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Application of high temperature resistant beta-glucosidase gene in plant gene conversion

A glucosidase and high temperature resistant technology, which is applied in the fields of plant genetic improvement, application, genetic engineering, etc., can solve the problems of deviation, interference, and high cost of research results, and achieve the effects of reducing detection costs, increasing reliability, and improving sensitivity

Inactive Publication Date: 2009-04-29
SHANGHAI ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the detection of GUS reporter genes in GMOs is often interfered by the endogenous GUS activity of the target organism, which limits the scope of application of GUS reporter genes and reduces the reliability of GUS as a reporter gene; in addition, because X-Gluc is very expensive, this Also caused the application of GUS reporter gene to be limited to a certain extent
Moreover, due to the obvious endogenous GUS activity in fruits, seed coats, endosperms and embryos, the research results of tissue-specific regulation are biased; in the process of transgenic plant detection, vegetative bodies are often selected for staining, but endogenous GUS is also unavoidable. Background, so in the daily staining process, high concentrations of methanol are often added to the staining solution to inhibit the endogenous GUS activity in the vegetative stage tissue, even so, the sensitivity of GUS detection is greatly reduced
Endogenous GUS activity is more prevalent in microorganisms, making it difficult to apply GUS genes in transgenic microorganisms
At present, the commercialization of transgenic plants often needs to obtain a large number of independent transgenic transformants, and large-scale histochemical staining is required; in addition, special tissue-specific regulation studies require staining of the entire transgenic plant. At this time, the staining substrate X-Gluc The high cost of

Method used

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  • Application of high temperature resistant beta-glucosidase gene in plant gene conversion
  • Application of high temperature resistant beta-glucosidase gene in plant gene conversion
  • Application of high temperature resistant beta-glucosidase gene in plant gene conversion

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1: Construction of Agrobacterium Transformation Binary Vectors pYG8557 and pYG8555

[0046] (1) Test method:

[0047] The Agrobacterium transformation binary vectors pYG8557 and pYG8555 of high temperature resistant GUS gene gus-tr3337 and Escherichia coli wild-type GUS gene gus-wt were constructed on the basis of binary vector pYF7716. The binary vector pYF7716 constructed by Biotechnology Research Institute of Shanghai Academy of Agricultural Sciences is constructed on the basis of vector pCAMBIA-1304. The binary vector pYF7716 has the following features: double CAMV35S promoter and Nos-ter terminator to control the expression of the target gene, CAMV 35S+TMVOmega leader sequence and CAMV 35S PolyA to control the expression of the intron Km resistance gene, as a transgenic plant screening marker genes. When constructing the Agrobacterium transformation binary vectors pYG8557 and pYG8555 of the high-temperature-resistant GUS gene gus-tr3337 and the Escherichi...

Embodiment 2

[0052] Embodiment 2: Transformation of Agrobacterium by electric shock method

[0053] (1) Test method:

[0054] 1) Prepare Agrobacterium AG211 competent, the method refers to MicroPulser TM Electroporation Apparatus Operating Instructions and Application Guide (BIO-RAD Corporation).

[0055] 2) Take 50 μL of AG211 competent cells, add 1 μL of DNA (plasmids pYG8555 and pYG8557), and transfer to a 0.2 cm electric shock cup for transformation (400Ω, 2.5KV, 25 μf). Add 1 ml of LB medium containing 1% mannitol to restore the culture for 2 hours (28° C., 250 rpm). Take 10 μL and 100 μL respectively and smear LB plates (rifampicin 50 μg / ml, gentamicin 50 μg / ml, chloramphenicol 100 μg / ml).

[0056] (2) Test results:

[0057] After 2 days of cultivation, Agrobacterium single clones were grown on LB plates.

Embodiment 3

[0058] Example 3: Arabidopsis Transformation

[0059] (1) Test method:

[0060] 1. Preparation of Agrobacterium

[0061] 1) A single Agrobacterium was picked and inoculated in 5 ml of LB liquid medium (rifampicin 50 μg / ml, chloramphenicol 100 μg / ml), and cultured at 28°C and 250 rpm for 20 hours.

[0062] 2) Transfer 1ml of bacterial liquid into 20-30ml LB liquid medium (rifampicin 50μg / ml l, chloramphenicol 100μg / ml), culture at 28°C, 250 rpm for about 12h, measure OD600≈1.5.

[0063] 3) The cells were collected by centrifugation at 8000 rpm at 4°C for 10 min, resuspended in Agrobacterium transformation permeate (5% sucrose, 0.05% Silwet L-77) and diluted to OD600≈0.8.

[0064] 2. Transformation of Arabidopsis by dipping flowers

[0065] 1) Immerse the flower moss of Arabidopsis thaliana in the permeate, stir gently for about 10 seconds and take it out. After all the transformation is completed, add water to the tray, cover the Arabidopsis with plastic wrap to maintain a h...

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Abstract

The invention relates to application that a high-temperature resistant beta-glucuronide gene is obtained through combining the evolvement of oriented molecules of a gene in vitro and the mutation of a fixed point and is used as a report gene in plant gene transformation, and belongs to the field of plant gene engineering. The invention discloses a method for using the high-temperature resistant beta-glucuronide gene in the plant gene transformation and effect thereof. The high-temperature resistant beta-glucuronide gene can prevent interference of entogenous background of a transgenic organism and increase the reliability of transgenic detection; the high-temperature resistant beta-glucuronide gene is also used to obviously improve the sensitivity of transgenic detection so that the research result of expression and regulation induced by spatial specificity, biotic environment or abiotic environment has larger difference and more accurate data, thereby greatly reducing the use amount of a primer and reducing detection cost.

Description

technical field [0001] The invention relates to a reporter gene in the field of plant genetic engineering, in particular to a high-temperature-resistant beta-glucosidase gene obtained by combining gene in vitro directional molecular evolution and site-directed mutation. Especially the application of the gene as a reporter gene in plant gene transformation. Background technique [0002] In transgenics, marker genes and reporter genes are selected to facilitate the rapid and convenient screening and identification of transgenic materials. As a reporter gene, the following conditions should be met in genetic selection and screening detection: (1) It has been cloned and sequenced; (2) The expression product does not exist in the recipient cell or has no similar endogenous expression product , that is, there is no background; (3) its expression product can be quantitatively determined. [0003] Currently commonly used screening marker genes include: antibiotic resistance gene, ...

Claims

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Application Information

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IPC IPC(8): C12N15/56C12N15/82A01H1/00
Inventor 熊爱生姚泉洪彭日荷高峰薛永付晓燕李贤田永生赵伟
Owner SHANGHAI ACAD OF AGRI SCI
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