Amplification primer of cardiidae shellfish mitochondria 16S rRNA (16small-subunit ribosomal RNA) gene

Abstract

According to the invention, 53 cardiidae 16S rRNA (16small-subunit ribosomal RNA) sequences amplified by the use of a universal primer are subjected to comparative analysis through CLUSTALW software, 3 pairs of primers are designed by primer designing software Primer Premier5 in a determined conserved region, 218 individuals of 24 species of cardiidae are repeatedly subjected to PCR reaction test in 10mu l system, and finally a specific amplification primer of cardiidae shellfish mitochondria 16S rRNA gene sequence is screened out through agarose gel electrophoresis detection. The amplification primer provided by the invention is characterized by NG16Sa5'-GCCTGTTTATCAAAAACATCTC-3'NG16Sb5'-TCTGAACTCAGATCACGTAAGGAT-3'. By adopting the cardiidae shellfish mitochondria 16S rRNA primer provided by the invention, the target fragments of different species of cardiidae shellfish can be quickly and efficiently amplified, and the amplification efficiency reaches 100%, thereby providing support to the identification of cardiidae species, identification of geographical population and research on germplasm resource and genetic diversity and laying a foundation for further developing artificial breeding of various important economic shellfishes of cardiidae.

Description

technical field [0001] The invention relates to a cockle shellfish mitochondrial 16S rRNA [16S ribosomal RNA (16small-subunit ribosomal RNA)] gene amplification primer. Background technique [0002] The family Cardiidae belongs to the class Bivalvia, the subclass Heterodonta, and the order Veneroida. There are about 250 species of cockle shellfish in the world, and about 50 species have been found in my country. The diameter of the small one is about 1 cm, and the diameter of the large one can reach 15 cm (such as the smooth giant cockle in California). Cockles are distributed worldwide, often inhabiting the shallow seas of the Indian Ocean-Western Pacific Ocean, and are distributed in Singapore, Japan, Australia, Malaysia, Indonesia, mainland China, Taiwan and the South China Sea Islands. Cockle family shellfish include slip-top thin-shelled cockles, California flat cockles, yellow-edged rough cockles, and Chinese cockles, which are of great value in aquaculture and fishin...

AI Technical Summary

Problems solved by technology

[0004] However, the mutation rate of shellfish 16S rRNA sequence is significantly higher than that of other groups of animals. The 16S rRNA sequence variation among different species of molluscs, even among closely related species or even different groups within a species, is very large, so its non-specific amplification primers The amplification efficiency is low, and some species cannot be expanded at all

The lack of primers with high amplification efficiency has seriously hindered the application of this molecular marker. Therefore, screening the specific amplification primers for the mitochondrial 16S rRNA gene of Cochinidae is of great significance for the identification of Cochinidae species and phylogenetic analysis using molecular methods. At the same time, it can also provide technical conditions for the use of 16S rRNA gene to study the phylogeography of cockles, protect the germplasm resources of cockles and select suitable breeding species

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