Method for preparing leaf receptor material for sugarcane transgene transient expression

A technology of transient expression and receptor materials, applied in other methods of inserting foreign genetic materials, plant cells, recombinant DNA technology, etc., can solve the problems of high cost, long time, low efficiency, etc., and achieve consistent physiological state and smooth surface Effect

Inactive Publication Date: 2013-08-07
FUJIAN AGRI & FORESTRY UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The method of the present invention provides a fast, efficient and convenient preparation method for the receptor material required for the study of transient expression of sugarcane transgenes, and overcomes the existing time for the research system of transient expression of transgenes using sugarcane callus as the receptor material long, low efficiency and high cost

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  • Method for preparing leaf receptor material for sugarcane transgene transient expression
  • Method for preparing leaf receptor material for sugarcane transgene transient expression
  • Method for preparing leaf receptor material for sugarcane transgene transient expression

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1: The leaves of sugarcane variety CP72-1210 were used as the recipient material, and GUS was used as the reporter gene to study the activities of different promoters.

[0028] 1. Transient expression of exogenous genes

[0029] 1.1 Tungsten powder preparation:

[0030] 1.1.1 Weigh 30 mg tungsten powder (1.1 μm, Bio-Rad Company) into a sterilized 1.5 mL Eppendorf tube.

[0031] 1.1.2 Add 500 μL of absolute ethanol.

[0032] 1.1.3 Vortex on a vortex for 1 min, then centrifuge at 8000 g for 1 min.

[0033] 1.1.4 Remove the ethanol supernatant.

[0034] 1.1.5 Repeat steps 1.1.2 to 1.1.4.

[0035] 1.1.6 Add 500 μL sterile water and wash twice.

[0036] 1.1.7 Add 500 μL sterile water, the final concentration of tungsten powder solution is 60 ng / μL.

[0037] 1.2 Coating of exogenous gene DNA (calculated according to the bombardment amount of 10 times for each test treatment)

[0038] 1.2.1 Pipette 83.3 μL of the solution containing tungsten powder into a steril...

Embodiment 2

[0048] Example 2: The leaves of sugarcane variety CP72-1210 were used as the recipient material, and EYFP was used as the reporter gene to study the activities of RNA silencing suppressors (P19, γb) encoded by different plants.

[0049] 1. Transient expression of exogenous genes

[0050] 1.1 Preparation of tungsten powder (the method is as the preparation of tungsten powder in Example 1);

[0051]1.2 Encapsulation of exogenous gene DNA (method as in Example 1 for exogenous gene DNA encapsulation);

[0052] 1.3 Bio-Rad bombardment:

[0053] The leaf receptor material of the sugarcane transgene transiently expressed in the present invention is transferred to fresh MS0.6 solid medium after 3-5 days of dark culture in small cube young leaves. Pipette 10 μL of the plasmid-coated tungsten powder-ethanol mixture to coat the particle slide, and use high-pressure helium to introduce the plasmid into the young sugarcane leaf cells. The bombardment parameters were helium pressure ...

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Abstract

The invention relates to a method for preparing a leaf receptor material for sugarcane transgene transient expression. The method includes taking a sample from a sugarcane leaf explant field; disinfecting surfaces of sugarcane leaf explants; preparing the sugarcane leaf explants; and pre-culturing the sugarcane leaf explants. The method has the advantages that the traditional sugarcane callus tissues are replaced by the explants, a quick, efficient, simple and convenient technology for preparing the sugarcane leaf receptor material is created, and the method can be widely applied to sugarcanetransgene transient expression research in the aspects of high-throughput exogenous gene expression, promoter analysis, gene silencing and the like.

Description

technical field [0001] The invention relates to a preparation method of a plant transgene receptor material, in particular to a preparation method of a sugarcane transgene transient expression leaf receptor material. Background technique [0002] Transgenic research is an important way to identify plant gene functions, usually using tobacco or Arabidopsis as model plants. Tobacco or Arabidopsis transgene stable expression research technology has matured. In recent years, the transient expression system mediated by Agroinfiltration has been widely used in Nicotiana benthamiana ( Nicotiana benthamian ) and its transgenic line 16C. The reporter gene and the RNA silencing suppressor were introduced into the leaf cells of Nicotiana benthamiana or 16C plants by mixed injection of Agrobacterium, and the expression of the reporter gene was analyzed to identify the activity of the RNA silencing suppressor encoded by the plant virus. [0003] Sugarcane is a monocotyledonous gramineo...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N15/09C12N15/87C12N5/04
Inventor 高三基米尔科夫艾瑞克陈如凯林艺华付为林傅华英陈永武
Owner FUJIAN AGRI & FORESTRY UNIV
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