Universal primer for identifying mitochondrial 16S rRNA (ribosomal Ribonucleic Acid) gene of intestinal gill species and application of universal primer

Abstract

The invention discloses a universal primer for identifying mitochondrial 16S rRNA (ribosomal Ribonucleic Acid) genes of intestinal gill species and application of the universal primer. The universal amplification primer disclosed by the invention is H16SF-R, the sequence of a forward primer of the universal amplification primer is 5 '-CGCCTGTTTACCAAAAACATGCCCC-3', and the sequence of a reverse primer of the universal amplification primer is 5 '-AATGCGTAGATAGAAGCCGACCTGGCT-3'. The universal primer provided by the invention can be used for amplifying a single strip, has high specificity and can meet various research requirements of species identification, phylogenetic relationship analysis and the like of intestinal gill species.

Description

Technical Field

[0001] The invention belongs to molecular biology, and in particular relates to a universal primer for mitochondrial 16S rRNA gene for identifying enterobranchial species and an application thereof. Background Art

[0002] Enteropneusta are a class of benthic marine animals in the phylum Hemichordata, which have characteristics such as oral cords, dorsal and ventral nerve cords and gill slits. These animals play a key transitional role in the evolution of invertebrates to chordates, and therefore have important evolutionary biological significance. Currently, there are about 130 known hemichordate species in the world, of which Enteropneusta accounts for more than 83%. These species vary in size from a few centimeters to two meters according to their morphological changes, and are widely distributed in tropical reefs and temperate intertidal zones.

[0003] In China, eight species of hemichordates have been recorded, all of which belong to the class Enterobranchiata. Among them, Saccoglossus hwangtauensis and Glossobalanus polybranchioporus are first-class protected wild animals in China, and the other five species are also listed as second-class protected animals in China. This regulation highlights the urgency of protecting these precious creatures in my country. Since the 1960s, Chinese scholars have identified three new species of Enterobranchiata, revealing the diversity of species in this class and the potential for exploration. Therefore, it is urgent to carry out the classification and identification of Enterobranchiata species.

[0004] With the rapid development of molecular biotechnology, its application in species classification, identification and phylogenetic analysis is becoming more and more extensive. Mitochondrial genes have become ideal molecular markers due to their unique genetic characteristics and structural features. Species identification using mitochondrial 16S rRNA gene fragments has been widely practiced, and its high intraspecific conservation makes it a key tool for species identification. At the same time, PCR-based detection technology has become the core method of species identification in current taxonomy with its advantages of simplicity, rapidity and accuracy.

[0005] In this context, it is particularly important to design universal amplification primers for partial gene fragments of mitochondrial 16S rRNA in enterobranch species. Such primers can effectively improve the species identification efficiency of enterobranch animals, meet the urgent needs of identifying endangered species and studying biodiversity, and provide strong support for biodiversity conservation research under the influence of climate change and human activities. By developing universal amplification primers suitable for enterobranch animals, we can better understand the evolutionary history of these animals, which has important scientific value and practical application prospects for biodiversity conservation and ecological protection. Summary of the invention

[0006] The purpose of the present invention is to provide a universal primer for mitochondrial 16S rRNA gene for identifying enterobranchial species and its application. The purpose of the present invention is to achieve rapid identification of enterobranchial animal samples so as to better compare the differences between different enterobranchial animals.

[0007] The first object of the present invention is to provide a primer pair H16SF-R for identifying enterobranchial species. The forward primer sequence of the primer pair H16SF-R is shown in SEQ ID NO.1, and the reverse primer sequence is shown in SEQ ID NO.2.

[0008] The second object of the present invention is to provide a PCR kit for identifying Enterobranchial species, comprising the primer pair H16SF-R and a PCR amplification reagent.

[0009] The third object of the present invention is to provide the use of the primer pair H16SF-R or the PCR kit in identifying enterobranchial species.

[0010] A fourth object of the present invention is to provide a method for identifying Enterobranchiata species, comprising the following steps:

[0011] S1. Extract DNA of the Enterobranchial species to be identified;

[0012] S2. Using the DNA from step S1 as a template, perform PCR amplification on H16SF-R or the PCR kit using the primers to obtain a PCR product;

[0013] S3. Compare the PCR product with the 16S rRNA gene sequence of the published Enterobranchia species. If the similarity of the 16S rRNA gene sequence reaches more than 95%, the species to which the Enterobranchia species to be identified belongs is determined; if there is no Enterobranchia species with a 16S rRNA gene sequence similarity of more than 95%, the Enterobranchia species to be identified is a new species.

[0014] Preferably, the PCR amplification in step S2 has a system of 25 μL, including 12.5 μL of 2×Taq Master Mix, 1 μL of forward primer of primer pair H16SF-R, 1 μL of reverse primer of primer pair H16SF-R, 1 μL of DNA template, and the balance is ddH2O.

[0015] Preferably, the PCR amplification in step S2 has the following amplification procedure: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 65°C for 20s, and extension at 72°C for 45s, for 30 cycles; and final extension at 72°C for 3min.

[0016] Beneficial effects of the present invention:

[0017] The universal primers provided by the present invention can amplify clear and single bands using DNA extracted by conventional methods as samples, and have good sensitivity; compared with the existing 16S rRNA universal primers of invertebrates, they have stronger specificity and better amplification effect. The universal primers and methods designed by the present invention are fast, accurate and simple, and provide strong technical support for the classification, identification and protection of enterobranchial species. BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 It is the amplification electrophoresis diagram of H16SF-R primer, invertebrate 16S rRNA universal primer and blank control; EM is the blank control; AR-BR is the band amplified by invertebrate 16S rRNA universal primer; H16SF-R is the universal primer for Enterobranchiata species.

[0019] Figure 2 The sequence and its peak diagram are obtained by PCR amplification of the enterobranch sample (Acorus trisakii) using H16SF-R primers, amplification of the PCR product, and then Sanger sequencing. DETAILED DESCRIPTION

[0020] The following examples are provided to further illustrate the present invention, rather than to limit the present invention.

[0021] Example 1: Screening of universal primers for amplification of mitochondrial 16S rRNA genes in enterobranchial species

[0022] The invention discloses a method for screening universal primers of mitochondrial 16S rRNA gene suitable for identification of enterobranchiate species, comprising the following steps: comparing and analyzing the 16S rRNA gene sequences of enterobranchiate species such as Balanoglossus misakiensis, Glandiceps qingdaoensis, Ptychodera flava and Saccoglossus kowalevskii, designing primers in conservative regions and synthesizing them artificially; using genomic DNA of Balanoglossus misakiensis as a template, using the synthesized primers and another universal primer AR-BR (forward primer: 5'-CGCCTGTTTATCAAAAACAT-3' (SEQ ID NO. 3); reverse primer: 5'-CCGGTCTGAACTCAGATCACGT-3' (SEQ ID NO. 4)) of 16S rRNA of another invertebrate to perform PCR amplification respectively, and detecting the amplified products by agarose gel electrophoresis.

[0023] The reaction system for PCR amplification was: 2×Taq Master Mix 12.5 μL (Novozyme, P111-01), 1 μL each of forward primer and reverse primer, 1 μL of template, and double distilled water to make up to 25 μL. The reaction procedure was: 98°C pre-denaturation for 30 s; 98°C denaturation for 10 s, 65°C annealing for 20 s, 72°C extension for 45 s, for a total of 30 cycles; 72°C final extension for 3 min.

[0024] Figure 1 The results showed that when the genomic DNA of Acornis trisakiensis was used as a template, the universal primer AR-BR for 16S rRNA of invertebrates could not amplify a clear band, while the primer H16SF-R could amplify a clear band of about 620 bp. The sequence and peak diagram of the PCR product amplified by primer H16SF-R were obtained by Sanger sequencing. Figure 2 shown.

[0025] The universal primer H16SF-R for the mitochondrial 16S rRNA gene of Enterobranchiata species has the following sequence:

[0026] Forward primer: 5'-CGCCTGTTTACCAAAAACATCGCCCC-3' (SEQ ID NO. 1);

[0027] Reverse primer: 5'-AATGCGTAGATAGAAGCCGACCTGGCTCT-3' (SEQ ID NO. 2).

[0028] Example 2

[0029] Two worm-like animals with unique appearance (species A and species B) were collected from the intertidal muddy beaches in Qingdao and Hainan, respectively. The length and body color of the collected samples all showed obvious differentiation of the snout, collar and trunk. These characteristics are consistent with the typical morphological characteristics of enterobranchia. Since enterobranchia species have weak mobility and narrow distribution areas, based on the geographical characteristics and morphology of the collection site and the description of known enterobranchia species [Study on enterobranchia in intertidal zones along the coast of China (Liang Xianyuan, 1984); Enterobranchia species and distribution in Jiaozhou Bay (Zhang Baolin, 2007)], it can be preliminarily identified that species A and species B may belong to the family Ptychoderidae of enterobranchia. The universal primer H16SF-R will be used for PCR amplification in the future, and the amplified products will be sequenced and compared with the 16S rRNA gene sequences of existing enterobranchia species to determine whether they belong to the final species or are new species.

[0030] Specifically, the genomic DNA of species A and species B are respectively extracted as templates, and amplified by the universal primer H16SF-R for Enterobranchia, and a stable single band of about 620 bp can be obtained. The PCR amplified product is compared with the 16S rRNA gene sequence of the published Enterobranchia species. If the comparison result shows that the similarity of the 16S rRNA gene sequence of the species to be tested and the published Enterobranchia species reaches more than 95%, the species to which the Enterobranchia species to be tested belongs can be finally determined; otherwise, it may be a new species.

[0031] The sequencing results showed that the 16S rRNA gene sequence of species A collected in Qingdao was shown as SEQ ID NO.5, and the 16S rRNA gene sequence of species B collected in Hainan was shown as SEQ ID NO.6.

[0032] After blast comparison, it was found that the matching rate of species A with the polybranchial pores of the lingula-formis distributed in Qingdao (16S rRNA gene sequence as shown in SEQ ID NO.7) was 99.8%, the matching rate with the Qingdao oak-headed worm (16S rRNA gene sequence as shown in SEQ ID NO.8) was 76.3%, and the matching rate with the three-saki acorn worm (16S rRNA gene sequence as shown in SEQ ID NO.9) was 87.2%. Finally, the molecular biological data supported the identification of species A as polybranchial pores of the lingula-formis. In addition, species B is distributed in Hainan and has a match rate of 83.3% with the published 16S rRNA sequence of Glossobalanus (16S rRNA gene sequence as shown in SEQ ID NO.10) and a match rate of 80.9% with the fleshy acorn worm (Balanoglossus carnosus, 16S rRNA gene sequence as shown in SEQ ID NO.11). Therefore, it is judged that species B collected in Hainan is an unnamed new species of Enterobranchiata [the new species has been published in the article: A new species of the genus Glossobalanus (Hemichordata, Enteropneusta, Ptychoderidae) from China].

Claims

1. A primer pair H16SF-R for the identification of Enterobranchiata species, characterized in that: The forward primer sequence of the primer pair H16SF-R primer pair is shown as SEQ ID NO.1, and the reverse primer sequence is shown as SEQ ID NO.

2.

2. A PCR kit for the identification of Enterobranchial species, characterized in that: The method comprises the primer pair H16SF-R according to claim 1 and a PCR amplification reagent.

3. Use of the primer pair H16SF-R according to claim 1 or the PCR kit according to claim 2 in identifying Enterobranchial species.

4. A method for identifying enterobranchial species, characterized in that: The following steps are involved: S1. Extract DNA of the Enterobranchial species to be identified; S2. Using the DNA of step S1 as a template, PCR amplification is performed using the primers of claim 1 for H16SF-R or the PCR kit of claim 2 to obtain a PCR product; S3. Compare the PCR product with the 16S rRNA gene sequence of the published Enterobranchia species. If the similarity of the 16S rRNA gene sequence reaches more than 95%, the species to which the Enterobranchia species to be identified belongs is determined; if there is no Enterobranchia species with a 16S rRNA gene sequence similarity of more than 95%, the Enterobranchia species to be identified is a new species.

5. The method according to claim 4, characterized in that The PCR amplification in step S2 is carried out in a system of 25 μL, including 12.5 μL of 2×Taq Master Mix, 1 μL of the forward primer of the primer pair H16SF-R, 1 μL of the reverse primer of the primer pair H16SF-R, 1 μL of the DNA template, and the balance is ddH2O.

6. The method according to claim 4, characterized in that The PCR amplification in step S2 has the following amplification procedure: pre-denaturation at 98°C for 30s; denaturation at 98°C for 10s, annealing at 65°C for 20s, and extension at 72°C for 45s, for 30 cycles; and final extension at 72°C for 3min.

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