Coding gene HOIL2 related to soybean oil synthesis and application thereof

Abstract

The invention discloses a soybean coding gene HOIL2 and application thereof in regulating and controlling the oil content of soybean seeds. The protein provided by the invention is the following (a), (b) or (c): (a) a protein consisting of an amino acid sequence as shown in SEQ ID NO.1; (b) a protein which is obtained by substituting and / or deleting and / or adding one or more amino acid residues of the amino acid sequence as shown in the SEQ ID NO.1, is related to the oil content of the plant seeds and is derived from the SEQ ID NO.1; and (c) a protein which is composed of an amino acid sequence having at least 75% homology with the amino acid sequence as shown in SEQ ID NO.1 and is related to the oil content of the plant seeds. The regulatory protein and the coding gene of the regulatory protein disclosed by the invention have important significance in improving the oil content of plant seeds and cultivating high-oil plant varieties.

Description

Technical Field

[0001] The present invention relates to the field of biotechnology and is related to the application of plant coding genes, in particular to soybean coding gene HOIL2 and its application in regulating soybean seed oil content and the like. Background Art

[0002] 71% of the oil in human diet comes from plants. Among the major oil-producing crops in the world, soybean accounts for about 30% of the total oil production, ranking first in the world's vegetable oil production.

[0003] The oil content of soybean seeds determines the economic value of its oil crops. The synthesis of oil in plant seeds during development is regulated by multiple metabolic pathways. Current research focuses on the three metabolic pathways of fatty acid, triacylglycerol (TAG) and oil body synthesis. At present, several important genes related to regulating oil synthesis have been identified in soybean, including GmMYB73, GmZF351 and GmWRI1. Soybean GmWRI1a and GmWRI1ab and their alternative splicing products GmWRI1a' and GmWRI1b' are highly expressed in developing seeds and nodules. GmWRI1s targets 15 genes containing AW domains in their promoters and participates in pathways including glycolysis, fatty acid (FA) and TAG biosynthesis. In addition, GmZF351 can activate the expression of lipid biosynthesis-related genes GmBCCP2, GmKASIII, GmDGAT1 and GmOLEO2, enhance the transcriptional activity of WRI1, positively regulate lipid biosynthesis and promote oil accumulation. The protein and its encoding gene involved in the present invention will have broad application prospects in the breeding of high-oil soybean varieties. Summary of the invention

[0004] The purpose of the present invention is to provide an application of a coding gene HOIL2 related to soybean oil synthesis.

[0005] The purpose of the present invention can be achieved through the following technical solutions:

[0006] The protein provided by the present invention has a gene number of Glyma.18G013300 and is named HOIL2. It is a coding gene derived from soybean (Glycine max (L.) Merrill), and is the application of any of the following (a), (b) or (c) in soybean grain weight and seed oil content:

[0007] (a) a protein consisting of the amino acid sequence shown in SEQ ID NO.1;

[0008] (b) a protein derived from SEQ ID NO.1, wherein the amino acid sequence shown in SEQ ID NO.1 is substituted and / or deleted and / or added with one or more amino acid residues and is associated with plant grain weight and oil content;

[0009] (c) A protein consisting of an amino acid sequence that is 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% homologous to the amino acid sequence of SEQ ID NO. 1 and that is associated with plant grain weight and oil content.

[0010] SEQ ID NO. 1 consists of 545 amino acid residues.

[0011] The substitution and / or deletion and / or addition in (b) above may be caused by natural variation or artificial mutagenesis.

[0012] The proteins in (a), (b) or (c) above can be synthesized artificially, or their encoding genes can be synthesized first and then expressed biologically.

[0013] At the same time, the gene HOIL2 encoding the protein also falls within the protection scope of the present invention.

[0014] The gene may be a DNA molecule as follows (1) or (2) or (3):

[0015] (1) DNA molecule shown in SEQ ID NO.2;

[0016] (2) a DNA molecule that hybridizes with the DNA sequence defined in (1) under stringent conditions and encodes a protein related to plant grain weight and oil content;

[0017] (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 to the DNA sequence defined in (1) and encodes a protein related to plant grain weight and oil content.

[0018] SEQ ID NO. 2 consists of 1,635 nucleotides, all of which are the coding sequence of HOIL2 protein.

[0019] In addition, an overexpression vector containing the gene, a knockout vector for the gene, an expression cassette, a transgenic cell line or a recombinant bacterium all fall within the protection scope of the present invention.

[0020] The overexpression vector is obtained by using pFGC5941 as the original plasmid and inserting the gene HOIL2 according to claim 3 or 4 between the BamHI and NcoI restriction sites after the CaMV 35S promoter.

[0021] In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vector used can be processed, such as adding genes that can be expressed in plants and encode enzymes or luminescent compounds that can produce color changes (GUS gene, luciferase gene, etc.), antibiotic markers with resistance (gentamicin marker, kanamycin marker, etc.) or chemical agent resistance marker genes (such as herbicide resistance genes). For example, the nptII gene that confers resistance to kanamycin and related antibiotics, the bar gene that confers resistance to the herbicide phosphinothricin, the hph gene that confers resistance to the antibiotic hygromycin, the dhfr gene that confers resistance to methatrexate, the EPSPS gene that confers resistance to glyphosate, and the mannose-6-phosphate isomerase gene that provides the ability to metabolize mannose.

[0022] Primer pairs for amplifying the full length of the gene or any fragment thereof also fall within the scope of protection of the present 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) A primer pair consisting of the DNA shown in SEQ ID NO.3 and the DNA shown in SEQ ID NO.5.

[0026] The present invention also protects a method for cultivating transgenic plants, which is to introduce the editing vector of the gene into the target plant to obtain a transgenic plant with an oil content higher than that of the target plant. The transgenic plant is understood to include not only the first generation of transgenic plants obtained by transforming the target plant with the editing vector of the gene, but also its progeny. For transgenic plants, the gene can be propagated in the species, and the gene can also be transferred into other varieties of the same species using conventional breeding techniques, especially including commercial varieties. Introducing the gene editing vector of the gene into the target plant will inhibit 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 specifically introduced into the target plant through the recombinant vector. The recombinant vector carrying the gene can transform plant cells or tissues by conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, electroporation, Agrobacterium-mediated, and the transformed plant tissues are cultivated into plants. The target plant can be either a monocot or a dicot. The target plant can be a leguminous plant (such as soybean, Lotus japonica, alfalfa, Pongamia chinensis, etc.) or an oil crop (such as rape, sunflower, corn, etc.) or an oil tree species, etc. The dicot can be Arabidopsis thaliana, such as the Columbia ecotype Arabidopsis thaliana.

[0028] The application of the gene HOIL2 in regulating the oil content of soybean seeds is characterized in that overexpression of the gene can significantly increase the oil content in soybean seeds and effectively improve the economic benefits of soybean crops.

[0029] The overexpression vector, expression cassette, transgenic cell line or recombinant bacteria of the gene HOIL2 of the present invention are used to increase the weight and oil content of soybean grains. The oil content is the total oil content in the seeds.

[0030] Beneficial effects:

[0031] The present invention provides a coding gene HOIL2 related to the oil content of plant tissues. The present invention amplifies the coding sequence of the gene by using specific primers, and uses an overexpression vector to transfer the specific sequence into Williams 82 (Wm82) soybeans to obtain a transgenic soybean strain; compared with the wild-type control, the oil content in the seeds of the transgenic soybean strain is significantly increased.

[0032] The experimental results show that in a small-scale repeated field test, overexpression of the HOIL2 gene can significantly increase the oil content of soybean seeds, with an average seed oil content of 22.512%; compared with the wild-type control, the oil content increased by about 8.32%. The protein and its encoding gene involved in the present invention will have broad application prospects in the cultivation of high-oil plants.

[0033] The present invention is further described in detail below with reference to specific embodiments. BRIEF DESCRIPTION OF THE DRAWINGS

[0034] Figure 1 . Amplification of the coding nucleic acid sequence of gene HOIL2. Lane 7 represents the amplified fragment of HOIL2.

[0035] Figure 2 .Expression levels of gene HOIL2 in different soybean tissues.

[0036] Figure 3. Schematic diagram of the plant overexpression vector pFGC5941-Glyma.18G013300.

[0037] Figure 4 .Molecular identification of soybean transgenic lines overexpressing HOIL2-1 / 2.

[0038] Figure 5 .Plant types of the control strain and the transgenic strain overexpressing HOIL2-1 / 2.

[0039] Figure 6 .Differences in grain weight and oil content between the control strain and the overexpression strain HOIL2-1 / 2. DETAILED DESCRIPTION

[0040] The following examples are provided to facilitate a better understanding of the present invention, but are not intended to limit the present invention.

[0041] The experimental methods in the following examples are all conventional methods unless otherwise specified.

[0042] The test materials used in the following examples, unless otherwise specified, can all be purchased from commercial sources. The primers used were synthesized by Beijing Qingke Biological Company; the transgenic transformation process was completed by Jiangsu Weimi Biotechnology Co., Ltd. The % in the following examples, unless otherwise specified, are all mass percentages. The T2 generation refers to the seeds produced by self-pollination of the T1 generation and the plants grown from it, and the T3 generation refers to the seeds produced by self-pollination of the T2 generation and the plants grown from it.

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

[0044] Example 1: Cloning of soybean encoding gene HOIL2

[0045] 1. Design specific primers for cloning genes

[0046] The starting position of the gene Glyma.18G013300 on the genome was searched on the SoyBase website as Gm18:931992-939554, and the reference genome sequence of the gene was obtained. In order to amplify the coding region sequence of the gene, primers were designed at the start and terminator, and the sequence was as follows:

[0047] HOIL2-F1:5'-ATGATAAATCTGCAATGCTGCG-3';

[0048] HOIL2-R1:5'-TTAAAGTCCATTGTTAGACCTTAACAATGC-3';

[0049] 2. Cloning of the coding sequence of the HOIL2 gene

[0050] RNA was extracted from soybean Williams 82 leaves by the TriZol method and reverse transcribed to obtain the cDNA sequence of the gene; the coding sequence of the gene was amplified by primer pair HOIL2-F1 / R1 using cDNA as a template. Agarose gel electrophoresis was used to detect whether the amplified gene fragment met the sequence size ( Figure 1 ) and can be used for subsequent experiments. The amplified sequence was sent to the company (Qingke, Beijing, China) for Sanger sequencing to verify that the sequence was cloned correctly.

[0051] Example 2: Tissue expression characteristics of soybean coding gene HOIL2

[0052] Using the transcriptome library data of soybean variety Williams 82 in different tissues, we found that the expression level of gene HOIL2 was different in different tissues ( Figure 2 ). By comparison, it can be found that HOIL2 has the highest expression level in roots, followed by relatively high expression levels in organs related to fruiting, such as flowers and seeds, and relatively low expression levels in organs related to vegetative growth, such as stems and leaves. Analysis of tissue expression levels found that the gene is expressed in all tissues, and the expression level in pods and seeds is higher. It can be further inferred that the gene is involved in more plant development processes and is involved in soybean grain development, so the correlation between the gene and the oil content phenotype was studied.

[0053] Example 3: Construction of HOIL2 plant overexpression vector

[0054] 1. Extract RNA from soybean variety Williams 82 leaves and reverse transcribe it into cDNA.

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

[0056] HOIL2-F2: 5'-tacatttacaattaccatggATGATAAATCTGCAATGCTGCG-3';

[0057] HOIL2-R2: 5'-ctctagactcacctaggatccTTAAAGTCCATTGTTAGACCTTAACA ATGC-3'.

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

[0059] 4. Double digest the PCR product with restriction endonucleases BamHI and NcoI, and recover the digestion product.

[0060] 5. Double-digest the pFGC5941 vector with restriction endonucleases BamHI and NcoI to recover the vector backbone.

[0061] 6. Connect the enzyme digestion product of step 4 and the vector backbone of step 5 to obtain a connection product.

[0062] 7. The ligation product was sequenced, and the results showed that the recombinant expression vector pFGC5941-Glyma.18G013300 was obtained (the original plasmid was the pFGC5941 plasmid purchased from Prodin Biotechnology Co., Ltd., and the DNA shown in sequence 2 of the sequence table was inserted between the BamHI and NcoI restriction sites after the CaMV 35S promoter). Figure 3 shown.

[0063] Example 4: Acquisition and identification of HOIL2 transgenic soybeans

[0064] 1. Identification of Transformed Soybean and Transgenic Plants

[0065] 1. Introduce the recombinant expression vector pFGC5941-Glyma.18G013300 into Agrobacterium EHA101 to obtain recombinant Agrobacterium.

[0066] 2. The recombinant Agrobacterium was transferred into Williams 82 soybean by infecting the axillary meristem of the cotyledon node with Agrobacterium. After the growth was stable, it was transferred into nutrient soil for cultivation. At the same time, the transformation efficiency was identified, and the positive single plant T0 was harvested and propagated.

[0067] 3. Harvest T1 generation plants, sow seeds of each plant separately, and continue screening to observe the segregation of T2 generation. Repeat this process until T3 generation to obtain a genetically stable homozygous overexpression strain HOIL2-1 / 2.

[0068] 2. Molecular identification of HOIL2 transgenic plants

[0069] 1. Molecular identification of HOIL2 transgenic soybean overexpression lines (HOIL2-1 / 2)

[0070] The leaf RNA of the T3 homozygous line (HOIL2-1 / 2) and the control plant at the V2 stage was extracted and reverse transcribed into cDNA. The cDNA was used as a template and the primer pair consisting of HOIL2-F1 and HOIL2-R3 was used for RT-PCR identification. The results are shown in Figure 4 . The sequences of the primers used are as follows:

[0071] HOIL2-F1: 5'-ATGATAAATCTGCAATGCTGCG-3';

[0072] HOIL2-R3: 5'-TTAAAGTCCATTGTTAGACCTTAACAATGC-3';

[0073] 3. Phenotypic analysis of HOIL2 transgenic soybean

[0074] The experimental samples were as follows: T3 transgenic soybean overexpression line of HOIL2 gene (HOIL2-1 / 2) and control line Williams 82.

[0075] 1. Determination of grain weight phenotype

[0076] When the transgenic lines and the control lines were planted in the same field environment, it was observed that there were no significant differences in plant type, branch number and other agronomic traits between the transgenic lines and the control lines ( Figure 5 ). These materials were harvested individually and placed in an oven at 45°C for 48 hours to be completely dried. The number of soybean seeds per plant, seed length, seed width, and 100-seed weight were counted using an intelligent seed analysis system (Zhejiang Top Yunnong Technology Co., Ltd.).

[0077] 2. Determination of seed oil content

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

[0079] The method for determining the oil content is as follows: after the seeds are fully ground, weigh 100 mg, add 500 ul of isopropanol solution, mix thoroughly, and rotate at 37 degrees overnight; centrifuge at 3000 rpm for 3 minutes, collect the supernatant into a new centrifuge tube (the weight of the centrifuge tube is weighed in advance W0); add 500 ul of isopropanol to the remaining powder, mix thoroughly, centrifuge at 3000 rpm for 3 minutes, collect the supernatant into the above centrifuge tube, and then put the centrifuge tube into a fume hood (24 hours) to allow the isopropanol to completely evaporate; finally, weigh the centrifuge tube again (W1). The change in weight before and after the centrifuge tube is the weight of the extracted lipids (W1-W0).

[0080] 3. Phenotypic analysis of transgenic soybeans with HOIL2 gene

[0081] The phenotypic data of transgenic soybean materials and control strains were analyzed and it was found that the average oil content of the HOIL2 overexpressing transgenic strains reached 22.512%. Compared with the control strains, the seed oil content of the overexpressing strains was significantly increased by about 8.32%, which verified the biological function of this gene in regulating the oil content of soybean seeds ( Figure 6). Compared with domestic soybean varieties, the oil content is relatively excellent.

Claims

1. A protein HOIL2 derived from soybean (Glycine max (L.) Merrill), selected from the proteins shown in any one of the following (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 which is related to the plant oil content and has one or more amino acid residues substituted and / or deleted and / or added to the amino acid sequence shown in SEQ ID NO.1; (c) A protein consisting of an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to the amino acid sequence shown in SEQ ID NO. 1 and associated with plant oil content.

2. The protein HOIL2 according to claim 1, characterized in that The substitution and / or deletion and / or addition in (b) is caused by natural variation or artificial mutagenesis.

3. The gene HOIL2 encoding the protein HOIL2 according to claim 1.

4. The gene HOIL2 according to claim 3, characterized in that Choose any of the following: (1) DNA molecule shown in SEQ ID NO.2; (2) a DNA molecule that hybridizes with the DNA sequence defined in (1) under stringent conditions and encodes a protein related to plant oil content; (3) A DNA molecule that has at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% homology to the DNA sequence defined in (1) and encodes a protein related to plant oil content.

5. An overexpression vector containing the gene HOIL2 according to claim 3 or 4.

6. The overexpression vector according to claim 5, characterized in that The overexpression vector is obtained by using pFGC5941 as the original plasmid and inserting the gene HOIL2 according to claim 3 or 4 between the BamHI and NcoI restriction sites after the CaMV 35S promoter.

7. An expression cassette, transgenic cell line or recombinant bacterium containing the gene HOIL2 according to claim 3 or 4.

8. Use of the gene HOIL2 according to claim 3 or 4 in regulating the oil content of soybean seeds, characterized in that Overexpression of this gene can increase the oil content of soybean seeds.

9. Use of the knockout vector of the HOIL2 gene according to claim 5 or 6, the expression cassette, the transgenic cell line or the recombinant bacteria according to claim 7 in increasing the oil content of soybean seeds.

10. A method for cultivating transgenic plants, characterized in that The overexpression vector of the gene HOIL2 according to claim 5 or 6 is introduced into a target plant to obtain a transgenic plant having an oil content higher than that of the target plant.

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