CN104178569A - AFLP primer, linker and method for identifying variety of Branchiostoma - Google Patents

Abstract

The invention discloses an AFLP primer for identifying the variety of Branchiostoma, an adapter sequence and an identifying method. The AFLP primer and the adapter sequence are as shown in SEQ ID NO:1-8; and the variety of Branchiostoma can be identified by applying the AFLP primer and the adapter sequence. In the result, the Branchiostoma with 109bp specific bands are Branchiostoma belcheri; and the Branchiostoma with b120bp specific bands are Japanese Branchiostoma japonicum. When the primer disclosed by the invention is used for identifying, an amplification band result is clear, and the sizes of the bands are conveniently observed; and meanwhile, the method is simple and convenient to operate.

Description

A kind of AFLP primer, joint sequence and authentication method thereof of identifying lancelet kind

Technical field

The present invention relates to genetically engineered field, relate in particular to a kind of AFLP primer and authentication method thereof of identifying lancelet kind.

Background technology

Lancelet (lancelet), being subordinate to Chordata, Cephalochordata, Branchiostoma, is the middle transition type of evolving to vertebrates in invertebrates, has the title of " living fossil ", be the model animal of research vertebrates origin and evolution, there is high scientific research and be worth.Meanwhile, lancelet also has very high nutritive value, and protein content is high and lipid content is low, and also has higher indispensable amino acid, local flavor amino acid and the abundant liposoluble vitamin of content in body.Due to Habitat destruction and overfishing etc., lancelet natural resources amount sharply reduces, and has been listed in national second class protection animal.Nearly ten years, China's lancelet artificial breeding and cultural technique have obtained impressive progress, for lancelet large-scale cultivation is laid a good foundation.

China coast has two kinds of lancelets to distribute, this day this lancelet and Bai Shi lancelet, from distribution range, between these two kinds of lancelets, there is no obvious boundary, there is report to point out to have this two kinds of lancelets in Xiamen sea area, and these two kinds of lancelets are extremely similar in form, be difficult to profile, they be separated.Therefore need a kind of stable, sensitive technology to differentiate lancelet kind.

Its gene order of biology not of the same race has more fixing difference conventionally, therefore can according to the difference of genomic dna sequence, carry out the discriminating of species, and this method is not subject to the impact of biological growth and development state and ecotope, and the accuracy of discriminating is very high.Wherein the most reliable method is to clone out certain gene, but checks order complicated operation, and cost is also higher.For fish, someone has set up extracting Mitochondrial DNA, with multiple restriction enzyme, Mitochondrial DNA is cut, according to enzyme, cut the difference identification and the method for identifying fingerling class of result, but this method needs more tissue sample to carry out extracting Mitochondrial DNA.Round pcr is a kind of very sensitive external DNA cloning technology, but has very high specificity simultaneously, and primer and the suitable reaction conditions of control by appropriate design, utilize it to identify and be subject to sequence difference small on identification of dna.

Summary of the invention

The object of the present invention is to provide a kind of AFLP primer and authentication method thereof of identifying lancelet kind.

For achieving the above object, the invention provides a kind of AFLP primer and joint sequence for the identification of lancelet kind, it is characterized in that, described sequence is SEQ ID NO1-8.

The present invention also provides a kind of method of identifying lancelet kind, it is characterized in that, described method has been used described primer and joint sequence thereof.

Comprise the steps,

Lancelet DNA extraction Mse I and EcoR I double digestion obtain enzyme and cut product;

The preparation of joint sequence obtains being connected product with connection;

Pre-amplification obtains connecting product;

Selective amplification;

Electrophoresis detection;

Result judgement, there is specific band at 109bp place, is Bai Shi lancelet; At 120bp place, there is specific band, be Japanese lancelet.

The preparation of described joint sequence and being connected to, balanced mix EcoR I adapter A is that SEQ ID NO:1 and EcoR I adapter B are SEQ ID NO:2,95 ℃ of sex change 5min, obtain EcoR I adapter in 37 ℃ of renaturation 1h immediately; Balanced mix Mse I adapter A is that SEQ ID NO:3 and Mse I adapter B are SEQ ID NO:4, and 95 ℃ of sex change 5min, obtain Mse I adapter in 37 ℃ of renaturation 1h immediately; Connect, linked system is as follows again, 37 ℃ of reaction 3.5h;

Described EcoR I adapter is prepared as: balanced mix SEQ ID NO:1 and SEQ ID NO:2, and 95 ℃ of sex change 5min, immediately in 37 ℃ of renaturation 1h;

Described Mse I adapter is prepared as: balanced mix SEQ ID NO:3 and SEQ ID NO:4,95 ℃ of sex change 5min, immediately in 37 ℃ of renaturation 1h.

The system of described pre-amplification is

Pre-amplification PCR response procedures is 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 20 circulations; 72 ℃ are extended 10min.

Described selective amplification system is,

Selective amplification PCR response procedures is 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 65-56 ℃ of annealing 30s, each circulation declines 1 ℃, and 72 ℃ are extended 1min, totally 10 circulations; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 25 circulations; 72 ℃ are extended 10min.

The inventive method step is:

1. extract lancelet muscle tissue genomic dna.

2. with Mse I and EcoR I restriction enzyme, lancelet genomic dna is carried out to double digestion.

3. the joint of synthetic is connected under the effect of T4 ligase enzyme to the two ends that enzyme is cut product fragment, forms the fragment with joint.EcoR I joint sequence is: 5 '-CTCGTAGACTGCGTACC-3 ' (SEQ ID NO:1), 5 '-AATTGGTACGCAGTCTAC-3 ' (SEQ ID NO:2); Mse I joint sequence is: 5 '-GACGATGAGTCCTGAG-3 ' (SEQ ID NO:3), 5 '-TACTCAGGACTCAT-3 ' (SEQ ID NO:4).Joint is the form formation sticky end with DNA double chain when synthetic, could under the effect of T4 ligase enzyme, be connected with double digestion fragment.Sequence is synthetic by Shanghai JaRa biotech firm.

4. increase in advance, the pre-amplimer of EcoR I is 5 '-GACTGCGTACCAATTCA-3 ' (SEQ ID NO:, 5), and the pre-amplimer of Mse I is 5 '-GATGAGTCCTGAGTAAC-3 ' (SEQ ID NO:6).Reaction system is 10-20 μ L, is specially: connect product 0.5-1 μ L; 1 * PCR damping fluid is (containing Mg ²⁺); DNTPs 0.2mM; Pre-each 0.25 μ M of amplimer; Taq enzyme 0.5-1U.Response procedures: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 20 circulations; 72 ℃ are extended 10min.

5. selective amplification, carries out selective amplification after pre-amplified production dilutes 10 times, by E-AGT/M-CTG selectivity combination of primers, carries out selective amplification.Amplified reaction is totally 10-20 μ L, is specially pre-amplified production 0.1-0.2 μ L; 1 * PCR damping fluid is (containing Mg ²⁺); DNTPs 0.2mM; Each 0.25 μ M of Mse I and EcoR I selective amplification primer; Taq enzyme 0.5-1U.Response procedures: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 65-56 ℃ of annealing 30s (each circulation declines 1 ℃), 72 ℃ are extended 1min, totally 10 circulations; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 25 circulations; 72 ℃ are extended 10min.

6.PCR product detects, and resulting selective PCR amplimer is carried out to electrophoresis, observe, and has specific band at 109bp place, is Bai Shi lancelet; At 120bp place, there is specific band, be Japanese lancelet.

Primer of the present invention be contriver by just screening for a long time, utilize 45 pairs of selectivity primer sets to test, remove the primer of result badly, such as band lacks serious result; Finishing screen is selected 9 pairs of combination of primers that amplified band is clear, abundant.

Apply primer of the present invention and detect, result is clear, and stripe size is convenient to observe.While method is simple, convenient operation.

Accompanying drawing explanation

Fig. 1 is that different sources lancelet DNA identifies that silver dyes electrophorogram.

Embodiment

Describe embodiments of the invention below in detail, the example of described embodiment is shown in the drawings, and wherein same or similar label represents same or similar element or has the element of identical or similar functions from start to finish.Below by the embodiment being described with reference to the drawings, be exemplary, be intended to for explaining the present invention, and can not be interpreted as limitation of the present invention.Unreceipted concrete technology or condition person in embodiment, according to the described technology of the document in this area or condition or carry out according to product description.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.

Embodiment 1:

1, lancelet extracting genome DNA

Lancelet sample extraction DNA with picking up from Qingdao, Xiamen and Beihai area, gets lancelet muscle tissue 30mg, grinds, and with Proteinase K-benzene phenol-chloroform method or DNA extraction test kit, extracts total DNA, then with ultrapure water, dissolves.

2, enzyme is cut and is connected

(1) enzyme is cut

Use restriction enzyme Mse I and EcoR I to carry out double digestion to the DNA of said extracted.The first step enzyme is cut, system 18 μ L, 65 ℃ of reaction 3h.Specific as follows:

Enzyme is cut second step, system 20 μ L, 37 ℃ of reaction 3h.Specific as follows

Enzyme is cut 80 ℃ of deactivation 20min of product, with 1% agarose gel electrophoresis inspection enzyme, cuts product.

(2) joint is prepared and is connected

Joint preparation: EcoR I adapter preparation, balanced mix EcoR I adapter A and EcoR I adapter B, 95 ℃ of sex change 5min, immediately in 37 ℃ of renaturation 1h.The preparation method of Mse I adapter is the same.

The total system 20 μ L of ligation, 37 ℃ of reaction 3.5h, obtain connecting product.Specific as follows:

Joint sequence:

3, amplification in advance

Pre-amplified reaction is totally 20 μ L, specific as follows:

Pre-amplification PCR response procedures: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 20 circulations; 72 ℃ are extended 10min.

Pre-amplified production detects with 1% agarose gel electrophoresis, uses ddH ₂10 times of O dilutions, 4 ℃ save backup.

EcoR I pre-expansion primer GACTGCGTACCAATTCA SEQ ID NO:5

Mse I pre-expansion primer GATGAGTCCTGAGTAAC SEQ ID NO:6

4, selective amplification

Selective amplification reaction is totally 20 μ L, specific as follows:

Selective amplification PCR response procedures: 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 65-56 ℃ of annealing 30s (each circulation declines 1 ℃), 72 ℃ are extended 1min, totally 10 circulations; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 25 circulations; 72 ℃ are extended 10min.Obtain selective amplification product.

Selective amplification primer:

E-AGT/M-CTG?GACTGCGTACCAATTCAGT?SEQ?ID?NO:7

GATGAGTCCTGAGTAACTG?SEQ?ID?NO:8

Also be that SEQ ID NO:7 is that primer is expanded in EcoR I choosing, SEQ ID NO:8 is that primer is expanded in Mse I choosing.

5, denaturing polyacrylamide gel electrophoresis

(1) electrophoresis prerunning: electrophoretic buffer is 1 * TBE, is heated to 45 ℃ of left and right by damping fluid, and then firm power 100W makes offset plate temperature rise to 55 ℃ of left and right.Loading: add 4 μ L methane amide loading indicator respectively in 20 μ L selective amplification products, 95 ℃ of sex change 5min, are placed on ice immediately.Electrophoresis: put after sample, 90W power runs 2h left and right.

(2) it is fixing that silver dyes colour developing: after electrophoresis finishes, pull down sheet glass, more than sheet glass is placed on to the middle 1h of stationary liquid (1% Glacial acetic acid).Silver dyes: after fixedly completing, offset plate is placed in the 2L silver dye liquor (2g AgNO3,3mL formaldehyde, 400 μ L1%NaS2O3) configuring, shakes up gently, silver rinses the offset plate several seconds gently with distilled water after dying 30min.Colour developing: after having dyeed, offset plate is placed on to 2L nitrite ion (10g NaOH, 3mL formaldehyde,) in, jiggle until there is clear band on gel, on daylight lamp box, observe, take pictures and obtain Fig. 1, according to specific band on electrophoretogram, lancelet is carried out to Identification of Species.In primer E-AGT/M-CTG electrophoretogram, at 109bp place, there is specific band, be Bai Shi lancelet (being Xiamen colony, the North Sea colony in Fig. 1); At 120bp place, there is specific band, be Japanese lancelet (being the Qingdao colony in Fig. 1).

Although illustrated and described embodiments of the invention above, be understandable that, above-described embodiment is exemplary, can not be interpreted as limitation of the present invention, those of ordinary skill in the art can change above-described embodiment within the scope of the invention in the situation that not departing from principle of the present invention and aim, modification, replacement and modification.

Claims (6)

1. for the identification of AFLP primer and the joint sequence of lancelet kind, it is characterized in that, described sequence is SEQ ID NO1-8.

2. a method of identifying lancelet kind, is characterized in that, described method has been used primer and the joint sequence of claim 1.

3. described in claim 2, identify the method for lancelet kind, it is characterized in that, comprise the steps,

Lancelet DNA extraction Mse I and EcoR I double digestion obtain enzyme and cut product;

The preparation of joint sequence obtains being connected product with connection;

Pre-amplification obtains connecting product;

Selective amplification;

Electrophoresis detection;

Result judgement, there is specific band at 109bp place, is Bai Shi lancelet; At 120bp place, there is specific band, be Japanese lancelet.

4. described in claim 3, identify the method for lancelet kind, it is characterized in that, the preparation of described joint sequence and being connected to, balanced mix EcoR I adapter A is that SEQ ID NO:1 and EcoR I adapter B are SEQ ID NO:2,95 ℃ of sex change 5min, obtain EcoR I adapter in 37 ℃ of renaturation 1h immediately; Balanced mix Mse I adapter A is that SEQ ID NO:3 and Mse I adapter B are SEQ ID NO:4, and 95 ℃ of sex change 5min, obtain Mse I adapter in 37 ℃ of renaturation 1h immediately; Connect, linked system is as follows again, 37 ℃ of reaction 3.5h;

Described EcoR I adapter is prepared as: balanced mix SEQ ID NO:1 and SEQ ID NO:2, and 95 ℃ of sex change 5min, immediately in 37 ℃ of renaturation 1h;

Described Mse I adapter is prepared as: balanced mix SEQ ID NO:3 and SEQ ID NO:4,95 ℃ of sex change 5min, immediately in 37 ℃ of renaturation 1h.

5. described in claim 3, identify the method for lancelet kind, it is characterized in that, the system of described pre-amplification is,

Pre-amplification PCR response procedures is 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 53 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 20 circulations; 72 ℃ are extended 10min.

6. described in claim 3, identify the method for lancelet kind, it is characterized in that, described selective amplification system is,

Selective amplification PCR response procedures is 94 ℃ of sex change 5min; 94 ℃ of sex change 30s, 65-56 ℃ of annealing 30s, each circulation declines 1 ℃, and 72 ℃ are extended 1min, totally 10 circulations; 94 ℃ of sex change 30s, 56 ℃ of annealing 30s, 72 ℃ are extended 1min, totally 25 circulations; 72 ℃ are extended 10min.