CN104946645A

CN104946645A - Specific primers for amplifying plecoptera insect mitochondrion COI (cytochrome c oxidase I) gene

Info

Publication number: CN104946645A
Application number: CN201510443567.0A
Authority: CN
Inventors: 杜予州; 陈志腾; 陆明星
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2015-07-24
Filing date: 2015-07-24
Publication date: 2015-09-30

Abstract

The invention relates to primers used for amplifying plecoptera insect mitochondrion COI (cytochrome c oxidase I) gene sequences, the plecoptera insect mitochondrion COI gene sequences are as shown in SEQ ID No.1 and SEQ ID No.2. The primers for amplifying are adopted, so as to amplify COI genes of various plecoptera species effectively, be conducive to accurately identifying close species difficult to indentify in some forms, and an important tool for classification and identification, phylogeny, population genetic structures and geographical population identification of plecoptera insects in China is provided.

Description

For the Auele Specific Primer of Kuo Zeng Plecoptera Mitochondria In Developing Flight Muscle of Insects COI gene

Technical field

The present invention relates to a pair for Kuo Zeng Plecoptera Mitochondria In Developing Flight Muscle of Insects Cytochrome c oxidase I (cytochrome c oxidase I; COI) Auele Specific Primer of gene.

Background technology

Plecoptera (Plecoptera) is also known as perlid, such insect is each in the world except the South Pole landly greatly all has distribution, but be mainly distributed in oriental region, palaearctic region and neoarctic region, especially abundant with China's kind across ancient north and Japan two large animal geographic region.Plecoptera insect is distributed in polytype waters more widely, is important water quality monitoring (WQM) indicator organism, strengthens the sort research of this monoid, can better for water quality environment monitoring provides service.

A crucial monoid from the angle ， Plecoptera of systematics and theory of evolution.The phylogeny of Sui Ran Plecoptera is widely studied, but still needs to be proved by other more features.And the data obtained by Molecular tools is by the sibship that more contributes between each section of Tui Duan Plecoptera and and other object sibships.The research of China You Guan Plecoptera protomala class insect is only limitted to traditional form, and molecules research is still blank at present.Deeply enter the origin of separating Plecoptera insect, evolution and Phylogenetic Relationships, all significant to the evolutionary relationship rebuilding Plecoptera phylogeny and even research Insecta.This carries out the research of association area with regard to needing by Molecular tools Dui Plecoptera insect fast and accurately.

In recent years, utilize molecular engineering to classify to species and Phylogenetic Analysis has become a kind of general and effective means, wherein mitochondrial COI gene has become owing to having higher evolutionary rate the desirable gene of one distinguishing species and research spore relation.Therefore, utilizing COI Ji to carry out species identification and Phylogenetic Analysis Yin Dui Plecoptera insect is a kind of method preferably, at present, in insect genetic research process, amplification Mitochondria In Developing Flight Muscle of Insects COI gene is generally adopt the universal primer reported, but due to its versatility stronger, specificity is more weak, in practical study process, while amplification insect COI gene, also often can amplify the COI gene fragment of miscellaneous bacteria in insect body surface and body and expanding fragment length is shorter.This will cause some insect, and the effect that the phylogeny as Dui Plecoptera insect is studied is undesirable, and the band that agarose gel electrophoresis detects is often two band even multi-ribbons, and order-checking peak figure mostly is bimodal.This Gei Dui Plecoptera insect carries out large-scale phylogenetic evolution and researchs and analyses and bring obstruction.There is no the relevant report of the special Auele Specific Primer for Kuo Zeng Plecoptera insect COI gene at present in the world.

Summary of the invention

The object of the invention is to provide the Auele Specific Primer of Plecoptera Mitochondria In Developing Flight Muscle of Insects COI gene amplification, and it can meet the demand of prior art.

The primer of a pair Yong Yu Plecoptera Mitochondria In Developing Flight Muscle of Insects COI gene order amplification, its sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.

The invention also discloses Yi Zhong Plecoptera Mitochondria In Developing Flight Muscle of Insects COI gene order amplification method, be adopt above-mentioned primer, under specific pcr amplification condition, carry out target fragment amplification by 50 μ l PCR system.

50 described μ l PCR system are: 5 μ l 10 × buffer (+Mg ²⁺), 2.5 μ l dNTP (2.5mM), 3 μ l primer LeCOI-F (10 μMs), 3 μ l primer LeCOI-R (10 μMs), 0.5 μ l rTaq enzyme (5 μ/μ l), 2 μ l DNA profilings (100ng), 34 μ l distilled waters.

Described pcr amplification condition is: 94 DEG C of denaturations 5 minutes, 94 DEG C of sex change 1 minute 15 seconds, and 51 DEG C of annealing temperatures 1 minute 15 seconds, 72 DEG C extend 1 minute 5 seconds, totally 35 circulations, last 72 DEG C of extensions 10 minutes.

Root of the present invention is the comparatively conservative and inefficient characteristic of non-specific amplification primer amplification according to Plecoptera COI gene order, leads to the COI gene order of ratio to the not equal species of Plecoptera and designs amplimer.Adopt the amplimer of Plecoptera mitochondrial COI gene of the present invention, effectively can expand the COI gene order increasing the not equal species of Plecoptera, fill up the blank that Plecoptera COI gene does not have special amplimer; insect that is not equal to Plecoptera and that belong to carries out large-scale COI order-checking, some form of precise Identification is difficult to the nearly edge species differentiated, for the taxonomic identification of Wo Guo Plecoptera insect, phylogeny, Genetic Constitution of Population and geographical population are differentiated to provide important tool.The amplimer that the present invention relates to comparatively passes by universal primer larger advantage, be embodied in: this primer effectively can amplify the COI gene fragment of nearly 2000bp, cover COI full length gene completely, and the effective amplification length of universal primer is generally 500-800bp in the past, and amplified fragments is mostly conservative region, be unfavorable for the data analysis of above-mentioned research.Utilize this Yin Wu Dui Plecoptera insect to carry out large-scale phylogenetic evolution analysis, significantly can reduce experimental cost, shorten experimental period, improve the accuracy of experiment.

Accompanying drawing explanation

Fig. 1 is the expanding effect figure of Yin Wu Dui Plecoptera 9 section insect COI gene of the present invention.1 Wei Juan Ji; 2 Wei Niu Ji; 3 Wei Kou Ji; 4 Wei Gou Ji; 5 Wei Jin Ji; 6 Wei Xin Ji; 7 Wei Ci Ji; 8 Wei Wang Ji; 9 Wei Lv Ji.

Fig. 2 is universal primer Dui Plecoptera 9 section insect COI gene amplification design sketch.1 Wei Juan Ji; 2 Wei Niu Ji; 3 Wei Kou Ji; 4 Wei Gou Ji; 5 Wei Jin Ji; 6 Wei Xin Ji; 7 Wei Ci Ji; 8 Wei Wang Ji; 9 Wei Lv Ji.

Embodiment

The amplimer screening method of Plecoptera COI gene of the present invention is divided into two steps: 1. in the Mitochondrial Genome Overview sequence recording Plecoptera insect COI gene sequence based on, the sequence fragment relatively guarded at COI gene two ends is found out by comparison, do not rely on existing insect universal primer, if meter Plecoptera primer special; 2. the Yin Wu Dui Plecoptera insect COI gene of synthesis is increased, detect the versatility in Qi Plecoptera and amplification efficiency; 3. once designed multipair primer during the course, showed that primer of the present invention was optimum through experiment show

Below in conjunction with example, invention is described further:

1. preparation of samples: sample all uses deposits in the not equal insect of the Plecoptera of depositing in 100% alcohol in Yangzhou University's application insect institute Specimen Room, comprises the Plecoptera insect of green Ji section, hook Ji section, flat Ji section, ChaJi Kedeng section.

2.DNA extracts: Axygen test kit extracts DNA, and concrete steps are with reference to specification sheets.

3. Data Source: data from US National Biotechnology Information center (NCBI, http:/www.ncbi.nlm.gov/).

4. sequence alignment: utilize ClustalX software the sequence at COI gene two ends in the mitochondrial genome complete sequence of Plecoptera insect to be compared, finds out the sequence fragment relatively more conservative, nucleotide variation degree is minimum.

5. primer synthesis: according to above-mentioned comparison result, utilize primer-design software Primer Premier 5 to design 1 pair of primer at the sequence fragment that nucleotide variation degree is minimum.Primer sequence is LeCOI-F:5 ' AAACTAATAGCCTTCAAAG 3 ' (SEQ ID NO.1); LeCOI-R:5 ' TATWTGGAGCTTAAATCCAT 3 ' (SEQ ID NO.2).In order to verify the specificity of primer of the present invention, we employ over simultaneously mtDNA COI universal primer in contrast.Its concrete sequence is: forward:5 '-CAACATTTATTTTGATTTTTTGG-3 ' (SEQ ID NO.3); Reword:5 '-TCCATGCACTAATCTGCCATATTA-3 ' (SEQ ID NO.4).

6. primer amplification: above-mentioned amplimer is applied to respectively the sample that pcr amplification gathers.Arranging TouchdownPCR thermal cycling annealing region is 40-60 DEG C.

7. amplification detects: the amplimer agarose gel electrophoresis of 1% detects.Result shows, and this primer can the mitochondrial COI gene of Kuo Zeng Plecoptera insect effectively, and amplification efficiency reaches 100%.This is utilized to be about 2000bp to the DNA fragmentation length that primer amplification goes out, the total length (total length is about 1500bp) of Fu Gai Plecoptera Mitochondria In Developing Flight Muscle of Insects COI gene, and agarose gel electrophoresis test strip comparatively single (Fig. 1).On the contrary, the DNA fragmentation length that universal primer amplifies is about 1000bp (Fig. 2), is less than the amplification length of primer of the present invention, and, agarose gel electrophoresis test strip poor (Fig. 2).Clearly, this universal primer is not suitable for the amplification of Bu Fen Plecoptera insect COI gene, and result is inaccurate.And primer of the present invention can expand the COI gene order increasing the not equal species of Plecoptera effectively, and sequencing result is more accurate.

8. primer amplification condition: above-mentioned primer 50 μ l PCR system is: 5 μ l 10 × buffer (+Mg ²⁺), 2.5 μ l dNTP (2.5mM), 3 μ l primer LeCOI-F (10 μMs), 3 μ l primer LeCOI-R (10 μMs), 0.5 μ l rTaq enzyme (5 μ/μ l), 2 μ l DNA profilings (100ng), 34 μ l distilled waters.

Claims

1. the primer of a pair Yong Yu Plecoptera Mitochondria In Developing Flight Muscle of Insects COI gene order amplification, it is characterized in that, its sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.

2. Yi Zhong Plecoptera Mitochondria In Developing Flight Muscle of Insects COI gene order amplification method, is characterized in that adopting primer according to claim 1, carries out target fragment amplification by 50 μ l PCR system under specific pcr amplification condition.

3. method as claimed in claim 2, it is characterized in that 50 described μ l PCR system are: 5 μ l are containing the 10 × buffer of Mg2+, the dNTP of 2.5 μ l 2.5mM, the upstream primer of 3 μ l 10 μMs, the downstream primer of 3 μ l 10 μMs, the rTaq enzyme of 0.5 μ l 5 μ/μ l, the DNA profiling of 2 μ l 100ng, 34 μ l distilled waters.

4. method as claimed in claim 2, is characterized in that described specific pcr amplification condition is: 94 DEG C of denaturations 5 minutes, 94 DEG C of sex change 1 minute 15 seconds, 51 DEG C of annealing temperatures 1 minute 15 seconds, 72 DEG C extend 1 minute 5 seconds, totally 35 circulations, and last 72 DEG C extend 10 minutes.