Agrobacterium-mediated spirodela polyrhiza stable transformation system establishment method

An Agrobacterium-mediated and stable technology, applied in the fields of molecular biology and genetic engineering, can solve problems such as unreported

Inactive Publication Date: 2016-10-12
INST OF AQUATIC LIFE ACAD SINICA
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  • Abstract
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  • Claims
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Problems solved by technology

At present, many species of Duckweed have established their gene transformation systems, such as Spirodelapunctata (Edelman et al., 1998), Spirodela oligorrhiza (Rival et al., 2008; Vunsh et al., 2007), Lemna gibba (Stomp and Rajbhandari , 2000), Lemna minor (Gasdaska et al., 2003), and Wolffia arhiza (Khvatkov et al., 2015), and the establishment of the Ziping (Spirodelapolyrhiza) gene transformation system has not been reported

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  • Agrobacterium-mediated spirodela polyrhiza stable transformation system establishment method
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  • Agrobacterium-mediated spirodela polyrhiza stable transformation system establishment method

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Embodiment 1

[0065] A method for the establishment of a stable transformation system of Zipinkia gene mediated by Agrobacterium {auxin reporter gene (DR5::GUS) and cytokinin reporter gene (TCS::GUS)}. DR5::GUS is a synthetic auxin-responsive construct (Ulmasov et al., 1997). DR5 is composed of multiple 11-base repeat sequences, including the auxin response element TGTCTC sequence, which is fused with the GUS reporter gene to mark the auxin response; TCS::GUS is also a synthetic construction that marks the distribution of cytokinins, and is produced by cells The mitogen B-type response regulatory site and the minimal 35S promoter (minimal promoter), which are fused with the GUS reporter gene (Müller and Sheen, 2008). The specific implementation steps are as follows:

[0066] 1. Transformation of plant expression vector pKGWFS7.0 in Escherichia coli (DH5α):

[0067] The plant expression vector pKGWFS7.0 (purchased from Shanghai Maiqi Biotechnology Co., Ltd., the same below) with auxin repo...

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Abstract

The invention discloses an agrobacterium-mediated spirodela polyrhiza stable transformation system establishment method, which comprises the following steps: A, inducing calli of spirodela polyrhiza; B, connecting a target gene with a T vector; C, connecting the target gene with a plant expression vector; D, transforming the plant expression vector in agrobacterium tumefaciens LBA4404; E, preparing an infective bacteria solution; F, co-culturing the calli and the agrobacterium tumefaciens; G, performing regeneration and selective culturing: determining that the target gene is successfully integrated into a genome of the spirodela polyrhiza if a GUS (glucuronidase) staining result of thalli is blue, and determining that a part which is stained to be blue by thallus tissues is a part expressed by the target gene. The target gene is inserted into the plant expression vector and then transferred into the agrobacterium tumefaciens LBA4404 for callus infection, and the concentration of antibiotics is finally controlled to screen a positive plant to obtain transgenetic spirodela polyrhiza, and an established transformation system can still ensure continuous and stable inheritance after asexual reproduction of the spirodela polyrhiza, and has bio-safety and development and application value.

Description

technical field [0001] The invention relates to the technical fields of molecular biology and genetic engineering, in particular to a method for establishing a stable transformation system of Spirodela polyrhiza mediated by Agrobacterium. The establishment of the Ziping stable transformation system is for the development and utilization of Ziping as a bioreactor to produce proteins (medicinal vaccines and antibodies), enzymes, sugars and lipids and other secondary metabolites, and to enhance the ability of high The tolerance to low temperature, salt, heavy metals, nitrogen and phosphorus has laid the foundation for bioremediation. Background technique [0002] Since the first transgenic tobacco was obtained in the world in 1983, plant transgenic technology has developed rapidly and has been widely used in the world. Its huge production potential has brought great economic and social benefits to mankind. Plant transgenic technology is mainly used in the fields of agriculture...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H4/00
CPCC12N15/8205A01H4/001A01H4/008
Inventor 杨晶晶侯宏伟
Owner INST OF AQUATIC LIFE ACAD SINICA
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