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

A CPB-BAN-GFP, transgenic alfalfa technology, applied in the direction of using vectors to introduce foreign genetic material, genetic engineering, plant gene improvement, etc., can solve the problem of no related reports on the construction strategy, so as to avoid the increase of production cost and reduce the environment The pressure of pollution, the effect of reducing pollution

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

At present, there is no relevant report on the construction strategy of the plant expression vector of the condensed tannin synthase BAN gene with the GFP marker gene and the herbicide-resistant bar gene

Method used

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

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Embodiment 1

[0062] 1. Extraction of total RNA of Ormosia japonica:

[0063] 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, and 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 ...

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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 BAN gene plant expression vector CPB-BAN-GFP with green fluorescent protein (GFP) marker genes and herbicide resistant bar genes, and a construction method of the condensed tannin synthetase BAN gene plant expression vector CPB-BAN-GFP is provided. The beneficial effects are that the BAN genes in the vector are from Onobrychis viciaefolia, and condensed tannin synthetase BAN genes are separated and cloned from the Onobrychis viciaefolia; the bar genes are added to the constructed vector, so that the double-marker genes with a herbicide resisting property and a gene marking function are obtained and the efficiency of screening transgenic plants is greatly improved. Through the transgenic plants transformed through the vector, production cost increase caused by adoption of a large amount of adopted herbicide can be avoided, and the pressure of environmental pollution can be reduced; meanwhile, the content of condensed tannin in forage can be increased and finally new pasture cultivars with the advantages of resisting herbicide and tympany can be cultivated.

Description

technical field [0001] The invention relates to a method for obtaining transgenic alfalfa and its special expression vector CPB-BAN-GFP. Background technique [0002] Onobrychis viciaefolia (Onobrychis viciaefolia) is a perennial herb of the Leguminosae genus. It is also known as donkey hi bean and donkey bean. It is an excellent pasture grass with good palatability and high nutritional value. , et al. Journal of Inner Mongolia University for Nationalities (Natural Science Edition), 2011, 6(25): 640-642; (2) Li Suqun, et al. Journal of Caoye, 1993, 6(2): 52-55. ]. During various growth stages of red bean grass, the leaves of red bean grass can produce bloat disease preventive agent - condensed tannin (CT), which can cause soluble protein to coagulate. Under the action of the bacterium, the bloat caused by violent decomposition and gas generation increases the content of rumen-passing protein and improves the nutritional value of forage grass [Li Suqun, et al. Journal of He...

Claims

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Application Information

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IPC IPC(8): C12N15/84C12N15/65A01H5/00
Inventor 陈春艳马晖玲董文科杨江伟
Owner GANSU AGRI UNIV
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