阅读说明：本技术 与油脂代谢调控相关的大豆转录因子GmMYB395及其编码基因与应用 (Soybean transcription factor GmMYB395 related to lipid metabolism regulation and control as well as coding gene and application thereof ) 是由 张劲松 陈受宜 白文婉 张万科 马彪 林晴 何锶洁 于 2019-04-12 设计创作，主要内容包括：本发明公开了一种与油脂代谢调控相关的大豆转录因子GmMYB395及其编码基因与应用。本发明提供的蛋白质,命名为GmMYB395蛋白,又称为与油脂代谢调控相关的转录因子GmMYB395,是序列表中序列1所示的蛋白质。编码GmMYB395蛋白的基因也属于本发明的保护范围。本发明还保护一种培育转基因植物的方法,包括如下步骤：将GmMYB395基因导入受体植物中,得到油脂含量高于所述受体植物的转基因植物。本发明对提高和改良作物油脂成份、特别是对于提高大豆等油料植物籽粒中油脂成份,培育高油脂品种具有重要的理论和现实意义。(The invention discloses a soybean transcription factor GmMYB395 related to oil metabolism regulation and application thereof as well as a coding gene and application thereof. The protein provided by the invention is named as GmMYB395 protein, is also named as a transcription factor GmMYB395 related to oil metabolism regulation, and is a protein shown in a sequence 1 in a sequence table. The gene coding the GmMYB395 protein also belongs to the protection scope of the invention. The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: and (3) introducing the GmMYB395 gene into a receptor plant to obtain a transgenic plant with the oil content higher than that of the receptor plant. The invention has important theoretical and practical significance for improving the oil and fat components of crops, particularly for improving the oil and fat components in oil plant seeds such as soybeans and the like and cultivating high-oil varieties.)

1. A protein which is (a) or (b) below:

(a) protein shown as a sequence 1 in a sequence table;

(b) the protein shown in the sequence 1 in the sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and is a protein related to the content of vegetable oil and fat and derived from the protein.

2. A gene encoding the protein of claim 1.

3. The gene of claim 2, wherein: the gene is (1) or (2) or (3) as follows:

(1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(2) a DNA molecule which is hybridized with the DNA molecule defined in the step (1) under strict conditions and codes the vegetable fat content related protein;

(3) a DNA molecule which is derived from soybean, 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 molecule defined in (1) and encodes a protein related to vegetable fat content.

4. A recombinant vector, expression cassette or recombinant bacterium comprising the gene of claim 2 or 3.

5. The recombinant vector of claim 4, wherein: the recombinant vector is a recombinant expression vector for expressing the protein of claim 1, which is obtained by inserting the gene of claim 2 or 3 into an expression vector.

6. A primer set for amplifying the full length of the gene of claim 2 or 3 or any fragment thereof.

7. Use of the protein of claim 1 for regulating the content of vegetable fats and oils.

8. Use of the gene of claim 2 or 3, or the recombinant vector of claim 4 or 5; the application is to culture transgenic plants with increased oil content.

9. A method of breeding a transgenic plant comprising the steps of: introducing the gene of claim 2 or 3 into a recipient plant to obtain a transgenic plant with a higher lipid content than the recipient plant.

10. A method of plant breeding comprising the steps of: increasing the content and/or activity of the protein of claim 1 in the target plant, thereby increasing the content of the vegetable oil.

Technical Field

The invention belongs to the technical field of biology, and particularly relates to a soybean transcription factor GmMYB395 related to oil metabolism regulation and control, and a coding gene and application thereof.

Background

71% of the fat in the human diet comes from plants. In several major oil-producing crops in the world, the total oil production of soybeans accounts for about 30%, and the first of the world plant oil production (see table 1 for major oil-producing crops in the world).

TABLE 1

Species of	Production capacity (million tons)	Percentage of total yield	Relative order
				Soybean (Soybean)	15.50	29.1	1
Palm (Palm)	8.52	16.0	2
				Rapeseed (Rapesed)	7.03	13.2	3
Sunflower (Sunflower)	7.00	13.1	4
				Cotton seed (cottonsed)	3.31	6.2	5
Coconut (Coconut)	2.71	5.1	6
				Peanut (Peanout)	2.69	5.0	7
Olive (Olive)	1.63	3.1	8

Fatty acid synthesis is one of the most important metabolic pathways in plants, and it is present in any cell of a plant and is essential for growth and development. Blocking it leads to cell death, so that a plant mutant which blocks fatty acid synthesis has not been found so far.

Plants differ greatly from other eukaryotes in the enzymes involved in the fatty acid synthesis pathway. The synthesis of fatty acids of 16 or 18 carbon atoms from acetyl-CoA and malonyl-CoA requires at least 30 different enzyme-catalyzed reactions, which in animals, fungi and some bacteria are carried out by a multi-enzyme complex present in the cytoplasm. In plants, the enzymes involved in fatty acid synthesis are present in the cytoplasm of plastids in soluble form.

In most plants, lipids are stored in the form of Triacylglycerols (TAGs), the content of which is a very important agronomic trait, the biosynthesis of TAG is called Kennedy pathway, as in the synthesis of membrane glycerides in eukaryotes, fatty acids are transferred to the 1 and 2 positions of 3-phosphoglycerol after removal of CoA, forming the intermediate product PA. Dephosphorylation of PA produces DAG. In the last step of TAG synthesis, a third fatty acid molecule is transferred to the empty DAG 3' -OH position, a reaction catalyzed by diacylglycerol acetyltransferase (DGAT), which is considered to be the only rate-limiting step in TAG biosynthesis. The lipid synthesis pathway has been recognized and many enzyme genes involved in lipid synthesis have been cloned. However, in plants, the mechanisms controlling lipid synthesis and their associated genes are still poorly understood.

Disclosure of Invention

The invention aims to provide a soybean transcription factor GmMYB395 related to oil metabolism regulation and application thereof as well as a coding gene and application thereof.

The protein provided by the invention is obtained from soybean (Glycine max (L.) Merrill) in the genus of soybean, is named GmMYB395 protein, is also named as a transcription factor GmMYB395 related to oil metabolism regulation, and is (a) or (b) as follows:

(a) Protein shown as a sequence 1 in a sequence table;

(b) the protein shown in the sequence 1 in the sequence table is subjected to substitution and/or deletion and/or addition of one or more amino acid residues, and is a protein related to the content of vegetable oil and fat and derived from the protein.

The one or several amino acid residues refer to 10 or less amino acid residues.

The gene coding the GmMYB395 protein also belongs to the protection scope of the invention. The gene encoding the GmMYB395 protein is named GmMYB395 gene.

The gene is (1) or (2) or (3) as follows:

(1) the coding region is a DNA molecule shown as a sequence 2 in a sequence table;

(2) a DNA molecule which is hybridized with the DNA molecule defined in the step (1) under strict conditions and codes the vegetable fat content related protein;

(3) a DNA molecule which is derived from soybean, 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 molecule defined in (1) and encodes a protein related to vegetable fat content.

The stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄And 1mM EDTA, and rinsed at 50 deg.C in 2 XSSC, 0.1% SDS. The stringent conditions may also be: 50 ℃ in 7% SDS, 0.5M NaPO ₄And 1mM EDTA, and rinsed at 50 ℃ in 1 XSSC, 0.1% SDS. The stringent conditions may also be: 50 ℃ in 7% SDS, 0.5M NaPO₄And 1mM EDTA, and rinsed at 50 deg.C in 0.5 XSSC, 0.1% SDS. The stringent conditions may also be: 50 ℃ in 7% SDS, 0.5M NaPO₄And 1mM EDTA, and rinsed at 50 deg.C in 0.1 XSSC, 0.1% SDS. The stringent conditions may also be: 50 ℃ in 7% SDS, 0.5M NaPO₄And 1mM EDTA, and rinsed at 65 ℃ in 0.1 XSSC, 0.1% SDS. The stringent conditions may also be: in a solution of 6 XSSC, 0.5% SDS at 65 ℃ and then washed once with each of 2 XSSC, 0.1% SDS and 1 XSSC, 0.1% SDS.

The recombinant vector, expression cassette or recombinant bacterium containing the GmMYB395 gene belongs to the protection scope of the invention.

The recombinant vector may specifically be a recombinant expression vector. The existing expression vector can be used for constructing a recombinant expression vector containing the GmMYB395 gene. When the GmMYB395 gene is used for constructing a recombinant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters can be added in front of a transcription initiation nucleotide, and can be used independently or combined with other plant promoters; in addition, when using the GmMYB395 gene to construct a recombinant expression vector, enhancers can be used, including translational or transcriptional enhancers, and these enhancer regions can be ATG initiation codons or initiation codons of adjacent regions, etc., but must be in the same reading frame as the coding sequence to ensure proper translation of the entire sequence. The translational control signals and initiation codons are widely derived, either naturally or synthetically. The translation initiation region may be derived from a transcription initiation region or a structural gene. In order to facilitate identification and screening of the transgenic plant or the transgenic microorganism, an expression vector to be used may be processed, for example, a gene for expressing an enzyme or a luminescent compound which produces a color change in the plant or the microorganism, a gene for an antibiotic marker having resistance or a chemical-resistant agent marker, etc. From the viewpoint of safety of transgenes, the transformed plants or microorganisms can be directly screened phenotypically without adding any selectable marker gene.

The recombinant expression vector is a recombinant expression vector for expressing GmMYB395 protein, which is obtained by inserting the GmMYB395 gene into an expression vector. The expression vector may specifically be the vector pGWB 412.

Primer pairs for amplifying the full length of the GmMYB395 gene or any fragment thereof also belong to the protection scope of the invention. The primer pair can be specifically a primer pair consisting of a primer shown in a sequence 3 of a sequence table and a primer shown in a sequence 4 of the sequence table.

The invention also protects the application of the GmMYB395 protein in regulating and controlling the content of the vegetable oil. The modulation is a forward modulation. The vegetable oil content is regulated and controlled to be improved.

The invention also protects the application of the GmMYB395 gene or a recombinant vector containing the GmMYB395 gene; the application is to culture transgenic plants with increased oil content. The application is to culture transgenic plants with increased seed oil content.

The invention also provides a method for cultivating the transgenic plant, which comprises the following steps: and (3) introducing the GmMYB395 gene into a receptor plant to obtain a transgenic plant with the oil content higher than that of the receptor plant. The 'oil content is higher than that of the receptor plant' is that the oil content of the seed is higher than that of the receptor plant. The GmMYB395 gene can be specifically introduced into a receptor plant through any one of the recombinant expression vectors. The recombinant expression vector transforms plant cells or tissues by using conventional biological methods such as Ti plasmid, Ri plasmid, plant virus vector, direct DNA transformation, microinjection, conductance, Agrobacterium mediation, etc., and the transformed plant tissues are cultivated into plants.

The invention also provides a plant breeding method, which comprises the following steps: the content and/or activity of GmMYB395 protein in the target plant is increased, so that the oil content of the target plant is increased. The increased oil content is the increased oil content of the seeds.

Any one of the above vegetable oil contents may specifically be an oil content of a plant seed.

The content of any one of the above oils is the total oil content.

Any of the above plants is a monocot or a dicot. The dicotyledonous plant can be specifically leguminous plant, and more specifically can be plant of genus Glycine. The dicotyledonous plant may specifically be a crucifer, more specifically an arabidopsis plant, such as a columbia ecotype arabidopsis thaliana.

The inventor obtains a transcription factor GmMYB395 which is highly expressed in seed oil accumulation in the analysis of transcriptome in the development process of soybean seeds and belongs to MYB transcription factors. Further functional identification shows that the soybean oil-fat content regulating agent can regulate and control the accumulation of the oil-fat content of soybean seeds. The invention has important theoretical and practical significance for improving the oil and fat components of crops, particularly for improving the oil and fat components in oil plant seeds such as soybeans and the like and cultivating high-oil varieties.

Drawings

FIG. 1 shows a vector

8/GW/TOPO diagramSpectra.

Fig. 2 is a result of the relative expression levels of the GmMYB395 gene in example 1.

Fig. 3 is a result of the relative expression levels of the GmMYB395 gene in example 2.

FIG. 4 shows the results of the total oil content of the seeds in example 2.

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The quantitative tests in the following examples, all set up three replicates and the results averaged.

Soybean melanong 44(HN 44): the first breeder of the soybean variety approved by the soybean research institute of the academy of agricultural sciences of Heilongjiang province in 2002 is a Duwei Guangdong researcher; the patent numbers are: CNA 20020216.2; the approval number is: black bean 2002003; the following documents are described: the group is full, the breeding and different planting modes of the new soybean variety Heilongjiang agricultural science 44 have influence on the yield and variety thereof, 5 th of Heilongjiang agricultural science 2004, 1-5; obtained from soybean institute of agri-sciences of Heilongjiang in 2006.

Agrobacterium GV3101, described in: lee CW et al, Agrobacterium tumefaciens proteins tissue transforming pathogenic in Arabidopsis, Plant Cell,2009,21(9), 2948-62.

Columbia ecotype Arabidopsis thaliana (col-0): arabidopsis Biological Resource Center (ABRC).

GmMYB395-up (sequence 3 of the sequence table): 5'-ATGGGAAGGAGTCCTTGCTGTA-3', respectively;

GmMYB395-dp (sequence 4 of the sequence table): 5'-CTACAGATACTGAATGTACTTC-3' are provided.