CN102687707B - CO I (cytochrome c oxidase I) gene sequence of dasyhelea species and molecular identification method thereof - Google Patents

Abstract

The invention discloses a novel dasyhelea species and a CO I (cytochrome c oxidase I) gene sequence thereof. The novel dasyhelea species belongs to dasyhelea and is named as dasyhelea arrhena Wang, Guan and Yu, sp. nov. The novel dasyhelea species is researched by methods of morphological identification, sequence analysis and the like, and results show that the novel dasyhelea species is novel in dasyhelea. In addition, a CO I gene sequence of the novel dasyhelea species is obtained, and basis for molecular identification of the novel dasyhelea species is provided.

Description

A kind of CO I gene order of moth fly novel species and the method for identifying molecules of moth fly novel species

Technical field

The present invention relates to a moth fly, be specifically related to a kind of moth fly novel species and CO I gene order thereof.

Background technology

Moth fly is miniature insect common in urban green belts and gardens, belongs to diptera (Diptera) Heleidae (Ceratopogonidae) moth fly subfamily.So far as is known, moth fly subfamily only comprise Psychoda ( dasyheleakieffer, 1911) 1 genus.Because moth fly is non-blood sucking midge class, to person poultry harmless, often get the brush-off.But the effect of moth fly in propagating plant pollen and environmentally friendlyization reaction paid close attention to by people gradually.

For a long time, to the evaluation of the midge classes such as moth fly, mainly rely on morphological feature to realize, but in recent years, progress along with technology such as DNA sequencings, people adopt molecular biology method to study the characteristic DNA of midge class successively, to setting up new sorting technique, mutually testify with traditional classification method.

Mitochondria CO I gene is the molecular labeling being most widely used in midge class identification research, be mainly used at present the Molecular Identification research of blood sucking midge, successfully identify the species in a plurality of kinds of groups such as greyish black storehouse midge and C.obsoletus, can be applied to equally in the Molecular Identification of moth fly.

Summary of the invention

The object of the present invention is to provide a kind of moth fly novel species and CO I gene order thereof.The diversity of world's species has been enriched in the discovery of this novel species, the acquisition of its CO I gene order, and the Rapid identification that can be these species provides scientific basis.

For achieving the above object, the present invention is achieved through the following technical solutions:

Gained sample is made to slide, under biomicroscope, observe, draw morphological feature figure, and consult domestic and international related documents, according to the morphological feature of sample, carry out species evaluation.This midge kind belongs to Psychoda, called after moth fly magnificent ( dasyhelea arrhenawang, Guan and Yu, sp. nov.).Type specimen is stored in Military Medical Science Institute's medical insect sample shop.

Described moth fly novel species has following Morphological Identification feature:

A, long 0.75 mm of ♂ wing, wide 0.24 mm;

B, body are brown, small-sized;

C, two compound eyes join, continuously distance.Between facet, pubescence is carefully small and dense;

D, volume sheet are fusiformis;

E, feeler flagellum rag are obvious, and wheel hair is sparse, and the 15th joint is without distal process, and it is 19:10:10:10:10:10:10:10:12:22:23:19:21 that each joint compares long; AR 1.08;

It is 5:6:15:10:6 that f, each joint of antenna compare long;

G, the nearly upper limb of chaperon have 2, hair on the neck, each 3 of every sides;

H, the prosperity of cercoids 9-tergum trailing edge pleurapophysis, embrace acra joint and be microbrachia shape, aed-aedeagus complex structure, and wherein prominent sturdy, the end of a pair of pleurapophysis carefully curves inwardly; Carefully short in paramere.

Adopt small insects trace DNA templet preparation method, by improved PCR reaction condition, by the CO I gene of this moth fly novel species of the primer amplification synthesizing, PCR product sequence is sent by professional biotech firm and is measured.Sequencing result by craft proofread, sequence assembly, and in NCBI, carry out Blast similarity searching, guarantee that institute's calling sequence is target sequence.The CO I gene order SEQ ID No.1 (not containing primer) as follows of described moth fly novel species:

tttaatatta ggagcccccg atatagcttt tcctcggata aataatataa gattttgaat 60

actccccccc tctttatctt tattactaat tagtagctta gtcgaaaatg gtgccggaac 120

aggatgaact gtttatccac ctttagctag taacatcgct catagcgggg catctgttga 180

tttggctatc ttttctttac atttagctgg tatttcatca attttagggg cagtaaattt 240

tattactacc attattaata tacgatcaaa tggaatctct tttgatcgta tacctctatt 300

tgtgtgatca gtatttatta cagctatcct tttactgtta tctctaccag ttctagcagg 360

agcaattaca atattattaa cagaccggaa tttaaataca tcattttttg accccgctgg 420

aggaggtgac cctattttat accaacatct attttgattt tttggacatc ct 472 。

The PCR primer of the CO I gene of described moth fly novel species, is characterized in that PCR primer sequence is:

Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 ';

Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '.

The method for identifying molecules of moth fly novel species of the present invention, comprising:

1) separation and Extraction DNA from moth fly novel species tissue to be measured;

2) take this DNA as template pair of primers, forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 ', reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 ', goes out this moth fly novel species CO I gene by polymerase chain reaction (PCR) amplification;

3) then get appropriate step 2) the CO I gene that goes out of polymerase chain reaction (PCR) amplification separated by agarose electrophoresis, after ethidium bromide staining, in gel imaging system, detect, according to the big or small result of determination of amplified production, if energy specific amplification goes out the band of 524bp, the order-checking of Ze Song biotech firm;

4) according to sequencing result, if with the autoploidy of described gene order SEQ ID NO.1 more than 98%, can judge that described tissue to be measured is moth fly magnificent.

The present invention can detect amplification by agarose gel electrophoresis detection method.

The present invention adopts traditional Morphological Identification to combine with molecular classification method, and described moth fly novel species is identified, can guarantee accuracy and reliability that species are identified.

Accompanying drawing explanation

Fig. 1 is moth fly feeler figure magnificent of the present invention.

Fig. 2 is moth fly antenna figure magnificent of the present invention.

Fig. 3 is moth fly volume sheet figure magnificent of the present invention.

Fig. 4 is moth fly chaperon figure magnificent of the present invention.

Fig. 5 is moth fly cercoids magnificent of the present invention (removing aed-aedeagus) figure.

Fig. 6 is moth fly aed-aedeagus figure magnificent of the present invention.

Fig. 7 is moth fly CO I gene PCR amplified production electrophoresis evaluation figure magnificent of the present invention.Numbering is described as follows: swimming lane 1-2 is the CO I gene of moth fly male worm magnificent, and detecting size is the band of 524bp.M is the DNA marker of DL-2000.

Fig. 8 is unknown moth fly CO I gene PCR amplified production electrophoresis evaluation figure.Numbering is described as follows: swimming lane 1 is the CO I gene of unknown moth fly male worm, and detecting size is the band of 524bp.M is the DNA marker of DL-2000.

embodiment

Embodiment 1

1, the collection of sample and preservation

Employing is waved net method and is gathered, and adopts the sample obtaining and is directly stored in 75% alcohol after freezing execution.

2, slide sample is made

Taking-up is stored in the sample in 75% alcohol, with dissecting needle, head, sexual organ part and wing is cut, and is convenient to film-making.Preparation of specimen carries out with reference to the described method of < < China Ceratopogonidae > > (Yu Yixin chief editor, 2005).

Sample remainder moves in PCR pipe, and 95% alcohol is preserved, one of every pipe, and carry out exclusive number, for DNA, extract.If need the long period to preserve, the PCR pipe that residue tissue site is housed should be placed in to-20 ℃ of refrigerators frozen.

3, DNA profiling preparation

adopt small insects trace DNA templet preparation method to extract moth fly DNA(Wen Lizhang chief editor magnificent. entomology research method and technology introduction [M]. Beijing: Science Press, 2010. 278-286), the DNA sample of extraction saves backup in-20 ℃.

4, primer is synthetic

The present embodiment the primer is as follows:

Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 '

Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '

5, pcr amplification

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

Table 1 is moth fly CO I gene PCR reaction system magnificent (50uL system)

Composition	Volume (uL)
		Template	10
Primer 1	1
		Primer 2	1
2×Mastermix	25
		ddH 2O	13

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

Table 2 is moth fly CO I gene PCR response procedures magnificent

Step	Reaction temperature (℃)	Time	Period
				Denaturation	94	3 min	—
Sex change	94	30 s	5
				Annealing	45	90 s	5
Extend	68	60 s	5
				Sex change	94	30 s	35
Annealing	51	90 s	35
				Extend	68	60 s	35
Extend	68	10 min	—

PCR product saves backup in 4 ℃.

6, pcr amplification product detects

Get 5 μ L pcr amplification products, with 1% agarose gel electrophoresis (120V voltage, electrophoresis 30 min).Ethidium bromide staining.Gel imaging system detects, and electrophoresis pattern is referring to accompanying drawing 7.

7, gene sequencing

After choosing 2-3 the good pcr amplification product Song You of effect biotech firm purifying, check order (order-checking the primer is identical with PCR the primer), after the calibrated splicing of gained gene order, be CO I gene order-SEQ ID No.1 of this moth fly novel species.

Embodiment 2

1, the collection of sample and preservation

Employing is waved net method and is gathered, and adopts the sample obtaining and is directly stored in 75% alcohol after freezing execution.

2, sample chooses and and pre-treatment

From the midge class sample collecting, random choose goes out a moth fly, with dissecting needle, head, sexual organ part and wing is cut, and is convenient to film-making and carries out Morphological Identification.Remainder moves in PCR pipe, and 95% alcohol is preserved, one of every pipe, and carry out exclusive number, for DNA, extract.

3, DNA profiling preparation

adopt small insects trace DNA templet preparation method to extract unknown moth fly DNA(Wen Lizhang chief editor. entomology research method and technology introduction [M]. Beijing: Science Press, 2010. 278-286), the DNA sample of extraction saves backup in-20 ℃.

4, primer is synthetic

The present embodiment the primer is as follows:

Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 '

Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '

5, pcr amplification

The PCR reaction system of the present embodiment is as shown in table 3.

Table 3 is unknown moth fly CO I gene PCR reaction system (50uL system)

Composition	Volume (uL)
		Template	10
Primer 1	1
		Primer 2	1
2×Mastermix	25
		ddH 2O	13

The response procedures of the present embodiment is as shown in table 4.

Table 4 is unknown moth fly CO I gene PCR response procedures

Step	Reaction temperature (℃)	Time	Period
				Denaturation	94	3 min	—
Sex change	94	30 s	5
				Annealing	45	90 s	5
Extend	68	60 s	5
				Sex change	94	30 s	35
Annealing	51	90 s	35
				Extend	68	60 s	35
Extend	68	10 min	—

PCR product saves backup in 4 ℃.

6, pcr amplification product detects

Get 5 μ L pcr amplification products, with 1% agarose gel electrophoresis (120V voltage, electrophoresis 30min).Ethidium bromide staining.Gel imaging system detects, and electrophoresis pattern is referring to accompanying drawing 8.The unknown moth fly of finding out embodiment 2 by electrophoretogram 8 also can specific amplification goes out the product of 524bp, can tentatively judge that described this unknown moth fly may be moth fly magnificent.In order more clearly to prove, be moth fly magnificent, willthe product of 524bp send biotech firm's order-checking, test result shows that with the autoploidy of gene order SEQ ID NO. 1 be 99.8%, thereby can confirm that this unknown moth fly is moth fly magnificent.

Claims (3)

1. a CO I gene for moth fly novel species is gene order SEQ ID No.1 as described below:

tttaatatta ggagcccccg atatagcttt tcctcggata aataatataa gattttgaat 60

actccccccc tctttatctt tattactaat tagtagctta gtcgaaaatg gtgccggaac 120

aggatgaact gtttatccac ctttagctag taacatcgct catagcgggg catctgttga 180

tttggctatc ttttctttac atttagctgg tatttcatca attttagggg cagtaaattt 240

tattactacc attattaata tacgatcaaa tggaatctct tttgatcgta tacctctatt 300

tgtgtgatca gtatttatta cagctatcct tttactgtta tctctaccag ttctagcagg 360

agcaattaca atattattaa cagaccggaa tttaaataca tcattttttg accccgctgg 420

aggaggtgac cctattttat accaacatct attttgattt tttggacatc ct 472 。

2. the CO of moth fly novel species claimed in claim 1

the PCR primer of gene, is characterized in that PCR primer sequence is:

Forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 ';

Reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 '.

3. a method for identifying molecules for moth fly novel species, comprising:

1) separation and Extraction DNA from moth fly novel species tissue to be measured;

2) take this DNA as template pair of primers, forward primer 5 ' GGA GGA TTT GGA AAT TGA TTA GTT CC3 ', reverse primer 5 ' CCC GGT AAA ATT AAA ATA TAA ACT TC3 ', goes out this moth fly novel species CO I gene by polymerase chain reaction (PCR) amplification;

3) then get appropriate step 2) the CO I gene that goes out of polymerase chain reaction (PCR) amplification separated by agarose electrophoresis, after ethidium bromide staining, in gel imaging system, detect, according to the big or small result of determination of amplified production, if energy specific amplification goes out the band of 524bp, the order-checking of Ze Song biotech firm;

4) according to sequencing result, if with the autoploidy of described gene order SEQ ID NO.1 more than 98%, can judge that described tissue to be measured is moth fly magnificent.

Images