阅读说明：本技术 蛋白GmRRM551在调控植物油脂代谢中的应用 (Application of protein GmRRM551 in regulation and control of vegetable oil metabolism ) 是由 张劲松 陈受宜 程彤 张万科 韦伟 林晴 何锶洁 于 2018-07-25 设计创作，主要内容包括：本发明公开了蛋白GmRRM551在调控植物油脂代谢中的应用。本发明提供了GmRRM551蛋白或其相关生物材料在调控植物油脂代谢中的应用；所述GmRRM551蛋白为SEQ ID No.1所示蛋白或其经一个或几个氨基酸残基的取代和/或缺失和/或添加,或序列具有99％以上、95％以上、90％以上、85％以上或者80％以上同源性且具有相同功能的蛋白,或其N端和/或C端连接标签后得到的融合蛋白。本发明证明GmRRM551蛋白可以调控植物种子中油脂含量,过表达后提高植物种子中油脂含量。该基因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 application of a protein GmRRM551 in regulation and control of vegetable oil metabolism. The invention provides an application of GmRRM551 protein or a related biomaterial thereof in regulation and control of vegetable oil metabolism; the GmRRM551 protein is a protein shown in SEQ ID No.1 or a protein which is substituted and/or deleted and/or added by one or more amino acid residues, or a protein with the sequence more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology and the same function, or a fusion protein obtained by connecting a label at the N end and/or the C end of the protein. The invention proves that the GmRRM551 protein can regulate and control the oil content in plant seeds, and the oil content in the plant seeds is improved after overexpression. The gene 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.)

The application of GmRRM551 protein or related biological materials thereof in regulation and control of vegetable oil metabolism;

the related biological material is a nucleic acid molecule capable of expressing the GmRRM551 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the GmRRM551 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

2, the GmRRM551 protein or the related biomaterial thereof is applied to the regulation of the oil content of plant tissues;

the related biological material is a nucleic acid molecule capable of expressing the GmRRM551 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule;

the GmRRM551 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

3. Use according to claim 2, characterized in that: the activity and/or expression quantity of the GmRRM551 protein or the coding gene thereof in the plant is increased, and the oil content of plant tissues is increased; the activity and/or expression of the GmRRM551 protein or the coding gene thereof in the plant is reduced, and the oil content of plant tissues is reduced.

4. A method for breeding a plant variety with an increased tissue oil content, comprising the step of increasing the expression level and/or activity of GmRRM551 protein in a recipient plant;

the GmRRM551 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

5. A method of breeding a transgenic plant with increased tissue oil content comprising the steps of: introducing a nucleic acid molecule capable of expressing GmRRM551 protein into a receptor plant to obtain a transgenic plant; the transgenic plant has increased tissue oil content as compared to the recipient plant;

the GmRRM551 protein is any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

6. The method of claim 5, wherein: the "introduction of a nucleic acid molecule capable of expressing the GmRRM551 protein into a recipient plant" is achieved by introducing a recombinant expression vector containing a gene encoding the GmRRM551 protein into the recipient plant.

7. The method of claim 6, wherein: the promoter for starting the transcription of the coding gene in the recombinant expression vector is a 35S promoter.

8. Use or method according to any of claims 1-7, wherein: the "nucleic acid molecule capable of expressing the GmRRM551 protein" is a coding gene of the GmRRM551 protein;

further, the coding gene of the GmRRM551 protein is a DNA molecule as described in any one of the following items:

(B1) DNA molecule shown in SEQ ID No. 2;

(B2) a DNA molecule which is hybridized with the DNA molecule defined by (B1) under strict conditions and encodes the GmRRM551 protein;

(B3) and (B) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the DNA sequence limited by (B1) or (B2) and encodes the GmRRM551 protein.

9. Use or method according to any of claims 2-8, wherein: the tissue is a seed.

10. The use or method according to claim 9, wherein: the plant is a dicotyledonous plant or a monocotyledonous plant;

further, the dicotyledonous plant is a crucifer or a leguminous plant;

still further, the crucifer is arabidopsis thaliana; the leguminous plant is soybean.

Technical Field

The invention relates to the technical field of biology, in particular to application of a protein GmRRM551 in regulation and control of vegetable oil metabolism.

Background

71% of the fat in the human diet comes from plants. In several major oil-producing crops in the world, the total oil yield of soybeans accounts for about 30%, and the first crop oil yield in the world is the first crop oil yield.

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.

The GmRRM551 belongs to the RNA binding protein family, contains an RNA recognition element (RRM), can bind to single or double stranded RNA in cells to form RNA-protein complexes (RBPs), and is widely present in organisms, mainly involved in the processes of mRNA stability, mRNA localization, translation, cleavage, and the like in organisms, and regulate each stage of growth and development. The protein is not reported to be involved in fatty acid synthesis at present.

Disclosure of Invention

The invention aims to provide application of protein GmRRM551 in regulation and control of vegetable oil metabolism.

In a first aspect, the invention claims the application of the GmRRM551 protein or related biomaterials thereof in regulating and controlling the metabolism of vegetable oil.

Wherein, the related biological material can be a nucleic acid molecule capable of expressing the GmRRM551 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule.

The GmRRM551 protein can be any one of the following proteins:

(A1) protein with an amino acid sequence of SEQ ID No. 1;

(A2) protein which is obtained by substituting and/or deleting and/or adding one or more amino acid residues to the amino acid sequence shown in SEQ ID No.1 and has the same function;

(A3) a protein having 99% or more, 95% or more, 90% or more, 85% or more, or 80% or more homology to the amino acid sequence defined in any one of (A1) to (A2) and having the same function;

(A4) a fusion protein obtained by attaching a tag to the N-terminus and/or C-terminus of the protein defined in any one of (A1) to (A3).

In (a2), the substitution and/or deletion and/or addition of one or several amino acid residues means substitution and/or deletion and/or addition of not more than ten amino acid residues.

SEQ ID No.1 consists of 551 amino acid residues.

In a second aspect, the invention claims the application of the GmRRM551 protein or the related biomaterial thereof in regulating and controlling the oil content of plant tissues.

Wherein, the related biological material can be a nucleic acid molecule capable of expressing the GmRRM551 protein or an expression cassette, a recombinant vector, a recombinant bacterium or a transgenic cell line containing the nucleic acid molecule. The GmRRM551 protein is a protein shown in any one of the above (A1) - (A4).

In the application, the activity and/or expression quantity of the GmRRM551 protein or the coding gene thereof in the plant is increased, and the oil content of plant tissues is increased; the activity and/or expression of the GmRRM551 protein or the coding gene thereof in the plant is reduced, and the oil content of plant tissues is reduced.

In a third aspect, the invention claims a method of breeding a plant variety with increased tissue oil content.

The method for cultivating the plant variety with the improved tissue oil content provided by the invention can comprise the step of improving the expression quantity and/or activity of the GmRRM551 protein in a receptor plant. The GmRRM551 protein is a protein shown in any one of the above (A1) - (A4).

Further, the present invention provides a method for breeding a transgenic plant with an increased tissue oil content.

The method for cultivating the transgenic plant with the improved tissue oil content provided by the invention specifically comprises the following steps: introducing a nucleic acid molecule capable of expressing GmRRM551 protein into a receptor plant to obtain a transgenic plant; the transgenic plant has increased tissue oil content as compared to the recipient plant. The GmRRM551 protein is a protein shown in any one of the above (A1) - (A4).

Further, the "introduction of a nucleic acid molecule capable of expressing the GmRRM551 protein into a recipient plant" may be achieved by introducing a recombinant expression vector containing a gene encoding the GmRRM551 protein into the recipient plant.

The recombinant expression vector can be constructed by using the existing plant expression vector. The plant expression vector includes, for example, Gateway system vector or binary Agrobacterium vector, such as pGWB411, pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301, pCAMBIA1300, pBI121, pCAMBIA1391-Xa, pCAMBIA1391-Xb (CAMBIA Co., Ltd.), or other derived plant expression vector. When the GmRRM551 is used for constructing a plant expression vector, any one of enhanced, constitutive, tissue-specific or inducible promoters, such as a cauliflower mosaic virus (CAMV)35S promoter, a Ubiquitin gene ubitin promoter (pUbi) and the like, can be added in front of transcription initiation nucleotides of the plant expression vector, and can be used alone or combined with other plant promoters; in addition, when the gene of the present invention is used to construct plant expression vectors, enhancers, including translational or transcriptional enhancers, may be used, and these enhancer regions may be ATG initiation codon or initiation codon 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 the identification and screening of transgenic plant cells or plants, plant expression vectors to be used may be processed, for example, by adding a gene encoding an enzyme or a luminescent compound which can produce a color change (GUS gene, luciferase gene, etc.), an antibiotic marker having resistance (gentamicin marker, kanamycin marker, etc.), or a chemical-resistant marker gene (e.g., herbicide-resistant gene), etc., which can be expressed in plants.

In the invention, the promoter for promoting the transcription of the gene coding for the GmRRM551 protein in the recombinant vector is a 35S promoter.

More specifically, the recombinant vector is a recombinant plasmid (named pGWB411-GmRRM551) obtained by inserting the encoding gene of the GmRRM551 protein into a recombination site of a pGWB411 vector.

Using Gateway system manufactured by Invitrogen corporation

According to the TA Cloning kit, both an entry vector TOPO and a target vector pGWB411 are provided with spectinomycin resistance markers, Escherichia coli can be efficiently screened, both the entry vector TOPO and the target vector pGWB411 are provided with homologous recombination sites attL1 and attL2, the vector TOPO connected with a target gene and the vector pGWB411 are subjected to homologous recombination under the action of recombinase, and a plant expression vector pGWB411-GmRRM551 is constructed.

In the above method, the recombinant expression vector carrying the encoding gene of the GmRRM551 protein is introduced into the recipient plant, and specifically, may be: plant cells or tissues are transformed by conventional biological methods using Ti plasmids, Ri plasmids, plant viral vectors, direct DNA transformation, microinjection, conductance, agrobacterium mediation, etc., and the transformed plant tissues are grown into plants.

In the above aspects, the "nucleic acid molecule capable of expressing the GmRRM551 protein" is a gene encoding the GmRRM551 protein.

Further, the encoding gene of the GmRRM551 protein can be any one of the following DNA molecules:

(B1) DNA molecule shown in SEQ ID No. 2;

(B2) a DNA molecule which is hybridized with the DNA molecule defined by (B1) under strict conditions and encodes the GmRRM551 protein;

(B3) and (B) a DNA molecule which has more than 99%, more than 95%, more than 90%, more than 85% or more than 80% of homology with the DNA sequence limited by (B1) or (B2) and encodes the GmRRM551 protein.

In the above genes, the stringent conditions may be as follows: 50 ℃ in 7% Sodium Dodecyl Sulfate (SDS), 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in2 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄And 1mM EDTAHybridization in the mixed solution, rinsing at 50 ℃ in 1 XSSC, 0.1% SDS; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.5 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 50 ℃; also can be: 50 ℃ in 7% SDS, 0.5M NaPO₄Hybridization with 1mM EDTA, rinsing in 0.1 XSSC, 0.1% SDS at 65 ℃; can 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.

In the above aspects, the tissue is specifically a seed.

In each of the above aspects, the plant may be a dicotyledonous plant or a monocotyledonous plant.

Further, the dicot may be a crucifer or a legume.

Still further, the crucifer may be arabidopsis; the leguminous plant may be soybean.

In a particular embodiment of the invention, the plant is in particular Columbia ecotype Arabidopsis thaliana (col-0).

Experiments prove that the RNA binding protein GmRRM551 related to the oil content of plant tissues and the coding gene thereof are provided, the coding gene is transferred into wild arabidopsis thaliana to obtain transgenic arabidopsis thaliana, and compared with the wild arabidopsis thaliana, the oil content of seeds of the transgenic arabidopsis thaliana is improved. The RNA binding protein GmRRM551 and the coding gene thereof can regulate and control the oil content in plant seeds, and the oil content in the plant seeds can be improved after overexpression. The gene 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 is a cloning vector

And a schematic diagram of the plant expression vector pGWB411-GmRRM 551. A is a cloning vector

B is a plant expression vector pGWB411-GmRRM 551.

FIG. 2 shows the expression analysis of GmRRM551 in different organs of soybean.

FIG. 3 shows the molecular characterization of the GmRRM551 transgenic pure line. The control was an empty vector control.

FIG. 4 shows the results of measuring the oil and fat content in the seeds of the GmRRM551 transgenic plants. Indicates significant differences at P <0.05 levels and indicates very significant differences at P <0.01 levels. The control was an empty vector control.

Detailed Description

The experimental procedures used in the following examples are all conventional procedures unless otherwise specified.

Materials, reagents and the like used in the following examples are commercially available unless otherwise specified.

Soybean melanong 44(HN 44): the method is described in the literature, "Heilongjiang agricultural science, 5 th.1-5 in 2004, and obtained from soybean institute of academy of agricultural sciences, Heilongjiang, in 2006. The first breeder of soybean variety approved by soybean institute of academy of agricultural sciences of Heilongjiang province in 2002 is Du Wei Guang researcher with patent numbers: CNA20020216.2, approval No.: black beans 2002003. The material is publicly available from the institute of genetics and developmental biology of the Chinese academy, but is only available for use in the duplication of the experiments of the invention.

Soybean ZYD 7: is provided for group researchers by soybeans of the agriculture institute of Heilongjiang. The expression "Xiang Lu, et al. APP2C-1 Allle exploiting a Quantitative train Qi cells Enhances Soybean 100-SeedWeight. molecular plant.2017". The material is publicly available from the institute of genetics and developmental biology of the Chinese academy, but is only available for use in the duplication of the experiments of the invention.

Expression vector pGWB 411: described in the literature "Department of Molecular and functional genomics, Shimane University, Aatsue, Shimane 690-. Available from the institute of genetics and developmental biology, of the institute of sciences, with the consent of doctor Tsuyoshi Nakagawa, and only available for use in the experiments of the present invention.

Agrobacterium GV 3101: the expression of the polypeptide is described in the documents "Lee CW et al, Agrobacterium tumefaciens protested or induction by modulation of pathogen damage in Arabidopsis thaliana, plantaCell, 2009,21(9), 2948-62". The material is publicly available from the institute of genetics and developmental biology of the Chinese academy, but is only available for use in the duplication of the experiments of the invention.