Complete sequence amplification method for mitochondrial genome of diptychus maculates steindachner

Abstract

The invention discloses a complete sequence amplification method for a mitochondrial genome of diptychus maculates steindachner. The complete sequence amplification method specifically comprises the following steps: (1) amplifying four gene segments Cyt b, COI, ND4 and 16S rRNA of the mitochondrial genome of the diptychus maculates steindachner; and (2) carrying out LA-PCR amplification on the mitochondrial genome of the diptychus maculates steindachner. According to the complete sequence amplification method, the four gene segments Cyt b, COI, ND4 and 16S rRNA of the mitochondrial genome of the diptychus maculates steindachner are amplified, four pairs of LA-PCR primers are designed according to an obtained sequence and are used for carrying out large-segment sequence amplification, and 28 sequencing primers are utilized for carrying out PCR amplification on the other parts of the mitochondrial genome of the diptychus maculates steindachner by virtue of a primer PCR-Walking method so as to obtain a whole-genome sequence, with a length of 16895bp, of a mitochondrion of the diptychus maculates steindachner. The inheritance and evolution of the diptychus maculates steindachner are researched in a molecular level, and particularly mtDNA is used as a molecular marker, so that the basic data and technical support are provided for the researches of species identification, population genetic structures and system evolution of the diptychus maculates steindachner.

Description

technical field [0001] The invention belongs to the technical field of molecular biology, and in particular relates to a method for amplifying the complete sequence of the mitochondrial genome of P. chinensis. Background technique [0002] Diptychus maculates Steindachner, also known as spotted cucumber fish, belongs to Cyprinidae family Cyprinidae, Schizothoracinae subfamily Schizothoracinae, Diptychus genus Diptychus, is a cold-water omnivorous fish, often inhabiting rivers and mountains with gravel in the tributary. The large lip fish has reproductive biological characteristics such as low fecundity, low fecundity, slow growth rate, and late sexual maturity. Once overfished, the resources will be difficult to recover. In recent years, due to the impact of environmental changes and human activities, the distribution area of ​​the spotted lip fish has been gradually shrinking, and the population has dropped sharply. It has been listed as a second-level aquatic wild animal ...

AI Technical Summary

Problems solved by technology

In order to ensure accuracy and readability, the length of each amplification sequence of conventional PCR is about 500-600bp, while the mitochondrial genome of vertebrates is mostly more than 15kb, so at least 20-30 pairs of primers need to be designed for amplification. This method Not only is it time-consuming and labor-intensive, but it is also prone to errors during the messy sequence splicing process in the later stage

However, the LA-PCR method involved in this patent has the characteristics of long amplified fragments and high fidelity. It is the first time in China to use this method to amplify the mitochondrial genome of the spotted heavy lip.

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