CN104164433B

CN104164433B - For expanding the specific primer of lepidopteran insects mitochondrial COI gene

Info

Publication number: CN104164433B
Application number: CN201410446927.8A
Authority: CN
Inventors: 陆明星; 汤小天; 杜予州
Original assignee: Yangzhou University
Current assignee: Yangzhou University
Priority date: 2014-09-03
Filing date: 2014-09-03
Publication date: 2016-09-14
Anticipated expiration: 2034-09-03
Also published as: CN104164433A

Abstract

The present invention relates to a kind of for expanding the primer pair of lepidopteran insects mitochondrial cytochrome c oxidase I (cytochrome c oxidase I, COI) gene order, its sequence is as shown in SEQ ID NO.1 and SEQ ID NO.2.Use the amplimer of the present invention, can effectively amplify the COI gene of multiple lepidopteran species, agriculture and forestry insect great in Lepidoptera is carried out large-scale Analysis of Genetic Background, precise Identification some be difficult to the nearly edge species that differentiate, differentiate to provide important tool for the Identification of Species of China's lepidopteran insects, phylogeny, Genetic Constitution of Population, group's mouth history of evolution and geographical population.

Description

For expanding the specific primer of lepidopteran insects mitochondrial COI gene

Technical field

The present invention relates to a pair for expanding lepidopteran insects mitochondrial cytochrome c oxidase I (cytochrome c oxidase I； COI) specific primer of gene.

Background technology

Lepidoptera (Lepidoptera) has the Superfamily of more than 40, the section of more than about 120 and 180,000 kinds, is to be only second to elytrum The second largest mesh of purpose, including butterfly class and moth class.It is biological that lepidopteran insects belongs to complete metamorphosis, growth course be divided into ovum, larva, In four periods of pupa and adult, be research insect mimicry and host plant relation, wing pattern developmental biology and insecticide physiology etc. The important model of research is biological.

The lepidopteran insects overwhelming majority is agriculture and forestry insects, the normal leaf roll of small person, sews leaf, weaves silk and knot or pierce plant tissue Taking food harm, the big person of build often eats most blade, bites root system broken or bore moth branch.On the other hand, a lot of butterfly classes and adult moth pests are Insect pollinator and resource insect, have huge economic implications.Either as agriculture and forestry primary pest, or as pollination matchmaker Being situated between and economic insects, lepidopteran insects all creates profound influence to human society.Understand the origin of lepidopteran insects in depth, enter Change and Phylogenetic Relationships, lepidopterous insect pest preventing and treating, economic insects germ plasm resource are utilized significant.This is accomplished by By quickly and accurately Molecular tools, lepidopteran insects is carried out the research of association area.

In recent years, utilize molecular engineering that species are classified and Phylogenetic Analysis has become a kind of effective method, its A kind of ideal that Mitochondria COI gene distinguishes species with research spore relation owing to having higher evolutionary rate to become Gene.Therefore, utilize COI gene pairs lepidopteran insects to carry out species identification and Phylogenetic Analysis be a kind of preferably method, At present, during insecticide genetic research, amplification Mitochondria In Developing Flight Muscle of Insects COI gene usually uses the universal primer reported, but by Relatively strong in its versatility, during practical study, while amplification insecticide COI gene, also tend to amplify insecticide body surface And the COI genetic fragment of internal miscellaneous bacteria and expanding fragment length shorter.This directly results in some insecticide, such as lepidopteran insects pink rice borer Expanding effect and sequencing result undesirable, the band of agarose gel electrophoresis detection be often double band even multi-ribbons, checks order Peak figure mostly is bimodal.This gives and lepidopteran insects carries out large-scale genetic background researchs and analyses and bring obstruction.The most in the world It is not specifically designed for expanding the relevant report of the specific primer of lepidopteran insects COI gene.

Summary of the invention

It is an object of the present invention to provide the specific primer of a kind of lepidopteran insects mitochondrial COI gene amplification, it can meet existing skill The demand of art.

A pair primer for lepidopteran insects mitochondrial COI gene sequence amplification, its sequence such as SEQ ID NO.1 and SEQ ID Shown in NO.2.

The invention also discloses a kind of lepidopteran insects mitochondrial COI gene sequence amplification method, be to use above-mentioned primer, use 50 μ l PCR system carry out target fragment amplification under specific PCR amplification condition.

50 described μ l PCR system are: 5 μ l 10 × buffer (+Mg2+), 2.5 μ l dNTP (2.5mM), 3 μ l primers 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 profiling (100ng), 34 μ l distilled waters.

Described PCR amplification condition is: 94 DEG C of denaturations 5 minutes, 94 DEG C of degeneration 1 minute 15 seconds, 53 DEG C of annealing temperature Spending 1 minute 15 seconds, 72 DEG C extend 1 minute 5 seconds, totally 35 circulations, and last 72 DEG C extend 10 minutes.

The present invention is the higher and inefficient characteristic of non-specific amplification primer amplification according to Lepidoptera COI gene order degree of variation, logical Cross comparison Lepidoptera the most equal species COI gene order and design amplimer.Use the Lepidoptera mitochondrial COI base of the present invention The amplimer of cause, can amplify the COI gene order of the most equal species of Lepidoptera, the essentially important evil of agriculture and forestry effectively Worm, having filled up Lepidoptera COI gene does not has the blank of special amplimer, loses typical case's lepidopteran insects on a large scale Pass context analyzer, precise Identification some be difficult to the nearly edge species that differentiate, for the Identification of Species of China's lepidopteran insects, phylogeny, Genetic Constitution of Population, group's mouth history of evolution and geographical population differentiate to provide important tool.The amplimer that the present invention relates to relatively mistake Go universal primer to have bigger advantage, be embodied in: this primer can effectively amplify the COI genetic fragment of 1200bp, long Degree is close to COI full length gene, and the effective amplification length of universal primer is generally 500-750bp in the past, and amplified fragments is mostly Conservative region, is unfavorable for the data analysis of the studies above；Meanwhile, the amplified production of this primer can directly check order, and surveys Sequence peak figure is good, it is not necessary to connect by carrier, converts and the complicated procedures such as clone, carries out typical case's lepidopteran insects on a large scale Analysis of Genetic Background, can significantly reduce experimental cost, shortens experimental period, improves the accuracy of experiment.

Accompanying drawing explanation

Fig. 1 is that primer of the present invention is to typical case's lepidopteran insects expanding effect figure.1,2 is Prodenia litura；3,4 is beet armyworm；5、6 For pink rice borer；7,8 is striped rice borer；9,10 is rice leaf roller；11,12 is fall webworms；13,14 is goldrimmed moth.

Fig. 2 is that primer of the present invention is to pink rice borer COI gene amplification design sketch.

Fig. 3 is primer extension product part sequencing result peak of the present invention spectrogram.

Fig. 4 is that universal primer is to pink rice borer COI gene amplification design sketch.

Fig. 5 is universal primer amplified production part sequencing result peak spectrogram.

Detailed description of the invention

The amplimer screening technique of the Lepidoptera COI gene of the present invention is the most in two steps: 1. with the Lepidoptera elder brother recorded In the mitochondrial genome sequence of worm based on the sequence of COI gene, find out, by comparison, the tract that COI gene is guarded relatively Section, designs primer；2. lepidopteran insects COI gene is expanded by the primer of synthesis, detect its general in Lepidoptera Property and amplification efficiency.

Below in conjunction with example, invention is described further:

1. sample collecting: in August, 2013, gathers the insecticide that Lepidoptera is the most equal, relates generally to the important pests in agriculture and forestry, Including Noctuidae, the insecticide such as Pyralidae and Arctiidae.

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

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

4. sequence alignment: utilize BioEdit software by the sequence of COI gene in the mitochondrial genome complete sequence of lepidopteran insects Compare, find out than sequence fragment more conservative, that nucleotide variation degree is minimum.

5. primer synthesis: according to above-mentioned comparison result, utilize primer-design software Primer Premier 5 at nucleotide variation degree Minimum sequence fragment designs 1 pair of primer.Primer sequence is LeCOI-F:5 ' GGAGCAGGAACTGGATGA 3 ' (SEQ ID NO.1)；LeCOI-R:5’GCAGGGGGTAGATTTTGA 3’(SEQ ID NO.2).In order to verify The specificity of primer of the present invention, the universal primer of we are simultaneously synthesizing in the past mtDNA COI is as comparison.Its particular 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 respectively applied to PCR and expands the sample gathered.Arrange PCR thermal cycle to move back Fire temperature range is 50-60 DEG C.

7. amplification detection: amplimer detects with the agarose gel electrophoresis of 1%.Result shows, this primer is permissible Effectively expanding the mitochondrial COI gene of lepidopteran insects, amplification efficiency reaches 100%.Utilize this DNA that primer amplification is gone out Fragment length is about 1200bp (Fig. 1), close to the total length (total length is about 1500bp) of lepidopteran insects mitochondrial COI gene, And agarose gel electrophoresis test strip the most single (Fig. 2), order-checking peak figure is preferably (Fig. 3).On the contrary, universal primer amplifies DNA fragmentation length be about 750bp (Fig. 4), less than the amplification length of primer of the present invention, and, agarose gel electrophoresis Test strip great majority are multi-ribbon (Fig. 4), and order-checking peak spectrogram is relatively disorderly (Fig. 5).It is obvious that this universal primer is not suitable for The amplification of part lepidopteran insects COI gene, and result is inaccurate.And primer of the present invention can amplify Lepidoptera not effectively The COI gene order of equal species, and sequencing result is more accurate.

8. primer amplification condition: above-mentioned primer 50 μ l PCR system is: 5 μ l 10 × buffer (+Mg²⁺), 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 profiling (100ng), 34 μ l distilled waters.

Claims

1. a pair primer for lepidopteran insects mitochondrial COI gene sequence amplification, it is characterised in that its sequence such as SEQ ID Shown in NO.1 and SEQ ID NO.2.

2. a lepidopteran insects mitochondrial COI gene sequence amplification method, is characterized in that using drawing described in claim 1 Thing, carries out target fragment amplification, described specific PCR amplification condition by 50 μ l PCR system under specific PCR amplification condition It is: 94 DEG C of denaturations 5 minutes, 94 DEG C of degeneration 1 minute 15 seconds, 53 DEG C of annealing temperatures 1 minute 15 seconds, 72 DEG C of extensions 1 minute 5 seconds, totally 35 circulations, last 72 DEG C extended 10 minutes.

3. method as claimed in claim 2, it is characterised in that 50 described μ l PCR system are: 5 μ l add Mg²⁺'s 10 × buffer, 2.5 μ l concentration are the dNTP of 2.5mM, and 3 μ l concentration are the primer SEQ ID NO.1,3 μ l of 10 μMs Concentration is the primer SEQ ID NO.2 of 10 μMs, and 0.5 μ l concentration is the rTaq enzyme of 5 μ/μ l, the DNA mould of 2 μ l 100ng Plate, 34 μ l distilled waters.