Multiplex-polymerase chain reaction (PCR) rapid screening method for target gene elements of transgenic sugarcane

Abstract

A multiplex-polymerase chain reaction (PCR) primer is designed by a sugarcane endogenous gene ALS and an exogenous gene element (including Ubiquitin promoter, NOS promoter, NOS terminator, a Bar gene and a Bt gene). After being optimized, a multiplex-PCR reaction system is established, wherein a template which has 60% Premixtaq<TM> volume, 0.3muM primer concentration and 150ng DNA is added into the reaction system, and the annealing temperature of the reaction system is set to be 56 DEG C; a sugarcane internal standard gene (ALS gene) and five exogenous transgenic elements (including the Ubi promoter, the Bt gene, the Bar gene, the NOS promoter and the NOS terminator) can be detected at the same time by one-time PCR reaction, and the detection limit reaches 1% of the DNA content of the template. The multiplex-PCR reaction system established by the experiment not only is capable of detecting the exogenous gene composition of transgenic sugarcane, but also is capable of effectively detecting other transgenic crops.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a multiplex PCR rapid screening method for target gene elements of transgenic sugarcane. Background technique [0002] Transgenic technology is becoming more and more mature, and the types of genetically modified sugarcane are becoming more and more diverse. At present, herbicide-resistant, insect-resistant, and disease-resistant genetically modified sugarcane has been field tested in 14 countries including the United States, Australia, India, and Brazil, creating conditions for the commercial cultivation of genetically modified sugarcane. In my country, sugarcane gene cloning and transgenic research only started in the mid-1990s. Although it started late, it has made rapid progress. The transgenic sugarcane that has been obtained includes insect-resistant genetically modified sugarcane, drought-resistant genetically modified sugarcane, and herbicide-tolerant geneticall...

AI Technical Summary

Problems solved by technology

Due to the complex chromosomes of sugarcane, the detection of inserted foreign genes is much more difficult than other crops

At present, nucleic acid PCR technology is the most widely used in domestic and foreign laboratories and national testing departments. A single PCR detection method is usually used for multiple target sequences, which is not only time-consuming, but also high cost of detection. At the same time, in the breeding of transgenic crops In order to avoid the "trans-inactivation" between the genes of multivalent transgenic crops, people avoid using the same promoter in the breeding of transgenic crops, which further increases the target sequence of multivalent transgenic crops detection

Although Dietrich et al. used real-time fluorescent quantitative PCR method and Chen Ru et al. used PCR-ELISA technology to establish a highly sensitive detection system for transgenic crops, but the operation process is complicated, the detection cost is high, and the requirements for operators are also high. Did not get rid of the way of detecting target sequences one by one

[0003]Sugarcane is an allopolyploid plant. Due to the large number of chromosomes and the complex genetic mechanism, it is still difficult to detect transgenic sugarcane. For transgenic sugarcane exogenous gene elements Multiplex PCR rapid screening has not been reported so far

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