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Method for transient expression of exogenous gene by cotton cotyledon

A technology of exogenous gene and transient expression, applied in genetic engineering, plant genetic improvement, botanical equipment and methods, etc., can solve the problems of high cost and complicated operation, and achieve the effect of clear dyeing and easy operation

Inactive Publication Date: 2013-09-04
SHAANXI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In recent years, the transient expression system in cotton is mainly completed by protoplasts formed by enzymatic hydrolysis of cotton callus. This method can quickly detect the transient expression and subcellular localization of cotton functional genes, but the operation is complicated and requires a gene gun. Gene transformation is costly

Method used

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  • Method for transient expression of exogenous gene by cotton cotyledon
  • Method for transient expression of exogenous gene by cotton cotyledon
  • Method for transient expression of exogenous gene by cotton cotyledon

Examples

Experimental program
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Effect test

Embodiment 1

[0040] The method for the transient expression of exogenous genes in cotton cotyledons of this embodiment consists of the following steps:

[0041] 1. Cotton planting

[0042] Soak 40 cotton seeds at 37°C for 12 hours, and mix the nutrient soil and vermiculite at a mass ratio of 3:1 to form a cultivation soil. The nutrient soil and vermiculite are commodities sold on the market. Sales by the company, soaked cotton seeds absorb water for 8 hours, planted in cultivation soil, planting depth is 1cm, covered with soil, cultivated in an incubator at 25°C, humidity 51±2%, 16 hours of light, 8 hours of darkness, and light intensity of 24000lux to germination of cotton seeds.

[0043] 2. Preparation of transformation solution

[0044] The foreign aid gene D7-pCambia1301 expression vector was constructed as follows, and PCR upstream and downstream amplification primers containing Nco I restriction sites were designed, and the primer sequences were:

[0045] F: ATTCCATGGCAGTTACACAATT...

Embodiment 2

[0065] The method for the transient expression of exogenous genes in cotton cotyledons of this embodiment consists of the following steps:

[0066] Cotton planting step 1 is identical with embodiment 1.

[0067] In step 2 of preparing the transformation solution, draw 200 μL of Agrobacterium GV3101 containing the D7-pCambia1301 expression vector and add it to 10 mL of LB liquid medium, add 50 mg / mL of kanamycin 10 μL, 50 mg / mL 10 μL of rifampicin, 10 μL of gentamicin at 50 mg / mL, the volume ratio of LB liquid medium to kanamycin at 50 mg / L, rifampicin at 50 mg / L, and gentamicin at 50 mg / L are 1: 0.001: 0.001: 0.001, 28 ° C, 180 rpm shaking culture for 15 to 18 hours, prepare a bacterial liquid with an OD value of 0.3 at 600 nm, centrifuge the bacterial liquid at 10,000 rpm for 1 minute, remove the supernatant, Collect the bacteria, add MS liquid medium and infection solution and mix well, the volume ratio of the bacteria to MS liquid medium and infection solution is 1:90:10 t...

Embodiment 3

[0071] The method for the transient expression of exogenous genes in cotton cotyledons of this embodiment consists of the following steps:

[0072] Cotton planting step 1 is identical with embodiment 1.

[0073] In step 2 of preparing the transformation solution, draw 200 μL of Agrobacterium GV3101 containing the D7-pCambia1301 expression vector and add it to 10 mL of LB liquid medium, add 50 mg / mL of kanamycin 10 μL, 50 mg / mL 10 μL of rifampicin, 10 μL of gentamicin at 50 mg / mL, the volume ratio of LB liquid medium to kanamycin at 50 mg / L, rifampicin at 50 mg / L, and gentamicin at 50 mg / L are 1: 0.001: 0.001: 0.001, 28 ° C, 180 rpm shaking culture for 15 to 18 hours, prepare a bacterial liquid with an OD value of 0.6 at 600 nm, centrifuge the bacterial liquid at 10,000 rpm for 1 minute, remove the supernatant, Collect the bacteria, add MS liquid medium and infection solution and mix well, the volume ratio of the bacteria to MS liquid medium and infection solution is 1:90:10 t...

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Abstract

The invention discloses a method for transient expression of an exogenous gene by cotton cotyledon, which consists of the steps of cotton planting, preparation of conversion liquid, injection and infection, cotton culture and GUS (glucuronidase) gene expression and detection. The method disclosed by the invention has the advantages of convenience, high speed, easiness in operation, clear dyeing and the like, and is suitable for detecting the expression of the exogenous gene from cotton, the subcellular localization and the promoter activity.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to the establishment of a transient expression system of cotton cotyledons mediated by Agrobacterium. Background technique [0002] Transient expression refers to a technique in which foreign genes are transcribed and translated as genetic material outside the genome after they are introduced into plant cells, and the expression products are accumulated in transformed cells. The time for transcription and translation is usually several days to more than ten days . The transient expression system has the advantages of high expression efficiency, short experimental cycle, simple operation and less restriction on materials. Common transformation methods for transient expression systems include particle gun bombardment, friction inoculation, protoplast transformation and Agrobacterium-mediated transient transformation. These transformation methods have their own advantages and...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00
Inventor 俞嘉宁王蕾何鹏周丹丹
Owner SHAANXI NORMAL UNIV
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