Alfalfa anthocyanin reductase gene and its encoded protein and application
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A technology of alfalfa and reductase, applied in the direction of oxidoreductase, application, genetic engineering, etc., can solve the problem of less research
Inactive Publication Date: 2014-10-22
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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Problems solved by technology
However, there are relatively few studies on the molecular mechanism of tannin synthesis in alfalfa
Method used
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Embodiment 1
[0041] Transformation of Alfalfa Plants with MsBAN Gene Mediated by Agrobacterium
[0042] 1. Materials and Reagents
[0043] 1. Plant material
[0044] The variety of alfalfa tested was Medicago sativa L.cv. Zhongmu No.1 (Zhongmu No.1, Beijing China Animal Science and Technology Co., Ltd.).
[0045] The cotyledons of sterile seedlings germinated for 7 days were the recipient materials for genetic transformation.
[0046] 2. Agrobacterium strains and plasmid vectors
[0047] The Agrobacterium strain used is Agrobacterium tumefaciens: LBA4404 (Beijing Tianenze Gene Technology Co., Ltd.)
[0048] Agrobacterium culture medium:
[0049]
[0050] Plasmid vector: pCAMBIA1302 (purchased from Shanghai Xipiji Biotechnology Co., Ltd.). The MsBAN gene was inserted into pCAMBIA1302, and the specific steps were: design the upstream primer of the NcoI restriction site at the 5' end and the downstream primer of the SpeI restriction site (F: 5'-CATGCCATGGATGATGGCTAGTATCAAAC-3', R: 5'-...
Embodiment 2
[0074] Detect the tannin content in the alfalfa of the alfalfa that is transformed into the pCAMBIA1302 carrier of the MsBAN gene obtained in the alfalfa that is transformed into the alfalfa that obtains in the comparative example 1 of 30 strains and the embodiment 1 of 30 strains, and specific method is as follows:
[0075] A. Preparation of Folin-Denis color reagent: 50g sodium tungstate, 10g phosphomolybdic acid, add 375mL distilled water to dissolve, then add 25mL 85% H 3 PO 4 , reflux in the water bath for 2 hours, and after cooling, set the volume to a 500mL brown bottle;
[0076] B. Extraction of the sample: After the test sample is dried and crushed, accurately weigh 1g, add 50mL of 1:5 ethanol solution in a 250mL conical flask, shake and extract for 60min, and filter with double-layer fast filter paper;
[0077] C. Draw a standard curve: Accurately weigh 50mg of tannin standard substance, set the volume to 500mL, draw tannin standard solution 0, 0.5, 1.0, 1.5, 2.0, 2...
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Abstract
The invention discloses a protein coded by a medicago sativa L. anthocyanin reductase gene, wherein the protein has an amino acid sequence shown in SEQ ID No:2 or an amino acid sequence which is obtained through substituting, deleting or adding one or a plurality of amino acid residues in the amino acid sequence shown in SEQ ID No:2 and still has anthocyanin reductase activity. The invention further relates to a coded gene of the protein, an expression vector and a cell which contain the gene, a method for culturing medicago sativa L. with high tannin content and application of the coded gene of the protein, the expression vector and the cell. Through the expression of the medicago sativa L. anthocyanin reductase gene (MsBAN) provided by the invention in the medicago sativa L., the tannin content of the medicago sativa L. can be obviously increased. The discovery of the gene provides a gene resource for overcoming the problem that main grass plants (in particular leguminous plants, such as the medicago sativa L.) easily cause livestock to suffer from tympanites, and plays an important role in the research of genetic engineering improved grasses.
Description
technical field [0001] The present invention relates to an alfalfa anthocyanin reductase-related gene, a protein encoded by the gene, an expression vector and a cell containing the alfalfa anthocyanin reductase gene, and a method for cultivating alfalfa The method, and the application of the alfalfa anthocyanin reductase gene and its encoded protein, the expression vector containing the gene and the cells in cultivating plants with high tannin content. Background technique [0002] Alfalfa is the most important leguminous forage in my country and even in the world, and is known as the "king of forage". For a long time, alfalfa has been mainly used to harvest hay and prepare silage, and it is not suitable for livestock to use it directly during grazing. This is because ruminant livestock tend to form a large amount of foam-like substances in the rumen after eating fresh alfalfa, which cannot be discharged, resulting in bloat disease, which limits the application of alfalfa i...
Claims
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