Application of GmDET2-1 protein and its encoding gene in increasing soybean seed oil content

Abstract

The present invention relates to the field of genetic engineering technology, and in particular to the use of the GmDET2-1 protein and its encoding gene in increasing the oil content of soybean seeds. The present invention is the first to discover that the soybean brassinosteroid synthase GmDET2-1 protein or its encoding gene can regulate the oil content of plant seeds. This has high practical application value in plant variety improvement or breeding.

Description

Technical Field

[0001] The present invention relates to the field of genetic engineering technology, in particular to application of GmDET2-1 protein and its encoding gene in improving the oil content of soybean seeds. 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] Soybean oil is a plant-based oil that, besides being used as food, is also used in industries such as energy, soaps, lubricants, and cosmetics. It also contains active ingredients such as fatty acids, phospholipids, and tocopherols, which have beneficial health effects on human health. While the metabolic pathways underlying oil biosynthesis are relatively well understood, their regulatory mechanisms remain largely unknown. While 186 QTLs associated with soybean oil content have been identified, only a few genes involved in soybean oil biosynthesis and regulation have been reported, including FAD2-1A, FAD2-1B, DGAT, LACS, AAPT1, Gmb ZIP123, and ACCase.

[0004] DET2 (DEETIOLATED2) is a key gene in the brassinosteroid (BR) biosynthesis pathway. Mutations in this gene result in plants that are BR-deficient. In darkness, det2 mutants exhibit dwarf stature, thin hypocotyls, anthocyanin accumulation, and excessive leaf expansion. In the light, det2 mutants are smaller and have greener leaves than wild-type plants. Cells in plant tissues (hypocotyls, cotyledons, and leaves) are smaller, and apical dominance and stamen fertility are reduced. det2 mutants also impair photoperiod responses, resulting in delayed flowering and a shortened circadian cycle for CAB (chlorophyll IIa / b-binding protein) gene expression. This leads to abnormal circadian gene expression and delayed leaf and chloroplast senescence. Our research demonstrates that overexpression of GmDET2-1 promotes seed size, increases 1000-seed weight, and improves yield. Our research findings have been granted a patent. Summary of the Invention

[0005] The present invention further studies the function of GmDET2-1 and finds that overexpression of the GmDET2-1 gene can not only increase seed size and yield, but also increase the oil content in soybean seeds.

[0006] First, the present invention provides the use of GmDET2-1 protein or its encoding gene, or biological materials containing its encoding gene in regulating plant seed oil content.

[0007] At the same time, the present invention also provides the use of GmDET2-1 protein or its encoding gene, or biological materials containing its encoding gene in breeding plants with different seed oil content.

[0008] The physiological processes of oil and protein synthesis in plant seeds are highly complex. In particular, 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 soybean brassinosteroid synthase GmDET2-1 in oil synthesis or accumulation in seeds during the late reproductive stages of plant growth remains unpredictable to those skilled in the art.

[0009] As a preferred embodiment of the present invention, the oil content of plant seeds is increased by promoting the expression of the GmDET2-1 gene; and / or the oil content of plant seeds is reduced by inhibiting the expression of the GmDET2-1 gene.

[0010] As a preferred embodiment of the present invention, the expression of the GmDET2-1 gene is promoted or inhibited by genetic engineering or gene editing technology.

[0011] As a preferred embodiment of the present invention, the GmDET2-1 protein has any one of the following amino acid sequences:

[0012] 1) the amino acid sequence shown in SEQ ID NO. 1; or

[0013] 2) The amino acid sequence of the amino acid sequence shown in SEQ ID NO.1 is obtained by replacing, deleting or inserting one or more amino acid residues, thereby obtaining an amino acid sequence having the same functional protein.

[0014] As a preferred embodiment of the present invention, the gene encoding the GmDET2-1 protein has any of the following nucleotide sequences:

[0015] (1) the nucleotide sequence shown in SEQ ID NO. 2, or

[0016] (2) A nucleotide sequence encoding a protein having the same function obtained by replacing, deleting or inserting one or more nucleotides of the nucleotide sequence shown in SEQ ID NO. 2;

[0017] (3) A nucleotide sequence that can hybridize with the nucleotide sequence shown in SEQ ID NO. 2 under stringent conditions.

[0018] As a preferred embodiment of the present invention, the biological material is an expression cassette, a vector, a host cell or a recombinant bacterium.

[0019] As a preferred embodiment of the present invention, the plant seeds are plant seeds of the genus Glycine max in the family Leguminosae, preferably soybean seeds.

[0020] Furthermore, the present invention also provides a method for preparing a transgenic plant with a high oil content in seeds, comprising: introducing or overexpressing a gene encoding the GmDET2-1 protein into the plant genome.

[0021] The present invention has found through experimental research that the transgenic plant seeds prepared using the above method have a significantly increased oil content of 0.9% to 3.06% compared to wild-type plant seeds. Therefore, the GmDET2-1 protein or its encoding gene can be used to improve plant quality.

[0022] In the specific implementation process, it includes but is not limited to applying biological materials containing genes encoding GmDET2-1 protein to prepare transgenic plants with high oil content seeds.

[0023] As a preferred embodiment of the present invention, the above method comprises introducing or overexpressing the nucleotide sequence shown in SEQ ID NO. 2 into the plant genome.

[0024] In a specific implementation process, when introducing or overexpressing the nucleotide sequence shown in SEQ ID NO. 2 into the plant genome, it is preferred to use a seed-specific promoter or the GmDET2-1 gene's own promoter.

[0025] Preferably, the transgenic plant is transgenic soybean.

[0026] The beneficial effects of the present invention are:

[0027] The present invention is the first to discover that soybean brassinolide synthase GmDET2-1 protein or its encoding gene can regulate the oil content of plant seeds, and has high practical application value in plant variety improvement or breeding. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 It is a schematic diagram of the structure of the cloning intermediate vector FU28 of Example 2 of the present invention.

[0029] Figure 2 It is a schematic diagram of the structure of the cloning intermediate vector FU76 of Example 2 of the present invention.

[0030] Figure 3 Schematic diagram of the structure of the plant expression vector pSoy2 of Example 3 of the present invention.

[0031] Figure 4 These are the results of determining the transcription level of the GmDET2-1 gene in transgenic soybeans overexpressing the soybean brassinolide synthase gene GmDET2-1 in Example 4 of the present invention; wherein, WT is the wild-type Walliams82, 29-8, 29-20, and 29-21 are transgenic soybeans overexpressing the GmDET2-1 gene via a seed-specific promoter, and 30-13, 30-22, and 30-26 are transgenic soybeans overexpressing the GmDET2-1 gene via its own promoter.

[0032] Figure 5 This is the result of measuring the oil content in transgenic soybean seeds using a near-infrared spectrometer in Example 5 of the present invention.

[0033] Figure 6 This is the result of measuring the protein content in transgenic soybean seeds using a near-infrared spectrometer in Example 6 of the present invention. DETAILED DESCRIPTION

[0034] To make the objectives, technical solutions, and advantages of the present invention more clear, the technical solutions of the present invention will be clearly and completely described below in conjunction with the accompanying drawings. Obviously, the embodiments described are only some of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts shall fall within the scope of protection of the present invention.

[0035] Where specific techniques or conditions are not specified in the examples, all methods were performed according to conventional methods, techniques or conditions described in literature in the field, or according to product specifications. Reagents and instruments used, for which the manufacturers are not specified, are conventional products that can be purchased through regular channels.

[0036] Example 1 Cloning of soybean brassinolide synthase gene GmDET2-1 The forward primer is shown in SEQ ID NO.3, and the reverse primer is shown in SEQ ID NO.4;

[0037] The GmDET2-1 gene was cloned and sequenced from Glycine max L. Tianlong 1 using the forward primer 5'-CCCAAGCTTATGATCCCAGAACACTACTC-3' and the reverse primer 5'-CCGGGGATCCATACAAGTAAGGAATAACAG-3'. The gene 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.

[0038] The PCR reaction program was as follows: pre-denaturation at 95°C for 5 min, 30 cycles of 94°C for 30 s, 55°C for 35 s, and 72°C for 1 min, and extension at 72°C for 10 min.

[0039] Example 2 Construction of Cloning Vector of Soybean Brassinolide Synthase Gene GmDET2-1

[0040] The PCR product obtained by amplification in Example 1 was directly cloned into Figure 1 The FU28 vector and PCR product were first digested with HindIII and BamHI, and the recovered products were ligated at 16°C. The ligated products were transformed into E. coli DH5α and amplified therein. Positive clones were screened and sequenced.

[0041] The promoter / seed promoter of the GmDET2-1 gene was also ligated to the Figure 2 On the intermediate carrier FU76 shown.

[0042] Example 3 Construction of a plant expression vector for the soybean brassinolide synthase gene GmDET2-1

[0043] The soybean brassinolide synthase gene GmDET2-1 obtained in Example 2, the cloning vector connected to the GmDET2-1 promoter, and the Figure 3 The plant expression vector pSoy2 shown above 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). This allowed the GmDET2-1 gene and its own promoter to be constructed on pSoy2. This was used to overexpress the soybean brassinolide synthase gene GmDET2-1 in plants and study its function. The plant expression vector overexpressing GmDET2-1 using a seed-specific promoter was constructed using the same method as above. Plant transformation was performed using Agrobacterium-mediated transformation. The selection marker in plants is Bar.

[0044] Example 4 Determination of the expression level of soybean brassinolide synthase gene GmDET2-1 in transgenic materials

[0045] Quantitative real-time RT-PCR was used to determine the expression level of the GmDET2-1 gene in wild-type and transgenic soybeans obtained in Example 3. Real-time RT-PCR was performed on an ABI StepOne instrument, and the fluorescence signal was detected using SYBR Green I. The reaction system was:

[0046]

[0047] The reaction parameters were a two-step method: 95°C for 10 seconds, hot start; 95°C for 5 seconds, 60°C for 1 minute, and 40 cycles. Gene expression was normalized and plotted using the gene chip data analysis software Genesis. GmDET2-1 was upregulated to varying degrees in the transgenic soybeans mentioned above, such as Figure 4 The primer sequences are shown in SEQ ID NO. 5 and SEQ ID NO. 6, wherein SEQ ID NO. 5 is the forward primer and SEQ ID NO. 6 is the reverse primer.

[0048] Example 5: Soybean brassinolide synthase gene GmDET2-1 increases oil content in seeds

[0049] 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)), three transgenic soybean lines overexpressing GmDET2-1 under the seed-specific promoter and three transgenic soybean lines overexpressing GmDET2-1 under the native promoter were obtained. The results of near-infrared spectroscopy are shown in Figure 2. Figure 5 It was shown that soybean transformation with the soybean brassinolide synthase gene GmDET2-1 resulted in a significant increase in the oil content in soybean seeds.

[0050] Example 6 Correlation between soybean brassinolide synthase gene GmDET2-1 and protein content in seeds

[0051] This example investigates the changes in protein content in the seeds of the aforementioned overexpressing transgenic material.

[0052] In this example, the protein content of the six overexpressed transgenic soybean lines obtained in Example 5 was determined using a near-infrared spectrometer. Figure 6 As shown, except for 30-22, the protein content of the other five transgenic materials decreased significantly.

[0053] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit it. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that they can still modify the technical solutions described in the aforementioned embodiments, or make equivalent replacements for some of the technical features therein. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the spirit and scope of the technical solutions of the various embodiments of the present invention.

Claims

1. Application of GmDET2-1 protein or its encoding gene, or biological materials containing its encoding gene, in regulating plant seed oil content, characterized in that: The oil content of plant seeds is increased by promoting the expression of the GmDET2-1 gene; or, the oil content of plant seeds is reduced by inhibiting the expression of the GmDET2-1 gene; the plant seeds are soybean seeds; the amino acid sequence of the GmDET2-1 protein is shown in SEQ ID NO.1; and the biological material is an expression cassette, a vector or a host cell.

2. Application of GmDET2-1 protein or its encoding gene, or biological materials containing its encoding gene in breeding plants with different seed oil content; the plant is soybean; the amino acid sequence of the GmDET2-1 protein is shown in SEQ ID NO.1; the biological material is an expression cassette, vector or host cell.

3. The use according to claim 1, characterized in that Promote or inhibit the expression of GmDET2-1 gene through genetic engineering or gene editing technology.

4. The use according to any one of claims 1 to 3, characterized in that The gene encoding the GmDET2-1 protein is shown in SEQ ID NO.2.

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