Method for improving instantaneous expression rate of agrobacterium tumefaciens-mediated sugarcane glucuronidase (GUS)

An Agrobacterium-mediated, transient expression technology, applied in the field of bioengineering, can solve the problems of low conversion rate, high operating cost, and high genetic stability of traits, and achieve the effects of increased transient expression rate, strong practicability, and simple operation

Inactive Publication Date: 2012-07-25
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

There are mainly two approaches to transgenic technology in a narrow sense, one is the gene gun bombardment method, and the other is the Agrobacterium-mediated method. The former has many application examples in sugarcane, but this technology has its inherent shortcomings. In addition to expensive equipment, high operating costs, and uncertain target gene source insertion sites, almost all transgenic lines obtained by this technology have multiple copies, which is not conducive to the stable inheritance of traits and transgenic safety, and is prone to gene silencing. The imported exogenous gene is easy to lose and other problems; the insertion site of the latter is clear, the frequen

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  • Method for improving instantaneous expression rate of agrobacterium tumefaciens-mediated sugarcane glucuronidase (GUS)
  • Method for improving instantaneous expression rate of agrobacterium tumefaciens-mediated sugarcane glucuronidase (GUS)
  • Method for improving instantaneous expression rate of agrobacterium tumefaciens-mediated sugarcane glucuronidase (GUS)

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Embodiment 1

[0025] Embodiment 1. A method for improving the transient expression rate of GUS in sugarcane mediated by Agrobacterium

[0026] A method for improving the transient expression rate of sugarcane GUS mediated by Agrobacterium, comprising the following steps:

[0027] 1. Material selection: the selected bacterial liquid is A bacterial liquid; the selected sugarcane variety is ROC22;

[0028] Verify that the A bacterial solution is a positive bacterial solution: the primer is GUSF - 5'- TCAGCGCGAAGTCTTTATAC- 3' GUSR -5'- TTCAGTTCGTTGTTCACACA- 3'; the program is 95°C pre-denaturation for 5min, 95°C denaturation for 30s, 56°C annealing for 30s, 72°C Extend for 30s, 30 cycles, and finally extend for 10min; the PCR test results of A bacteria solution are attached figure 1 As shown, the b and c lanes amplify the target band with the same size at the position of 210bp, and the A bacterial solution is a positive bacterial solution;

[0029] 2. Preparation of transformed bacteria solu...

Embodiment 2

[0038] Embodiment 2. A method for improving the transient expression rate of GUS in sugarcane mediated by Agrobacterium

[0039] A method for improving the transient expression rate of sugarcane GUS mediated by Agrobacterium, comprising the following steps:

[0040] 1. Material selection: the selected bacterial liquid is A bacterial liquid; the selected sugarcane variety is Funong 95-1702;

[0041] 2. Preparation of transformed bacteria solution: transfer 100 μl of A bacteria solution to 50 mL of LB liquid medium containing 35 mg / L rifampicin and 50 mg / L kanamycin, at 28°C, 150 r / min Shake culture to OD 600 =1.2, the bacteria were suspended in M1 after centrifugation at 5000 r / min for 10 min, after centrifugation, resuspended in M1 again and diluted to OD 600 =1.2, that is, the transformed bacterial liquid;

[0042] 3. Infection method: get the healthy and pest-free plant top part of the sugarcane variety Funong 95-1702, cut it off from the 30 cm below the highest hypertrop...

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Abstract

The invention relates to a method for improving the instantaneous expression rate of an agrobacterium tumefaciens-mediated sugarcane glucuronidase (GUS). The method comprises the following steps of: selecting materials, preparing conversion bacteria liquid, performing an infection method, performing cocultivation and selective cultivation, and chemically dyeing GUS active tissues. The method that the advantages and disadvantages of a transgenic technology method is measured by taking the degree of the GUS instantaneous expression rate as an index is nationally acknowledged. Therefore, the index which is nationally acknowledged is adopted to evaluate the technology method. By the method, the GUS instantaneous expression rate can be increased by 335.8 to 1,859.3 percent, the bottle neck problem about efficiently obtaining a transgenic sugarcane plant by using a agrobacterium tumefaciens-mediated way can be effectively solved, a key technology method for introducing an exogenous gene into a sugarcane is provided for improving the variety of the sugarcane by using transgenic technology. Meanwhile, the method is also applied to the genetic transformation for other plants based on an agrobacterium tumefaciens-mediated way.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for improving the transient expression rate of GUS in sugarcane mediated by Agrobacterium. Background technique [0002] sugar cane( Saccharum Complex) is the most important sugar crop. Sucrose accounts for 76% of the world's total sugar production and 92% of my country's total sugar production. Sugarcane is also one of the most important biomass energy crops. C4 plants with the largest amount and fuel ethanol production. The contribution rate of sugarcane variety improvement to industrial scientific and technological progress is as high as 60%, but due to the large genome of sugarcane, the genetic background is very complex, the number of chromosomes is more than 120, and the modern sugarcane cultivated species is the interspecific hybridization of 3 or 4 species of Succulus The complex belongs to allopolyploid plants. At the same time, the target object of sug...

Claims

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Application Information

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IPC IPC(8): C12N15/82A01H5/00
Inventor 许莉萍高世武林庆良陈如凯
Owner FUJIAN AGRI & FORESTRY UNIV
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