Construction method for economic plant effective root transgene system

A construction method and plant technology, applied in botany equipment and methods, plant products, angiosperms/flowering plants, etc., can solve the problems of lack of rapid screening system for gene function analysis and limited development of research fields

Active Publication Date: 2019-05-10
BEIJING FORESTRY UNIVERSITY
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

Due to their uniqueness or long growth cycle, most medicinal plants and woody plants lack efficient rapid sc

Method used

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  • Construction method for economic plant effective root transgene system
  • Construction method for economic plant effective root transgene system
  • Construction method for economic plant effective root transgene system

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Taking the economic plant edamame as an example to illustrate the construction method of effective root transgenic system

[0048] Pigeon pea seeds came from the Key Laboratory of Forest Plant Ecology, Ministry of Education, Northeast Forestry University. The surface of pigeonpea seeds is sterilized with 0.1% mercuric chloride for 5-10 minutes, and then washed five times with sterile water. Inoculate the sterilized pigeonpea seeds in MS medium and cultivate them for about 1-2 months. For pigeonpea, the bacterial solution can be injected into the subcultured plants after about 30 days of growth. For other plants, they should be moved to soil (10 cm (diameter) × 9 cm (height) pots, soil and sand, the volume ratio is 3:1), and grown in a high-humidity environment at 25°C. At 50 μmol photon m in a 16 h photoperiod before performing the injection experiment -2 ·s -1 glow everywhere. In order to screen a strain of A. rhizogenes with higher transformation efficiency in pi...

Embodiment 2

[0066] The effects of injection concentration of Agrobacterium solution on callus induction rate, hairy root induction rate and transgenic rate were tested. Taking the K599 strain as the infecting strain, in order to select the optimal concentration of the K599 strain suspension, the concentrations were set to 0.2, 0.3, 0.4, 0.5 and 0.6OD 600 Value, take pigeonpea as object, implement and calculate hairy root regeneration rate according to the method of embodiment 1.

[0067] RT-PCR (operating method is the same as in Example 1) and GFP signal detection experiments were used to verify the transfer of the target gene. Described detection GFP signal experiment is specifically as follows: get pigeon pea regeneration hairy root protein, through the protein sample that PAGE (polyacrylamide gel electrophoresis) separates, transfer on the solid phase carrier (NC film), add rabbit anti-GFP serum to start Immunoreaction, then reacted with horseradish peroxidase, detected the expressio...

Embodiment 3

[0070] The best injection sites for pigeonpea seedling plants were tested separately.

[0071] First, the stem located 0.1–1.0 cm above the original hairy root of the seedling was divided into three parts with a height of 0.5 cm; they were named positions A, B and C ( image 3 (a)). With K599 strain as the infection strain, implement according to the method of Example 1.

[0072] The result shows: the callus of B and C position and the induction rate of hairy root are all about 60%, relatively A position has higher efficiency ( image 3 (b)).

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Abstract

The invention provides a construction method for an economic plant effective root transgene system, and belongs to the technical field of plant transgene. A bacterial liquid containing a target recombinant vector agrobacterium rhizogene is subjected to solid-liquid separation, an obtained thallus containing the target recombinant vector agrobacterium rhizogene is subjected to resuspension, an obtained agrobacterium rhizogene suspension is injected into stems of economic plant seedlings, and 10-12 days after injection is conducted, calluses grow at wounds of the stems of the economic plant seedlings; the seedlings continue to be cultured for 28-35 days, and when the calluses are differentiated into regenerated hairy roots, original hairy roots are cut off. The construction method is applicable to various economic plants, and the regeneration rate of the hairy roots and the regeneration rate of the calluses are high. The construction method provides very rapid and effective gene system function analysis, secondary metabolite engineering and plant stress response studies. According to the constructed root transgene system, only the roots are transgenic roots rather than the whole plants, and the constructed system is a good system for studying signal transduction between the roots and the stems.

Description

technical field [0001] The invention belongs to the technical field of plant transgenics, and in particular relates to a method for constructing an effective root system transgenic system of economic plants. Background technique [0002] The Agrobacterium-mediated plant transformation system has been the most widespread and successful approach in plant genetic engineering in recent decades (Gelvin, 2003; Vain, 2007; Matveeva and Lutova, 2014). Agrobacterium tumefaciens can induce tumor growth in plants by injuring and transferring tumor-inducing (Ti) plasmids to the plant nucleus. Based on this theory, the tumor-inducing genes are transformed into desired genes by modifying and removing Ti plasmids, and carry any desired genes into the chromosomes of plant cells. Since the first widespread demonstration of the successful transfer of exogenous genes into tobacco using the Agrobacterium-mediated plant transformation method in 1983, gene function studies and plant genetic modi...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/06A01H6/54
Inventor 付玉杰孟冬牛丽丽杨清董碧莹宋治华
Owner BEIJING FORESTRY UNIVERSITY
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