 oxidase isolated from soybean. This gene regulates the content of bioactive gibberellins in soybean, affects the plant height of soybean, and can be used to achieve the breeding goal of semi-dwarf plant type in soybean. The CDS sequence of the soybean GmGA20ox4 gene is shown as SEQ ID NO: 1 in the sequence listing. The amino acid sequence of its encoded protein is shown as SEQ ID NO: 2. This gene participates in the regulation of the gibberellin biosynthesis process in soybean, affects the content of bioactive gibberellins in soybean, and ultimately affects the plant height of soybean. The present invention can be used to optimize the lodging resistance and density tolerance of soybean and achieve the breeding goal of semi-dwarf plant type in soybean.

Description

Technical Field

[0001] The present invention relates to the field of soybean genetic engineering, and particularly relates to a soybean GmGA20ox4 gene, its encoded protein, and applications thereof. Background Art

[0002] Gibberellin is a class of tetracyclic diterpenoid compounds and is one of the essential hormones in the process of plant growth and development. It plays an important role in regulating processes such as seed germination, stem elongation, floral transition, and the development of flowers and fruits. So far, more than 130 kinds of gibberellins have been identified, among which GA1, GA3, GA4, and GA7 are generally considered to have biological activity by the academic community. GA20ox is a key regulatory enzyme in the gibberellin biosynthesis pathway and acts as a rate-limiting enzyme in the final step of gibberellin biosynthesis to control the content of gibberellin.

[0003] Soybean is the most widely consumed food and cash crop. Plant height is one of the main traits affecting soybean yield and is closely related to the lodging resistance and yield of soybeans. The pods of soybeans are borne at the nodes, and the number of nodes determines the number of pods per plant, thus determining the yield of soybeans. Therefore, modifying the soybean plant type is an important strategy to increase soybean yield. Summary of the Invention

[0004] The present invention provides a GmGA20ox4 gene encoding gibberellin 20 oxidase isolated from soybeans. This gene regulates the content of bioactive gibberellin in soybeans, affects the plant height of soybeans, and can be used to achieve the breeding goal of a semi-dwarf soybean plant type.

[0005] The CDS sequence of the soybean GmGA20ox4 gene of the present invention is shown as SEQ ID NO: 1 in the sequence listing.

[0006] The amino acid sequence of the protein encoded by the soybean GmGA20ox4 gene of the present invention is shown as SEQ ID NO: 2.

[0007] The nucleotide sequence of the soybean GmGA20ox4 gene of the present invention is shown as SEQ ID NO: 3 in the sequence listing.

[0008] The present invention also provides the application of the soybean GmGA20ox4 gene in regulating the plant height of soybeans.

[0009] Furthermore, the soybean GmGA20ox4 gene can affect the content of gibberellin in soybeans.

[0010] Advantages of the Present Invention:

[0011] The present invention provides a GmGA20ox4 gene encoding gibberellin 20 oxidase derived from soybean, which participates in the regulation of the gibberellin biosynthesis process in soybean, affects the content of bioactive gibberellin in soybean, and further affects the elongation of stem cells in soybean plants, ultimately affecting the plant height of soybean.

[0012] The present invention can be used to optimize the lodging resistance and high-density planting ability of soybean, and achieve the breeding goal of semi-dwarf plant type of soybean. Brief Description of the Drawings

[0013] Figure 1 It is the whole plant phenotype of wild type and mutant at the 12th day after germination;

[0014] Figure 2 It is the length of each node of wild type and Gmga20ox4 mutant (**p-value < 0.01, ***p-value < 0.001, ****p-value < 0.0001);

[0015] Figure 3 It is the comparison of the number of branches of wild type and Gmga20ox4 mutant;

[0016] Figure 4 It is the comparison of the bottom pod height of wild type and Gmga20ox4 mutant (****p-value < 0.0001);

[0017] Figure 5 It is the comparison of the circumference of epicotyl of wild type and Gmga20ox4 mutant (****p-value < 0.0001);

[0018] Figure 6 It is the comparison of the circumference of the fourth node from bottom to top of wild type and Gmga20ox4 mutant (****p-value < 0.0001);

[0019] Figure 7 It is the comparison of the grain weight per plant of wild type and Gmga20ox4 mutant;

[0020] Figure 8 It is the comparison of 100-seed weight of wild type and Gmga20ox4 mutant (****p-value < 0.0001);

[0021] Figure 9 It is the epicotyl section of wild type and mutant plants at 9 days after germination;

[0022] Figure 10 It is the comparison of the content of bioactive gibberellin GA1 in the first internode of wild type and Gmga20ox4 mutant grown for 20 days (*p-value < 0.05; ***p-value < 0.001);

[0023] Figure 11 Comparison of bioactive gibberellin GA4 content in the first internode of wild type and Gmga20ox4 mutants grown for 20 days (*p-value < 0.05; ***p-value < 0.001). Detailed implementation manners

[0024] The following is a detailed description of the embodiments of the present invention. The following embodiments are implemented on the premise of the technical solution of the present invention, and detailed implementation schemes and specific operation processes are given. However, the protection scope of the present invention is not limited to the following embodiments.

[0025] Example 1: Isolation and structural analysis of GmGA20ox4 gene in soybean

[0026] (1) Isolation of the gene

[0027] mRNA was extracted from the young stems of the wild type soybean variety Henong 85, and using this as a template, the first strand of cDNA was synthesized with oligo(dT)17 as the primer. Then, using the first strand of this cDNA as the template, PCR amplification was carried out using the upstream primer F (5’-GGGTTCGAAATCGATGGATCCATGGCAATAGACTGCATAACAAG-3’) and the downstream primer R (5’-GTCCTAGGCTACGTAGGATCCTCAGTTACTTTTCCGGAGCC-3’), and a 1149bp cDNA fragment was obtained, which is the CDS sequence of the GmGA20ox4 gene. It was cloned into the myc-pBA vector and named myc-pBA-GmGA20ox4.

[0028] The CDS sequence of the GmGA20ox4 gene is shown as SEQ ID NO:1 in the sequence listing, with a total of 1149bp. It encodes a protein of 382 amino acids, and its amino acid sequence is shown as SEQ ID NO:2 in the sequence listing.

[0029] (2) Structural analysis of the gene

[0030] Using the young leaves of the wild type soybean variety Henong 85 as the material, DNA was extracted and used as a template to amplify the genomic fragment of GmGA20ox4. The DNA sequence of the GmGA20ox4 gene is shown as SEQ ID NO:3, with a total of 1616bp. It contains 2 introns and 3 exons. Among them, the first exon is 1 - 566bp, the second exon is 889 - 1210bp, and the third exon is 1356 - 1616bp; the first intron is 567 - 888bp, and the second intron is 1211 - 1355bp.

[0031] Example 2: Regulation of soybean plant height by the GmGA20ox4 gene in soybean

[0032] A related mutant library was obtained by gamma-ray mutagenesis of Henong 85, and a plant with a semi-dwarf phenotype was screened from it. After multiple generations of self-crossing to purify its background, it was crossed with the distant variety Dongnong 50. The ratio of wild-type phenotype plants to semi-dwarf mutant phenotype plants in the F2 generation after hybridization was in accordance with the segregation ratio of 3:1 through chi-square test. Therefore, it was considered that the semi-dwarf phenotype was controlled by a single recessive nuclear gene. Further, mutants and wild-type plants were collected for whole-genome sequencing and mapping verification, and the GmGA20ox4 gene was confirmed as the candidate gene, and the related semi-dwarf mutant was named the Gmga20ox4 mutant.

[0033] The plant heights of wild-type Henong 85 and the Gmga20ox4 mutant were compared. The whole-plant phenotypes of the wild-type and the mutant were recorded on the 12th day after seed germination. The results showed that after the GmGA20ox4 gene mutated, the height of the mutant plants was significantly shorter than that of the wild-type plants (as Figure 1 , the scale in the figure indicates 5 cm). The plant traits of fully mature wild-type Henong 85 plants and Gmga20ox4 mutant plants were measured (as Figures 2 - 8 , Figure 2 in which ■ represents WT, represents Gmga20ox4). In terms of plant height, except for the lengths of several top internodes that were not significantly different between the wild-type and the mutant, the lengths of the remaining internodes in the Gmga20ox4 mutant were significantly smaller than those in the wild-type, especially in the bottom several internodes, where the difference in internode length between the mutant and the wild-type was more obvious. In other aspects, there were no significant differences between the mutant and the wild-type in terms of the number of branches, grain weight per plant, etc.; the bottom pod height of the mutant was significantly shorter than that of the wild-type; the circumference of the epicotyl and the circumference of the fourth internode from the bottom up of the mutant were significantly longer than those of the wild-type, which also indicated that the stem diameter of the mutant plants was significantly larger than that of the wild-type; the 100-seed weight of the mutant plants was significantly lower than that of the wild-type, indicating that the seeds of the mutant plants were significantly smaller than those of the wild-type. According to Figure 9 the results of epicotyl cross-sections of the mutant and wild-type plants 9 days after germination in

[0034] Example 3: The GmGA20ox4 gene in soybean affects the content of bioactive gibberellins in plants

[0035] The first internode of wild-type Henong 85 and Gmga20ox4 mutants grown in the field for 20 days was collected to determine the gibberellin content. The content of biologically active gibberellins GA1 and GA4 in the mutants and wild types was detected by high-performance liquid chromatography. The GA1 content in the Gmga20ox4 mutant was significantly lower than that in the wild type (p-value<0.05), and the GA4 content in the Gmga20ox4 mutant was extremely significantly lower than that in the wild type (p-value<0.001) (e.g. Figure 10 and Figure 11 ). It can be seen that the dwarfing of the whole plant of the Gmga20ox4 mutant is due to the fact that the mutation of the GmGA20ox4 gene affects the gibberellin biosynthesis pathway, resulting in a decrease in the content of biologically active gibberellins in the plant, which in turn affects the elongation of the plant's stem cells, ultimately causing the dwarfing phenotype of the whole plant.

Claims

1. Application of soybean GmGA20ox4 gene in positively regulating soybean plant height, wherein the soybean GmGA20ox4 gene encodes a protein with the amino acid sequence shown in SEQ ID NO:

2.

2. The application according to claim 1, wherein: The soybean GmGA20ox4 gene can positively regulate the gibberellin content in soybeans.

Images