Internal standard gene suitable to exogenous gene detection such as transgene cotton and application thereof

Abstract

The present invention is internal standard gene suitable for quantitative and qualitative PCR detection of foreign gene of transgenic cotton, tomato, rice and rape and copy number analysis and its application. Based on the intraspecific non-specificity, interspecific specificity, monocopy or low copy number, and through bioinformatical analysis and biological experiment, four internal standard genes, including cotton SADI, tomato LAT52, rice SPS and rape HMGI / Y gene are found out separately in cotton, tomato, rice and rape. The detection sensitivity and precision of these internal standard genes are experiment proved in PCR method. At the same time, the quantitative and qualitative PCR detection systems for foreign gene of transgenic cotton, tomato, rice and rape are established separately based on these internal standard genes, and corresponding PCR detection and analysis platforms are established.

Description

technical field [0001] The invention relates to an internal standard gene suitable for qualitative and quantitative detection and copy number detection of exogenous genes in transgenic cotton, tomato, rice and rapeseed and its application. It belongs to the technical field of bioengineering. Background technique [0002] Modern biotechnology with genetic engineering technology as the core has widely penetrated into agriculture in the 20th century. High technology has changed the research and production mode of traditional agriculture and provided us with more abundant food and medical products. Take the United States, where the research and industrialization of agricultural transgenic technology is the most developed, for example, more than 50% of soybeans, corn, cotton and other crops in the American market are genetically modified varieties. [0003] (1) Research progress of internal standard genes in qualitative and quantitative detection of transgenic plants [0004] I...

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Problems solved by technology

[0018] But the following factors will affect its results: 1) The number of PCR cycles affects the formation of CE peaks, and it is easy to form hybrid molecules during the PCR plateau, adding new peaks

2) Sampling method: such as electromigration sampling, pressure sampling, which can cause deviations in results

3) The influence of electroosmotic flow, at the end of sample injection, electroosmotic flow will remove part of the sample molecules in the interface, affecting the quantitative results

Mainly include: 1) When FISH detects chromosomal abnormalities, it can still only target one or a few specific chromosomes; 2) The probe preparation technology is relatively difficult, and the types of probes are far from meeting the needs of endless changes in actual work; 3 ) FISH technology is relatively expensive to carry out; 4) FISH is mainly used for mapping plant DNA repeat sequences and multi-copy gene families, and is not suitable for single-copy gene FISH, because most single-copy genes have a low detection rate, Gustafson et al. used a 0.7kb single-copy DNA sequence as a probe, and the detection rate was only about 6%, and it was detected at a single point on the chromosome. Under such low detection rate conditions, if there is no large sample population and careful It is difficult to distinguish signal points from background noise points

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