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Identification method for insertion site of wheat exogenous gene

An identification method and a technology of exogenous genes, which are applied in the field of identification of insertion sites of exogenous genes in wheat, can solve the problems of low gene transformation efficiency, achieve the effect of low false positive occurrence probability and prevent interference

Pending Publication Date: 2017-12-01
TIANJIN AGRICULTURE COLLEGE
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Problems solved by technology

Due to the randomness of the insertion of the target gene and the blindness of gene expression at the insertion site in the transgenic technology, the safety of transgenic crops has aroused widespread public concern; and the gene transformation efficiency of cereal crops such as wheat is extremely low, and the search for an effective Method to identify GM wheat is crucial

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  • Identification method for insertion site of wheat exogenous gene
  • Identification method for insertion site of wheat exogenous gene
  • Identification method for insertion site of wheat exogenous gene

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Embodiment 1

[0036] 1. Extraction of Genomic DNA from Transgenic Wheat

[0037] 1) Material collection of transgenic wheat: After the transgenic wheat plants are obtained by the Agrobacterium-mediated method, at the seedling stage, select the green and stretched leaves, use sterilized scissors and tweezers, cut off the leaves of about 2cm, cut them into pieces and put them into 2mL Store in sterilized centrifuge tubes at -80°C.

[0038] 2) Extraction of genomic DNA: Use a sterile grinding rod to grind the leaves in liquid nitrogen until they are ground into a whitish powder, and use the kit Sangon Bioengineering (Shanghai) Co., Ltd. Ezup column plant genomic DNA extraction Kit model: B518261-0100, the operation steps are as shown in the kit instructions, and the genomic DNA of wheat leaves is extracted. The DNA solution was obtained, mixed gently, collected by short centrifugation to the bottom of the tube, and 1 μL of the extract was taken to measure the concentration with a micro-ultrav...

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Abstract

The invention relates to an identification method for an insertion site of a wheat exogenous gene, and belongs to the technical field of transgenic crop identification. The identification method provided by the invention comprises the following steps: 1) extracting a wheat genome; 2) designing three nested special primers SP1, SP2 and SP3; 3) combining the special primers SP1, SP2 and SP3 obtained in the step 2) with a chromosome walking kit to perform thermal asymmetric interlaced PRC reaction, thereby obtaining an amplified fragment; and 4) comparing the amplified fragment obtained in the step 3) with wheat genome data of the national wheat genome sequencing league to obtain the insertion site, in wheat, of the exogenous gene. The identification method provided by the invention is low in false positive occurrence probability, can precisely locate an insertion position of the exogenous gene, has profound significance in researching functions of later-period transgenetic seedlings, and lays a foundation for breeding new germplasm resources of wheat.

Description

technical field [0001] The invention relates to the technical field of identification of transgenic crops, in particular to an identification method for insertion sites of exogenous genes in wheat. Background technique [0002] Wheat is one of the most important food crops in the world, providing abundant protein and energy for the global population, and it is also the second largest food crop in my country. With the increase of population, the global demand for wheat will show a trend of substantial growth in the future. Due to genetic limitations, the growth of wheat yield is limited, and traditional cultivation techniques cannot meet the demand. Transgenic technology may become an important means to alleviate food security problems. In order to meet the demand, it is necessary to cultivate transgenic plants by means of genetic engineering. Transgenic technology is a flourishing means to help cultivate plants with desirable traits and has great potential. Due to the ra...

Claims

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Application Information

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IPC IPC(8): C12Q1/68
CPCC12Q1/6806C12Q1/6869G16B30/00C12Q2531/107C12Q2565/125
Inventor 丁博谢晓东郭雨李明曹雪松李杨詹瑶邢欣欣曹高燚佟德清
Owner TIANJIN AGRICULTURE COLLEGE
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