Method for obtaining transgenic alfalfa and special expression vector CPB-LAR-GFP thereof

A CPB-LAR-GFP, transgenic alfalfa technology, applied in the direction of introducing foreign genetic material, genetic engineering, plant genetic improvement using vectors, etc., to avoid the increase of production costs, reduce pollution, and reduce the pressure of environmental pollution.

Inactive Publication Date: 2015-09-16
GANSU AGRI UNIV
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Problems solved by technology

In the direction of genetic resource innovation, identifying and isolating condensed tannin synthase gene from red bean grass, and then usi

Method used

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  • Method for obtaining transgenic alfalfa and special expression vector CPB-LAR-GFP thereof
  • Method for obtaining transgenic alfalfa and special expression vector CPB-LAR-GFP thereof
  • Method for obtaining transgenic alfalfa and special expression vector CPB-LAR-GFP thereof

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Embodiment 1

[0045] 1. Extraction of total RNA of Ormosia japonica:

[0046] According to the characteristics of red bean grass rich in polyphenols, polysaccharides and secondary metabolites, the fresh young tissue of Gansu red bean grass plant was used to prepare RNA crude extract by CTAB method, and the extracted RNA was further purified to find out a suitable Extraction method of total RNA of Ormosia japonica. This method mainly prevents secondary metabolites from combining with RNA by adding 4% PVP to the extraction buffer; adds 2% β-mercaptoethanol to reduce the interference of polyphenols; utilizes water-saturated phenol: chloroform: isoamyl alcohol to extract Removing the interference of DNA, protein, chlorophyll, anthocyanin and other pigments on RNA not only saves experimental costs, but also reduces the probability of RNA degradation and improves the quality of RNA. RNA is efficiently separated from polysaccharides by selective precipitation of RNA using LiCl. Purification of t...

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Abstract

The invention discloses a method for obtaining transgenic alfalfa and a special expression vector thereof. A plant expression vector is a condensed tannin synthetase LAR gene plant expression vector CPB-LAR-GFP with a green fluorescent protein (GFP) marker gene and a herbicide resistant bar gene, and a construction method of the plant expression vector is provided. The beneficial effects are that a condensed tannin synthetase LAR gene is separated and cloned from onobrychis viciaefolia with a high condensed tannin content, and a double-marker selecting plant expression vector CPB-LAR-GFP with the green fluorescent protein (GFP) marker gene and the herbicide resistant bar gene is constructed. Through a genetic transformation method, the herbicide resistant gene and the condensed tannin gene are introduced into the alfalfa at the same time. Therefore, the amount of adopted herbicide can be reduced, pollution of a large amount of adopted pesticide to environment is reduced, the content of the condensed tannin in forage can be improved, and finally new pasture cultivars with the advantages of resisting herbicide and tympany are cultivated.

Description

technical field [0001] The invention relates to a method for obtaining transgenic alfalfa and its special expression vector CPB-LAR-GFP. Background technique [0002] Red bean grass (Onobrychis viciaefolia) is a perennial herb. Its aerial part is rich in crude protein and amino acid, and contains a certain amount of crude fat, vitamins, minerals and other chemical substances. The seed yield and grass yield of red bean grass are high, the seed setting performance is good, the nutrients are comprehensive and rich, and the palatability is excellent [Chen Baoshu. China Grassland, 1983, (2): 36-45.]. Gansu red bean grass (Onobrychis viciifolia cv.Gansu) is bred by single plant selection from the plants of common red bean grass and Caucasian red bean grass free hybrid multiple times mixed pollination [Chen Baoshu. Red bean grass [M]. Lanzhou: Gansu Science and Technology Press ,1992.]. The purpose of breeding is to establish artificial grassland for arid and semi-arid areas in m...

Claims

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Application Information

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IPC IPC(8): C12N15/82C12N15/65C12N15/84A01H5/00
Inventor 马晖玲陈春艳董文科牛奎举杨江伟
Owner GANSU AGRI UNIV
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