Gene HOIL1 encoding soybean oil synthesis and application thereof

Abstract

The application discloses a soybean coding gene HOIL1 and application thereof in regulating soybean seed oil content. The protein provided by the application is as follows: (a), (b) or (c): (a) a protein consisting of the amino acid sequence shown in SEQ ID NO. 1; (b) a protein derived from SEQ ID NO. 1 by substitution, deletion and / or addition of one or more amino acid residues and related to plant seed oil content; and (c) a protein consisting of an amino acid sequence having at least 75% homology with the amino acid sequence shown in SEQ ID NO. 1 and related to plant seed oil content. The regulating protein and the coding gene thereof have important significance in improving plant seed oil content and cultivating high-oil plant varieties.

Description

Technical Field

[0001] This invention relates to the field of biotechnology and to the application of plant-encoding genes, particularly to the HOIL1 gene, which is related to soybean oil synthesis, and its application in soybean grain weight and oil content. Background Technology

[0002] Soybean (Glycine max L.Merr.) is one of the world's most important food and oilseed crops, possessing high economic value and providing essential vegetable oils and proteins for humans and animals. Simultaneously, soybeans are also a crucial raw material for various industrial products, and their increasingly widespread and diversified uses have led to a continuous growth in demand.

[0003] The oil content of soybean seeds determines its economic value as an oilseed crop. In research on soybean oil synthesis, QTL mapping has identified 186 genes related to oil synthesis, including functional genes and transcription factors that regulate gene expression. Among these, genes such as GmFATA1B, FAD2, DGAT, GmSDP1, AAPT1, GmbZIP123, and ACCase have been extensively explored and studied. Oil synthesis in plant seeds during development is synergistically regulated by multiple metabolic pathways. Current research focuses on three pathways: fatty acid synthesis, triglyceride (TAG) synthesis, and oil body synthesis. Several important genes regulating oil synthesis have been identified in soybeans, including GmMYB73, GmZF351, and GmWRI1. Soybean GmWRI1a and GmWRI1ab, along with their alternative splice products GmWRI1a' and GmWRI1b', are highly expressed in developing seeds and root nodules. GmWRI1s target 15 genes containing AW domains in their promoters, participating in pathways including glycolysis, fatty acid (FA), and TAG biosynthesis. Furthermore, GmZF351 can activate the expression of lipid biosynthesis-related genes GmBCCP2, GmKASIII, GmDGAT1, and GmOLEO2, enhancing the transcriptional activity of WRI1 and positively regulating lipid biosynthesis, thereby promoting oil accumulation. In the process of breeding superior soybean varieties, combining multiple superior genes related to yield and quality is more conducive to improving soybean yield and quality. The proteins and their encoding genes involved in this invention have broad application prospects in the breeding of high-oil soybean varieties. Summary of the Invention

[0004] This invention provides an application of the gene HOIL1, which is related to soybean oil synthesis.

[0005] The protein provided by this invention, with gene number Glyma.15g212200 and named HOIL1, is a coding gene derived from soybean (Glycine max (L.) Merrill). It represents the application of any one of the following substances (a), (b), or (c) in soybean grain weight and seed oil content:

[0006] (a) A protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0007] (b) Proteins derived from SEQ ID NO.1 with one or more amino acid residues substituted and / or deleted and / or added, and which are related to plant grain weight and oil content;

[0008] (c) A protein consisting of an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the amino acid sequence shown in SEQ ID NO.1 and relating to plant grain weight and oil content.

[0009] SEQ ID NO.1 consists of 340 amino acid residues.

[0010] The substitutions and / or deletions and / or additions in (b) above may be caused by natural variation or artificial mutagenesis.

[0011] The proteins mentioned in (a), (b) or (c) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0012] Furthermore, the gene HOIL1 encoding the protein is also within the scope of protection of this invention.

[0013] The gene may be a DNA molecule as follows (1) or (2) or (3):

[0014] (1) The DNA molecule shown in SEQ ID NO.2;

[0015] (2) DNA molecules that hybridize with the DNA sequence defined in (1) under strict conditions and encode proteins related to plant grain weight and oil content;

[0016] (3) A DNA molecule that has at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% homology with the DNA sequence defined in (1) and encodes a protein related to plant grain weight and oil content.

[0017] SEQ ID NO.2 consists of 1,020 nucleotides, all of which are coding sequences for the HOIL1 protein.

[0018] An overexpression vector containing the HOIL1 gene.

[0019] As a preferred embodiment of the present invention, the overexpression vector is obtained by inserting the gene HOIL1 between the BamHI and NcoI restriction sites after the CaMV 35S promoter using pFGC5941 (Purutin Biotechnology Co., Ltd.) as the original plasmid.

[0020] To facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as by adding genes that can be expressed in plants, encoding enzymes that produce color changes or luminescent compounds (GUS genes, luciferase genes, etc.), antibiotic resistance markers (gentamicin markers, kanamycin markers, etc.), or chemical reagent resistance marker genes (such as herbicide resistance genes). Examples include the nptII gene for resistance to kanamycin and related antibiotics, the bar gene for resistance to the herbicide phosphinic acid, the hph gene for resistance to the antibiotic hygromycin, the dhfr gene for resistance to methatrexate, the EPSPS gene for resistance to glyphosate, and the mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose.

[0021] Expression cassettes containing the HOIL1 gene, transgenic cell lines, or recombinant bacteria.

[0022] Primer pairs that amplify the full length of the gene or any fragment thereof are also within the scope of protection of this invention.

[0023] The primer pair may specifically be as follows (Ⅰ) or (Ⅱ):

[0024] (I) A primer pair consisting of the DNA shown in SEQ ID NO.3 and the DNA shown in SEQ ID NO.4;

[0025] (II) Primer pair consisting of DNA shown in SEQ ID NO.3 and DNA shown in SEQ ID NO.5.

[0026] This invention also protects a method for cultivating transgenic plants, which involves introducing an overexpression vector of the stated gene into a target plant to obtain a transgenic plant with a higher oil content than the target plant. The transgenic plant is understood not only to include the first-generation transgenic plant obtained by transforming the target plant with the overexpression vector of the stated gene, but also to include its progeny. For transgenic plants, the gene can be propagated within the species, or it can be transferred into other varieties of the same species using conventional breeding techniques, particularly commercial varieties. Introducing the gene overexpression vector into the target plant inhibits the synthesis of the target protein in the plant, thereby improving the grain weight and oil content traits of the target plant.

[0027] The gene can be introduced into the target plant via the recombinant vector. The recombinant vector carrying the gene can be used to transform plant cells or tissues using conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrocoagulation, and Agrobacterium-mediated transformation, and the transformed plant tissues can be cultured into plants. The target plant can be either a monocotyledonous or dicotyledonous plant. The target plant can be a legume (such as soybean, birdsfoot, alfalfa, and water clover), an oilseed crop (such as rapeseed, sunflower, and corn), or an oilseed tree species. The dicotyledonous plant can be Arabidopsis thaliana, such as the Colombian ecotype Arabidopsis thaliana.

[0028] The application of the gene HOIL1 described in this invention in the synergistic regulation of soybean grain weight and oil content shows that overexpression of this gene can significantly increase the oil content in soybean seeds, thereby effectively improving the economic benefits of soybean crops.

[0029] The present invention relates to the application of the HOIL1 gene overexpression vector, expression cassette, transgenic cell line, or recombinant bacteria in increasing soybean grain weight and oil content. The oil content refers to the total oil content within the seed.

[0030] Beneficial effects:

[0031] This invention provides a gene encoding HOIL1 that is related to the oil content of plant tissues. The coding sequence of this gene was amplified using specific primers, and the specific sequence was transferred into Williams82 (Wm82) soybean using an overexpression vector to obtain transgenic soybean lines. Compared with the wild-type control, the oil content in the seeds of the transgenic soybean lines was significantly increased.

[0032] Experimental results showed that in small-scale field replicates, overexpression of the HOIL1 gene significantly increased the oil content of soybean seeds, with an average seed oil content of 22.770%; compared with the wild-type control, the oil content increased by approximately 9.57%. The protein and its encoding gene involved in this invention have broad application prospects in the cultivation of high-oil-content plants.

[0033] The present invention will be further described in detail below with reference to specific embodiments. Attached Figure Description

[0034] Figure 1 The coding nucleic acid sequence of the gene HOIL1 was amplified. Lane 5 represents the amplified HOIL1 fragment.

[0035] Figure 2 Expression levels of gene HOIL1 in different soybean tissues.

[0036] Figure 3 Schematic diagram of the plant overexpression vector pFGC5941-Glyma.15g212200.

[0037] Figure 4 Molecular identification of the soybean overexpression transgenic line HOIL1-1 / 2.

[0038] Figure 5 The plant types of the control line and the overexpressing transgenic line HOIL1-1 / 2.

[0039] Figure 6 Differences in seed oil content between the control line and the overexpression line HOIL1-1 / 2. Detailed Implementation

[0040] The following examples are provided to help better understand the present invention, but are not intended to limit the invention.

[0041] Unless otherwise specified, the experimental methods described in the following examples are conventional methods.

[0042] Unless otherwise specified, all experimental materials used in the following examples were commercially available. All primers used were synthesized by Beijing Qingke Biotechnology Co., Ltd.; the transgenic transformation process was performed by Jiangsu Weimi Biotechnology Co., Ltd. Unless otherwise specified, all percentages in the following examples refer to mass percentages. T2 generation represents seeds produced by self-pollination of T1 generation and the plants grown from them; T3 generation represents seeds produced by self-pollination of T2 generation and the plants grown from them.

[0043] The experimental materials used in the following examples were: Williams 82 (Wm82) soybean seeds, purchased from Jiangsu Weimi Biotechnology Co., Ltd.; the Agrobacterium transformation and transgenic experimental procedures were all completed by Jiangsu Weimi Biotechnology Co., Ltd.

[0044] Example 1: Cloning of the soybean-encoding gene HOIL1

[0045] I. Designing specific primers for cloning genes

[0046] The start position of the gene Glyma.15g212200 on the genome was found on the SoyBase website as Gm15:33871825-33887986, and the reference genome sequence of this gene was obtained. To amplify the coding region sequence of this gene, primers were designed at the start and stop points, with the sequences as follows:

[0047] HOIL1-F1:5'-ATGTCGCAGCAGGGCACA-3';

[0048] HOIL1-R1:5'-CTAGCGTGCATTACGGTTAAGCAATC-3';

[0049] II. Coding sequence of the HOIL1 cloning gene

[0050] RNA was extracted from soybean Williams 82 leaves using the TriZol method and reverse transcribed to obtain the cDNA sequence of the gene. Using the cDNA as a template, the coding sequence of the gene was amplified using primers HOIL1-F1 / R1 (SEQ ID NO.3 and SEQ ID NO.4). Agarose gel electrophoresis was used to detect whether the amplified gene fragment conformed to the sequence size (…). Figure 1 The amplified sequence was used for subsequent experiments. The amplified sequence was sent to the company (Qingke, Beijing, China) for Sanger sequencing to verify the correctness of the cloned sequence. Its nucleotide sequence is shown in SEQ ID NO.2, and the encoded protein amino acid sequence is shown in SEQ ID NO.1.

[0051] Example 2: Tissue expression characteristics of the soybean coding gene HOIL1

[0052] Analysis of transcriptome library data from different tissues of soybean variety Williams 82 revealed differences in the expression level of the gene HOIL1 across various tissues. Figure 2 Comparative analysis revealed that HOIL1 expression levels were relatively high in organs related to fruit setting, such as flowers, pods, and seeds, while expression levels were relatively low in organs related to vegetative growth, such as leaves. Tissue expression level analysis showed that the gene was expressed in all tissues, with higher expression levels in pods and seeds. This suggests that the gene is involved in many plant developmental processes, including soybean seed development. Therefore, a correlation study was conducted between this gene and seed oil content phenotypes.

[0053] Example 3: Construction of HOIL1 plant overexpression vector

[0054] 1. RNA was extracted from the leaves of soybean variety Williams 82 and reverse transcribed into cDNA.

[0055] 2. Design specific primer pairs containing BamHI and NcoI linker sequences as follows:

[0056] HOIL1-F2: 5'-tacatttacaattaccatggATGTCGCAGCAGGGCACA-3';

[0057] HOIL1-R2: 5'-ctctagactcacctaggatccCTAGCGTGCATTACGGTTAAGCAATC-3'.

[0058] 3. Using the cDNA from step 1 as a template, perform PCR with the specific primer pair from step 2, and recover the PCR product.

[0059] 4. Digest the PCR product with restriction endonucleases BamHI and NcoI, and recover the digested product.

[0060] 5. The pFGC5941 vector was digested with restriction endonucleases BamHI and NcoI, and the vector backbone was recovered.

[0061] 6. Ligate the enzyme digestion product from step 4 with the vector backbone from step 5 to obtain the ligation product.

[0062] 7. The ligation product was sequenced, and the results showed that the recombinant expression vector pFGC5941-Glyma.15g212200 was obtained (the original plasmid was pFGC5941, Puruting Biotechnology Co., Ltd., with the DNA shown in SEQ ID NO.2 inserted between the BamHI and NcoI restriction sites after the CaMV 35S promoter). The recombinant expression vector pFGC5941-Glyma.15g212200... Figure 3 As shown.

[0063] Example 4: Obtaining and identifying HOIL1 transgenic soybeans

[0064] I. Identification of Transformed Soybeans and Genetically Modified Plants

[0065] 1. The recombinant expression vector pFGC5941-Glyma.15g212200 was introduced into Agrobacterium EHA101 to obtain recombinant Agrobacterium.

[0066] 2. By infecting the axillary meristem of the cotyledonary node with Agrobacterium, recombinant Agrobacterium was transferred into Williams 82 soybean. After the growth stabilized, it was transferred to nutrient soil for cultivation. At the same time, the transformation efficiency was identified, and positive T0 plants were harvested and propagated.

[0067] 3. Harvest T1 generation individual plants, sow seeds from each individual plant separately, and continue screening to observe the segregation of T2 generation. Repeat this process until T3 generation to obtain the genetically stable homozygous overexpression line HOIL1-1 / 2.

[0068] II. Molecular Identification of HOIL1 Transgenic Plants

[0069] 1. Molecular identification of HOIL1 transgenic soybean overexpression lines (HOIL1-1 / 2)

[0070] RNA was extracted from leaves of T3 homozygous lines (HOIL1-1 / 2) and control plants at stage V2, and reverse transcribed into cDNA. Using the cDNA as a template, RT-PCR was performed using primer pairs composed of HOIL1-F1 and HOIL1-R3 for identification. Results are shown below. Figure 4 The primer sequences used are as follows:

[0071] HOIL1-F1: 5'-ATGTCGCAGCAGGGCACA-3';

[0072] HOIL1-R3: 5'-TGTTGAGGGTGGAGTCGGATT-3';

[0073] III. Phenotypic Analysis of HOIL1 Transgenic Soybeans

[0074] The experimental samples are as follows: T3 generation soybean overexpression line transgenic with HOIL1 gene (HOIL1-1 / 2) and control line Williams 82.

[0075] 1. Post-harvest processing of field materials

[0076] When both transgenic and control lines were planted in the same field environment, no significant differences were observed in plant type, number of branches, and other agronomic traits between the transgenic and control lines. Figure 5 These materials were harvested individually and placed in an oven at 45°C for 48 hours to dry completely.

[0077] Seed oil content determination

[0078] Oil content refers to the percentage of total fatty acids by mass in the seed.

[0079] The method for determining oil content is as follows: After thorough grinding of the seeds, weigh 100 mg, add 500 μL of isopropanol solution, mix thoroughly, and centrifuge overnight at 37 degrees Celsius; centrifuge at 3000 rpm for 3 minutes, and collect the supernatant into a new centrifuge tube (pre-weigh the centrifuge tube weight W0); add another 500 μL of isopropanol to the remaining powder, mix thoroughly, centrifuge at 3000 rpm for 3 minutes, collect the supernatant into the same centrifuge tube, and then place the centrifuge tube in a fume hood (24 h) to allow the isopropanol to completely evaporate; finally, weigh the centrifuge tube again (W1). The change in weight of the centrifuge tube before and after centrifugation is the weight of extracted lipids (W1-W0).

[0080] 3. Phenotypic analysis of soybeans transgenic with the HOIL1 gene

[0081] Analysis of phenotypic data from control lines and transgenic soybean materials revealed that the average seed oil content of HOIL1-overexpressing transgenic lines reached 22.770%, significantly increasing by approximately 9.57% compared to the control lines. This validates the biological function of this gene in regulating soybean seed oil content. Figure 6 According to the quality indicators of high-oil soybeans in the national standard for soybeans (GB1352-2009), a crude fat content of ≥22.0% is considered as Grade 1 high-oil soybean quality, indicating that the HOIL1 overexpression transgenic line has Grade 1 high-oil quality.

Claims

1. Genes HOIL1 Its application in regulating the oil content of soybean seeds is characterized by... Overexpression of this gene increases the oil content of soybean seeds. HOIL1 The nucleotide sequence is shown in SEQ ID NO.

2.

2. Containing the gene as described in claim 1 HOIL1 Application of overexpression vectors in increasing the oil content of soybean seeds.

3. Containing the gene described in claim 1 HOIL1 The application of expression cassettes, transgenic cell lines, or recombinant bacteria in increasing the oil content of soybean seeds.

4. A method for cultivating transgenic soybeans, characterized in that... The gene described in claim 1 HOIL1 The overexpression vector was introduced into the target plant to obtain a transgenic plant with a higher oil content than the target plant.

Images