CN103937802A - DNA barcoding standard gene sequence of Rizhao Blepharipoda liberata and species identification method based thereon - Google Patents

Abstract

Relating to the technical field of species identification, the invention specifically relates to a DNA barcoding standard gene sequence of Rizhao Blepharipoda liberata and species identification method based on the gene sequence. The DNA barcoding standard gene is COI gene, which has a gene sequence shown as SEQIDNO.1. The identification method includes: conducting extraction separation of DNA on a to-be-tested tissue, and amplifying a target gene by polymerase chain reaction. A pair of primers used in the amplification reaction are shown as the following: a forward primer 5'-GGTCAACAAATCATAAAGATATTGG-3', and a reverse primer 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'. The gene sequence obtained in the invention is in favor of realizing molecular identification of the Rizhao Blepharipoda liberata, and can effectively shorten the identification time of the Rizhao Blepharipoda liberata.

Description

The DNA bar code standards gene order of day Zhaohai cicada and the species authentication method based on this gene order

Technical field

The present invention relates to species authenticate technology field, particularly the DNA bar code standards gene order of day Zhaohai cicada and the species authentication method based on this gene order.

Background technology

Day Zhaohai cicada, be sunshine locality be commonly called as the one biology into " extra large cicada ", it be similar to land young cicada, formal name used at school, for the sufficient crab of liberation eyebrow, claims again extra large Hippidea, is a kind of ocean crustacean between shrimp and crab.Coastal in China's Mainland, only originate in From Shandong Rizhao region following the line of the sea, be orthodox school's special product at sunshine.Sea Hippidea after decocting stir-fry, entrance delicious and crisp, delicious abnormal, the delicious lobster that matches in excellence or beauty of its meat, is rich in the mineral substance such as calcium phosphorus potassium, nutritious.Put on the dining table because liberation eyebrow foot crab is used as seafood speciality end in recent years, human consumer increases greatly to the demand of liberation eyebrow foot crab, thereby has caused thereupon increasing of its amount of excavating; In addition the discharge of coastal pollutent has destroyed its living environment, has had a strong impact on the reproductive survival rate of liberation eyebrow foot crab; So the existence quantity of liberation eyebrow foot crab is always in downward trend.Therefore, the local endemic species rescue property of gegenschein sea cicada collects, protects very urgent.

DNA barcode (DNA barcoding) is a segment standard, can fast, efficiently identify the DNA sequence dna of species.The generation of this concept is the inspiration that is subject to bar codes technique application in retail trade.DNA bar codes technique is just as the commodity bar code extensively using in retail trade, scan by the one or more genes to a biological entities, identify species and find novel species, to setting up between DNA sequence dna and living species relation one to one.Along with the development of molecular biotechnology and computer technology; DNA barcode-this emerging technology oneself through having obtained increasing scientist's the concern in many fields; utilize DNA barcode to find new species or cryptic species; the work of qualification endangered species and discriminating Alien invasive species is in the ascendant, and for protecting species diversity to make huge contribution.

Species identification qualification is the first step of the knowledge of natural environment, is also the vital basic steps of research on taxonomy and even nearly all biological field.Therefore, be correctly familiar with and distinguish species, particularly those being had the kind of protection meaning, classification qualification work just seems and is even more important.Marine organisms are in occupation of a big chunk in global species amount, and oneself found marine organisms approximately have 210,000 kinds at present, and it is a very large order that huge marine organisms colony like this is classified.Since Hebert in 2003 conducts a research to DNA barcode, increasing research shows that DNA barcode has efficient feasibility on species taxonomy.In recent years, DNA bar codes technique oneself be applied in halobiontic sort research.

With respect to other marine organisms, DNA bar codes technique is more extensive in fish sort research and application.Oneself obtains the DNA bar code sequence of more than 5000 kind of fish at present, and wherein majority is marine fishes, and has set up special fish bar code data storehouse, and plan obtains the bar code data of more than the 1.5 ten thousand kind of marine fishes in the whole world, at least 5 records of each species.In other marine organisms, the research of DNA barcode is also carried out gradually.Liu Jun etc. have studied the application of DNA bar codes technique in Mytilidae Identification of Species, Wang He etc. have studied CHINESE OFFSHORE commonly see siphonopods DNA barcode and Molecular Phylogeny and Evolution thereof, Liang Huafang etc. belong to the molecular systematics research of 8 kinds of lobster COI gene DNA barcodes to coastal area of china lobster, Wang Linnan etc. are studied the Molecular Phylogeny of Chinese curtain clam order Main Economic shellfish, result shows, mitochondrial COI gene provides certain foundation as curtain clam order shellfish barcode in the suitability of species qualification, simultaneously also for typoiogical classification system provides necessary complement.

A large amount of results of study all shows that the DNA barcode taking CO gene as mark can accurately carry out species qualification to marine organisms, can select the standard bar code of COI gene as each marine organisms bar code data storehouse.The method and traditional sorting technique are proved each other, not only can solve in qualification the problems such as sample is incomplete, and be conducive to realize Rapid identification.At present, in Genbank, have the COI gene order of part Hippidea, but there is no so far the genes involved sequence of day Zhaohai cicada.

Summary of the invention

The object of the present invention is to provide the DNA bar code standards gene order of a kind of day Zhaohai cicada, to realize a day Rapid identification for Zhaohai cicada.

The DNA bar code standards gene order of provided by the invention day Zhaohai cicada, is characterized in that: described DNA bar code standards gene is COI gene, has the gene order as shown in SEQ ID NO.1.

The present invention also provides the species authentication method of a kind of day Zhaohai cicada, it is characterized in that comprising the following steps:

1) separation and Extraction DNA from tissue to be measured;

2) DNA extracting taking step 1) is template, goes out goal gene by polymerase chain reaction (PCR) amplification, and the sequence of the pair of primers using in amplified reaction is as follows:

Forward primer 5'-GGTCAACAAATCATAAAGATATTGG-3',

Reverse primer 5'-TAAACTTCAGGGTGACCAAAAAATCA-3';

3) get appropriate step 2) the goal gene agarose electrophoresis that goes out of polymerase chain reaction (PCR) amplification separate, after smelling the dyeing of second ingot, under ultraviolet lamp, observe, according to the big or small result of determination of amplified production, if energy specific amplification goes out the band of 709 bp, the order-checking of Ze Song biotech firm;

4) according to sequencing result, if with the homology of the gene order as shown in SEQ ID NO.1 more than 98%, can judge that this tissue to be measured is a day Zhaohai cicada.

Beneficial effect:

Compared with traditional Morphological Identification method, the gene order that the present invention obtains is conducive to realize a day Molecular Identification for Zhaohai cicada, can effectively shorten a day qualification time for Zhaohai cicada.

Brief description of the drawings

Fig. 1 is techniqueflow chart of the present invention;

Fig. 2 is the electrophoresis qualification figure of the goal gene that obtains after sample to be tested pcr amplification, and numbering is described as follows: swimming lane 1-6 is goal gene, detects the band that size is 709 bp, the DNA marker that M is DL2000.

Embodiment

Below in conjunction with the drawings and specific embodiments, the present invention is described in further detail, and following examples are explanation of the invention, and the present invention is not limited to following examples.

Embodiment 1

1, the collection of sample to be tested is preserved and is processed

In southern Shandong seashore forest park, (great Sha at sunshine low-lying area) seashore gathers sample to be tested, gets a part of muscle tissue, then by sample retention in the refrigerator-freezer of 95% alcohol or-20 DEG C.

2, DNA profiling preparation

Phenol-chloroform method is extracted the DNA in sample, saves backup in-20 DEG C.

3, primer is synthetic

Forward primer: 5'-GGTCAACAAATCATAAAGATATTGG-3'

Reverse primer: 5'-TAAACTTCAGGGTGACCAAAAAATCA-3'

4, pcr amplification

The PCR reaction system of the present embodiment is as shown in table 1.

Table 1 is sample to be tested goal gene PCR reaction system (50uL system)

Sterile purified water	37.75 μl
		10×Ex Taq Buffer	5 μl
dNTP Mixture (2.5 mM each)	4 μl
		LCO1490 (10 μM)	2.5 μl
HCO2198 (10 μM)	2.5 μl
		DNA profiling	2 μl
TaKaRa Ex Taq HS (5 units/μl)	0.25 μl

The response procedures of the present embodiment is as shown in table 2.

Table 2 is sample to be tested goal gene PCR response procedures

Cycle number	Sex change	Annealing	The extension time
				1	95℃ 5 min
35	95℃ 30 s	47℃ 30 s	72℃ 45 s
				1			72℃ 7 min

PCR product saves backup in 4 DEG C.

5, pcr amplification product detects

Get 5 μ L pcr amplification products, with 1.5% agarose gel electrophoresis (80V voltage, electrophoresis 40 min), ethidium bromide staining.Gel imaging system detects, and electrophoretogram, referring to accompanying drawing 2, detects the band that size is 709 bp, can preliminary judgement sample to be tested be very likely a day Zhaohai cicada.

6, gene sequencing

Choose 3-5 the good pcr amplification product of effect and send by checking order after biotech firm's purifying, sequencing result shows that with the homology of the gene order as shown in SEQ ID NO.1 be 98.8%, thereby can judge that this sample to be tested is a day Zhaohai cicada.

<110> Liu Feng inscription

The DNA bar code standards gene order of <120> day Zhaohai cicada and the species authentication method based on this gene order

<160> 1

<210> 1

<211> 658

<212> DNA

<213> liberation eyebrow foot crab (Blepharipoda liberata)

<400> 1

tacattatat tttatttttg gagcttgagc gggtatagta ggtacttcat taagtttaat 60

tattcgaaca gaactaggac aaccgggaag tttaattgga gacgatcaaa tctataatgt 120

agttgttaca gcacatgcat ttgttataat tttctttata gttataccta ttataattgg 180

aggatttgga aattgactag tcccattaat attaggagct cctgatatag catttccacg 240

aataaataac ataagatttt gattattacc cccttcttta acacttttat taactagagg 300

aatagttgaa agaggtgtag gtactggatg aactgtttac ccacctctct cagctgctat 360

tgcacatgct ggagcttcag tagatttagg aattttctca ttacatttag caggagtgtc 420

atcaatctta ggagcagtta attttataac tacagttatt aatatacgac cgcaaggaat 480

aactatagac cgtatacctt tatttgtatg atcagtattt attactgcaa ttcttttact 540

attatcttta cccgtattag caggtgctat tactatatta ttaactgacc gaaatcttaa 600

tacatccttt tttgaccctg ctggaggagg agaccctgtt ttataccaac acttattt 658

Claims (2)

1. the DNA bar code standards gene order of day Zhaohai cicada, is characterized in that: described DNA bar code standards gene is COI gene, has the gene order as shown in SEQ ID NO.1.

Day Zhaohai cicada a species authentication method, it is characterized in that comprising the following steps:

1) separation and Extraction DNA from tissue to be measured;

2) DNA extracting taking step 1) is template, goes out goal gene by polymerase chain reaction (PCR) amplification, and the sequence of the pair of primers using in amplified reaction is as follows:

Forward primer 5'-GGTCAACAAATCATAAAGATATTGG-3',

Reverse primer 5'-TAAACTTCAGGGTGACCAAAAAATCA-3';

3) get appropriate step 2) the goal gene agarose electrophoresis that goes out of polymerase chain reaction (PCR) amplification separate, after smelling the dyeing of second ingot, under ultraviolet lamp, observe, according to the big or small result of determination of amplified production, if energy specific amplification goes out the band of 709 bp, the order-checking of Ze Song biotech firm;

4) according to sequencing result, if with the homology of the gene order as shown in SEQ ID NO.1 more than 98%, can judge that this tissue to be measured is a day Zhaohai cicada.