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Method for synchronously identifying transgenic mannose gene, glucanase gene, xylanase gene and galactosidase gene plants

A technology of mannanase and galactosidase, which is applied in biochemical equipment and methods, measurement/testing of microorganisms, DNA/RNA fragments, etc., and can solve the problems of low amplification efficiency of interactions

Active Publication Date: 2014-04-30
INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although multiplex PCR has many advantages, it also has many disadvantages that cannot be ignored, including the interaction between multiple pairs of primers, low amplification efficiency, and the influence of different templates on primers.
These shortcomings inhibit the application of multiplex PCR

Method used

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  • Method for synchronously identifying transgenic mannose gene, glucanase gene, xylanase gene and galactosidase gene plants
  • Method for synchronously identifying transgenic mannose gene, glucanase gene, xylanase gene and galactosidase gene plants
  • Method for synchronously identifying transgenic mannose gene, glucanase gene, xylanase gene and galactosidase gene plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] Example 1 Four kinds of feed enzyme transgenic maize genome extraction

[0036] 1. Four methods of obtaining transgenic corn

[0037] The target gene obtained by codon optimization was connected to the plant expression vector pHP20754, and transformed into maize callus by the method of gene gun. The positive callus was screened by the selection medium, and the screened callus was regenerated to obtain the T0 generation carrier, and then hybridized with the non-transformed maize line Zheng 58 to obtain the transformed maize. The plant expression vector pHP20754, callus and Zheng 58 corn were all from Chen Rumei, a researcher at the Institute of Biology, Chinese Academy of Agricultural Sciences.

[0038] 2. Extraction of maize genome

[0039] 1) CTAB preparation

[0040] Element

final concentration

CTAB

2%

NaCl

1.4mol / L

[0041] EDTA-Na2 (pH8.0)

20mmol / L

Tris-HCl (pH8.0)

100mmol / L

β-mercaptoetha...

Embodiment 2

[0050] The primer-specific detection of embodiment 2 four kinds of feed enzyme genes

[0051] Using galactosidase aga primers A-F / R, glucanase glu primers G-F / R, mannanase man primers M-F / R, xylanase xyl primers X-F / R, respectively aga, glu, man and xyl The transgenic maize genome was used as a template to test the specificity of four pairs of primers. The non-transgenic Zheng 58 corn genome and ddH2O were used as templates for negative control.

[0052] Primer concentration: 20μmol / L; genome quantification: 1μg / μl

[0053] reaction system:

[0054]

[0055] PCR reaction parameters: 95°C for 5min, 95°C for 30sec, 60°C for 30sec, 72°C for 60sec, 30 cycles, 72°C for 10min.

[0056] Experimental results such as figure 1 As shown, when experimenting with primers A-F / R, there will be a 1028bp specific band when only using galactosidase transgenic corn as a template; when using primer G-F / R, only using glucanase transgenic corn as a template There will be a 783bp specific ba...

Embodiment 3

[0057] Example 3 Multiple Genome Templates Effect on the Specificity of Each Specific Primer

[0058] Using four feed enzyme transgenic maize genomes as templates, four pairs of primers, galactosidase aga primer A-F / R, glucanase glu primer G-F / R, mannanase man primer M-F / R, xylanase Enzyme xyl primers X-F / R were used for PCR amplification to detect the effect of the template mixture on the specificity of the four pairs of primers.

[0059] Primer concentration: 20 μmol / L;

[0060] Genome quantification: 1μg / μl

[0061] reaction system:

[0062]

[0063] PCR reaction parameters: 95°C for 5min, 95°C for 30sec, 60°C for 30sec, 72°C for 60sec, 30 cycles, 72°C for 10min.

[0064] Experimental results such as figure 2 As shown, four feed enzymes galactosidase, glucanase, mannanase and xylanase transgenic maize genome were used as templates, and primers A-F / R, G-F / R, M-F / R and X-F were used respectively When / R is used for amplification, it can and can only amplify the corre...

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Abstract

The invention relates to the field of transgenic plant detection, and in particular to a method for synchronously identifying transgenic mannose gene, glucanase gene, xylanase gene and galactosidase gene plants. A primer, designed according to the invention, of exogenous genes of transgenic galactosidase, glucanase, mannose and xylanase can be used for synchronously identifying the four exogenous genes in a transgenic corn feed, so that a method for rapidly identifying and detecting ingredients of a transgenic corn material for feeds is provided.

Description

technical field [0001] The invention relates to the field of detection of transgenic plants, in particular to a method for simultaneously identifying plants with transgenic mannanase genes, glucanase genes, xylanase genes and galactosidase genes. Background technique [0002] In addition to conventional insect-resistant and disease-resistant transgenic strains, with the development of genetic engineering technology, the breeding of new varieties of functional transgenic crops for feed has become a research hotspot, such as the phytase gene transgenic corn that has obtained safety certification in green feed It has a good application prospect in production. Transmannanase gene, transglucanase gene, transxylanase gene and transgalactosidase gene corn are new feeding functionalities developed to reduce anti-nutritional factors in feed and improve the nutritional performance of feeding animals Transgenic corn varieties, because the target enzyme can be specifically expressed in...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/68C12N15/11
Inventor 姚斌徐晓露孟昆杨培龙罗会颖王亚茹
Owner INST OF ANIMAL SCI OF CHINESE ACAD OF AGRI SCI