CN109735631A - A kind of pair of stick arteries and veins midge gene 16SrDNA fragment sequence combines the analysis method of its form - Google Patents
A kind of pair of stick arteries and veins midge gene 16SrDNA fragment sequence combines the analysis method of its form Download PDFInfo
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Abstract
The invention belongs to gene order Valence Analysis fields, disclose the analysis method that a kind of pair of stick arteries and veins midge gene 16SrDNA fragment sequence combines its form.The present invention is first depending on morphological classification method and classifies to 17 stick arteries and veins midges, it is divided into 7 kinds, by to 16SrDNA gene sequencing, there is no replace saturation to the gene order as the result is shown for analysis, it can be used for carrying out Phylogenetic Analysis, by analyzing the gene order, and the calculating of its genetic distance, it can obtain the genetic distance between not of the same race and the genetic distance in kind, the genetic distance of inter-species is greater than the genetic distance in kind, this experiment tentatively show that the more close then genetic distance of affiliation is small, the more remote big rule of then genetic distance of affiliation；It is modal it is same to gather be one, and the value of the confidence is 100, shares 7 molecular species, show that the Molecular Identification and Morphological Identification result based on 16SrDNA gene fit like a glove.
Description
Technical field
The invention belongs to gene order Valence Analysis field more particularly to a kind of couple of stick arteries and veins midge gene 16SrDNA
Fragment sequence combines the analysis method of its form.
Background technique
Chironomidae (Chironomidae, non-bitingmidges) is under the jurisdiction of Arthropoda.The larva of midge is in fishing
It is the food of diversified economy aquatic animal (a variety of fish, river crab etc.) in industry.There is extensive emergent stage in the history of life of midge,
It can make troubles to people's lives and production.
Midge figure is small to medium-sized, and adult mouthpart is degenerated.The distribution of chironomus larvas is very wide, in Different Waters
Have, larva type also compares more.It is one of maximum monoid of biomass in Freshwater Zoobenthos monoid.In water body environment
In, it plays good indicative function.In straight prominent Chironominae, part larva can be used as the biological monitoring and water quality of water environment
Evaluation, in chironomus larvas, many types be economic pest, therefore, to the monoid carry out research have highly important warp
Ji meaning.
In recent years, research of the research gradually in terms of molecules in terms of morphology combines, and forms a more section
Research method.Such experiment can make analysis it is more accurate, with more science, can avoid in the analysis process by
In people subjective factor and caused by error, keep experimental data more convincing.Stick arteries and veins midge form is smaller, and forefathers are to stick
The molecular systematics research of arteries and veins midge is less.
In conclusion problem of the existing technology is: because stick arteries and veins midge form is smaller, sample material is difficult to obtain；Before
People is based on morphological research to the research majority of stick arteries and veins midge, and molecular systematics research is less；In addition, the molecular studies of midge monoid
Main Mitochondrial cytochrome c oxidase subunit 1 (COI) base of choosing is studied, and it is molecule mark that 16SrDNA, which is rarely employed, in forefathers
Note；The success rate ratio 16SrDNA that the present invention tests research amplification of the discovery to stick arteries and veins midge based on COI gene is much lower, because
This, for the present invention premised on Senile Mouse, selection 16SrDNA is molecular labeling, is aided with molecular systematics research, ties classification
Fruit is relatively reliable accurate, while also providing effective methods and techniques for the Rapid identification of other species.
Summary of the invention
In view of the problems of the existing technology, the present invention provides a kind of pair of stick arteries and veins midge gene 16SrDNA fragment sequences
In conjunction with the analysis method of its form.
The invention is realized in this way a kind of pair of stick arteries and veins midge gene 16SrDNA fragment sequence combines the analysis of its form
Method, it is described to stick arteries and veins midge gene 16SrDNA fragment sequence combine its form analysis method the following steps are included:
Step 1, the production of midge slide；
Step 2, typoiogical classification
Step 3 extracts the gene of midge；
Step 4, target gene carry out PCR amplification；
Step 5, pcr amplification product detection；
Step 6, sequencing；
The PCR product of Successful amplification chosen in gel electrophoresis imaging to band is bright, clearly sample is numbered, is carried out
Sequencing；
Step 7, sequence alignment；
Sequence is converted into meg. format, imports in MEGA7.0 software, is compared completely, then comparison result is saved
Get off, carries out other analyses as basic data；
Step 8, the base composition of analytical sequence；
Various analyses are carried out to the base of sequence；Need the conserved positions of the sequence of calculation, variant sites, brief information bit
Point.And the case where composition of analytical sequence base, the composition of base-pair and codon use；
Step 9, sequence replace saturability analysis；
With MEGA7.0 software, conversion genetic distance (S), transversion genetic distance (V) the correction heredity of sequence are calculated separately out
Distance (D), is then mapped using resulting value in Excel, if done figure is linear, it was demonstrated that is unsaturated between sequence
, it is unsaturated between sequence if gained figure line sexual intercourse；
Step 10 calculates genetic distance and phylogenetic tree construction；
Using MEGA7.0 software, genetic distance between the sequence of calculation, and phylogenetic tree construction.Select p-
Distance, Kimura2-parameter Model Reconstruction Phylogenetic Relationships.
Step 11 is combined with typoiogical classification and is analyzed.
Further, the production of midge slide specifically includes:
(1) with tweezers by the midge being immersed in 85% ethyl alcohol take out, under anatomical lens, by the ipsilateral foot of midge, feeler,
Wing is removed, and is placed on glass slide, and extra ethyl alcohol is sucked with blotting paper, drips upper Arabic gum covered respectively；
(2) the foot separation of remaining chest and side is put into the centrifuge tube equipped with 85% ethyl alcohol and is saved, and post mark
Label；
(3) head and abdomen are put into the centrifuge tube equipped with Nei Shi liquid, and the rear taking-up of 80 DEG C of heating water bath 10min or so,
It is adhered to respectively on the glass slide in (1) with Euparal natural gum, covered is labelled；
(4) ready-made slide is put into baking oven and dries 55 DEG C, 15d, later with transparent nail sheet for oil seal, place into baking oven
Drying；
(5) slide of drying is placed under body formula fluorescence DIC imaging system, is taken pictures with suitable amplification factor, weight
Point claps the abdomen of midge, and carries out relevant record；
(6) midge is selected to be tested.
Further, the gene for extracting midge specifically includes:
(1) foot of the chest that saves when making sample and side is taken out, be respectively put into the 1.5mL of the sterilizing marked from
In heart pipe, 2h is impregnated with the pure water after sterilizing, changes a water per hour；It is abandoned finally, the water in centrifuge tube is sucked out with liquid-transfering gun
Fall, 100 μ lBufferATL are added, add 40 μ l protein kinase Ks, plays mixing with pipette tips suction, be put in 56 DEG C of thermostat water baths
Overnight；
(2) centrifuge tube in (1) is taken out, it is slight to shake 15S or more, the chest of midge and foot are chosen with the pipette tips of sterilizing
Out, it is put into the original centrifuge tube equipped with ethyl alcohol, into remaining liquid plus 200 μ lBufferAL, oscillation are mixed, added
200 μ l dehydrated alcohols, shaken well；
(3) will in (2) mixture move into filter column in, be centrifuged 2min under the conditions of 6000 × g (8000rpm), discard filtrate and
Centrifuge tube；
(4) filter column is put into new 2mL collecting pipe, 500 μ lBufferAW1, >=6000 × g is added and are centrifuged 2min, so
After discard filter liquor and collecting pipe；
(5) filter column is put into new collecting pipe, 500 μ lBufferAW2,20000 × g (14000rpm) centrifugation is added
Then 3min discards filter liquor and collecting pipe；
(6) filter column is moved to new 1.5mL centrifuge tube, 100 μ lBufferAE are added into filter column, takes DNA for eluting,
It is incubated for after five minutes under room temperature (15 DEG C -25 DEG C), 6000 × g (8000rpm) is centrifuged 1min；Again 6000 × g (8000rpm) from
Heart 2min；
(7) filter liquor (DNA profiling) is marked, is put into 4 DEG C and temporarily saves backup.
Further, pcr amplification product detection specifically includes:
(1) preparation of TBE solution: 18.612g disodium ethylene diamine tetraacetate is weighed, is placed in the beaker of 100mL.Take constant volume
Good solution 100mL uses ddH2O constant volume to 1000mL again, repeatedly once, TBE solution is made, with stand-by；
(2) Ago-Gel that compound concentration is 1%: weighing a certain amount of agar Icing Sugar in conical flask, is added corresponding
The TBE solution of amount makes the concentration 1% of agarose in solution, is heated to agar Icing Sugar and is completely dissolved, then solution is poured on glue
On plate, after waiting solution to solidify, comb is taken out；
(3) point sample: in second hole, the 2.5 μ lmarker of gel, the hole below sequentially adds 5 μ lPCR amplified productions
With the mixture of 6 × loadingBuffer；First is not added any substance with the last one hole；
(4) gel after point sample electrophoresis: is put into PCR amplification instrument, 120V electrophoresis 40min；After the completion of electrophoresis, it will coagulate
Glue is put into EB solution and dyes 10min；
(5) it observes gel-tape: after dyeing, placing the gel in gel imaging system ImageLab4.0 and see whether
It expands successfully, such as expands successfully, then save the picture of imaging.
Further, the correlative study situation before the present invention:
(1) research conditions of gene are commonly used before the present invention in midge Molecular Identification:
In midge Molecular Identification between common gene shown in following table, it is seen then that think in the research of forefathers: COI and
COII is suitble to study the Phylogenetic Relationships of genus and species and the following rank member of kind；16SrDNA is suitable for kind, belongs to the horizontal system of rank member
Research is learned, but is not suitable in kind；Correlative study without Molecular Identification in the kind based on 16SrDNA in midge.
The applicable cases of common karyogene and chondriogen in Chironomidae systematic growth research
(2) the specific research conditions before the present invention based on 16SrDNA gene-correlation technology in midge:
The prior art carries out species identification using 16SrDNA gene and being rarely reported, root it is documented that, only
Cranston etc. is in last century Mo, based on mitochondria 16SrDNA gene with Heleidae (Ceratopogonidae) and Simulidae
(Simuliidae) type has made Phylogenetic Analysis, result of study to the Chironomidae insect in Australian area as outer group
It discloses: monosystem group's problem of two category Archaeochlus and Afrochlus, but 16SrDNA is not inquired into the interior identification of kind
Validity.
(3) research conditions before the present invention in terms of stick arteries and veins Chironomous group's Molecular Identification:
Currently, only reporting Silva&Wiedenbrug in 2014 to the research of the molecular systematics of stick arteries and veins Chironomous group
Chondriogen COI is based on to the stick arteries and veins Chironomous of Brazil to be studied；Based on 16SrDNA gene pairs stick arteries and veins Chironomous group's
Research has not been reported.
Further, advantages of the present invention and good effect are as follows:
The prior art is compared, for the present invention for kind of a grade rank member, selection is the smallest stick arteries and veins Chironomous group of Chironomidae individual
(Corynoneura, Thienemanniella) is object, and successful application of the present invention in the monoid illustrates that the technology can be with
It can also be used in Chironomidae and other animal monoids with further genralrlization.
The genetic fragment of 17 kinds of present invention acquisition stick arteries and veins Chironomous group (Corynoneura, Thienemanniella)
(i.e. 16SrDNA gene), in conjunction with the existing gene order of experiment.16SrDNA genetic fragment is carried out with Custalx software more
Sequence alignment, MAGA7.0 is to the base composition of gene order, base replacement, codon usage frequency, amino acid composition, heredity
Distance etc. is statisticallyd analyze, and phylogenetic tree construction.By analyzing above, can to obtain conclusion as follows: (1) by 16SrDNA
Sequence carries out saturability analysis, in a linear relationship, illustrates the phenomenon that their sequence is saturated there is no replacement, it was demonstrated that these data
It can carry out Phylogenetic Analysis.(2) it by the calculating of classification and its genetic distance to midge in this experiment, can obtain
The genetic distance in genetic distance and kind between not of the same race.The genetic distance of inter-species is greater than the genetic distance in kind, this experiment
Tentatively show that the more close then genetic distance of affiliation is small, the more remote big rule of then genetic distance of affiliation.(3) in this experiment,
The average content of T, C, A, G are respectively 43.3%, 5.8%, 38.6%, 12.3% in 16SrDNA gene order.Wherein A+T
The content that content is 82%, G+C is that the content of 18%, A+T is apparently higher than G+C.Simultaneously when base is replaced, transversion
Frequency be greater than conversion.(4) Phylogenetic Analysis show that 16SrDNA segment can be used for Molecular Identification, and energy will not be of the same race separated.
Detailed description of the invention
Fig. 1 is the analysis side that its form is combined to stick arteries and veins midge gene 16SrDNA fragment sequence that the present invention implements to provide
Method flow chart.
Fig. 2 is that the present invention implements the gel electrophoresis images provided.
Fig. 3 is that the present invention implements the 16SrDNA transcription frequency transversion number provided and p-distance schemes.
Fig. 4 is that the present invention implements the ABGD assessment figure provided.
Fig. 5 is that the present invention implements NJ tree graph of the midge based on 16SrDNA gene provided.
Fig. 6 is that the present invention implements the stick arteries and veins midge gonosomite outline drawing provided.
Specific embodiment
In order to make the objectives, technical solutions, and advantages of the present invention clearer, with reference to embodiments, to the present invention
It is further elaborated.It should be appreciated that the specific embodiments described herein are merely illustrative of the present invention, it is not used to
Limit the present invention.
Application principle of the invention is further described with reference to the accompanying drawing.
As shown in Figure 1, the present invention provides the analysis that a kind of pair of stick arteries and veins midge gene 16SrDNA fragment sequence combines its form
Method the following steps are included:
Step S101: sample is selected；
Step S102, the production of midge slide；
Step S103, slide observation, morphometry and drawing；
Step S104, consulting literatures；
Step S105, Morphological Identification
Step S106 extracts the gene of midge；
Step S107, target gene carry out PCR amplification；
Step S108, pcr amplification product detection and sequencing；
The PCR product of Successful amplification chosen in gel electrophoresis imaging to band is bright, clearly sample is numbered, is sent to
Wuhan six directions Hua Da Gene science limited liability company is sequenced.
Step S109, sequence alignment；The editor of gene order and composition analysis；
Sequence is converted into meg. format, imports in MEGA7.0 software, is compared completely, then comparison result is saved
Get off, carries out other analyses as basic data.
Various analyses are carried out to the base of sequence.Need the conserved positions of the sequence of calculation, variant sites, brief information bit
Point.And the case where composition of analytical sequence base, the composition of base-pair and codon use.
Step S110, Genetic Distance Analysis；
With MEGA7.0 software, conversion genetic distance (S), transversion genetic distance (V) the correction heredity of sequence are calculated separately out
Distance (D), is then mapped using resulting value in Excel, if done figure is linear, it was demonstrated that is unsaturated between sequence
, it is unsaturated between sequence if gained figure line sexual intercourse.
Step S111, OUTs assessment；
Step 112, genetic distance and phylogenetic tree construction are calculated.
Using MEGA7.0 software, genetic distance between the sequence of calculation, and phylogenetic tree construction.Select p-
Distance, Kimura2-parameter Model Reconstruction Phylogenetic Relationships.
Laboratory apparatus provided by the invention includes:
Centrifuge (EppendofAG22331Hamburg), autoclave (winning fast in Shanghai), low speed centrifuge (SCI
LOGEXD1008E), electronic balance (SartoriusBS2245, Sai Duolisi scientific instrument (Beijing) Co., Ltd),
SCILOGEXD1008E low speed centrifuge, PCR amplification instrument (GeneAmpPCRsystem9700), micro-wave oven (Glanz
G70D20CSP-D2CS0), gel imager (UniversalHoodII, Bio-BadLa boratories)
(BIORADMolecularImagerGelDocTMXR+), thermostat water bath (HHS type, the medical treatment of Shanghai Bo Xun cause Co., Ltd
Instrument factory), anatomical lens (Beijing Fu Kai Instrument Ltd.), body formula fluorescence DIC imaging system (NikonAZ100), electrophoresis apparatus
(DYY-10C type), electrophoresis tank (DY CP-31DN type Beijing Liuyi Instrument Factory), plastic plate and comb, PCR pipe
(Genexbeta), liquid-transfering gun (10 μ L, 200 μ L, 1000 μ L), eight unions, graduated cylinder (100mL), conical flask (100mL) gel slab,
Tweezers, volumetric flask, beaker, 1.5mL centrifuge tube, dissecting needle, blotting paper, culture dish, glass slide, coverslip etc..
Drug and reagent provided by the invention include:
QIAGENDNeasy organize & blood DNA extracts kit (include BufferAL, BufferAW1, BufferAW2,
BufferAE, BufferATL, DNeasyMini filter column, 2mL collecting pipe, protein kinase K), 95% ethyl alcohol, 85% ethyl alcohol, 75%
Ethyl alcohol, 50% ethyl alcohol, Nei Shi liquid, Euparal natural gum, Shanghai raw work PCR kit (SterilizedddH2O, dNTP, MgCl2
(25mmol/L), 10 × TaqBuffer, 10 × TaqBuffer, TaqDNApolymerase, primer solution COI (on), COI
(under), disodium ethylene diamine tetraacetate, agar powder, DNAMarker (10kb), 6 × LoadingBuffer, EB dye liquor, pure water, surpass
Pure water, nail polish.
Embodiment:
1, materials and methods
1.1 experimental material
This experiment totally 17 head tremor mosquito sample.The immersion of collected midge sample is stored in 85% ethanol solution, in detail
It is shown in Table 1.1.
1.1 sample information of table
1.2 instrument and equipments, drug
1.2.1 laboratory apparatus
Centrifuge (EppendofAG22331Hamburg), autoclave (winning fast in Shanghai), low speed centrifuge (SCI
LOGEXD1008E), electronic balance (SartoriusBS2245, Sai Duolisi scientific instrument (Beijing) Co., Ltd),
SCILOGEXD1008E low speed centrifuge, PCR amplification instrument (GeneAmpPCRsystem9700), micro-wave oven (Glanz
G70D20CSP-D2CS0), gel imager (UniversalHoodII, Bio-BadLa boratories)
(BIORADMolecularImagerGelDocTMXR+), thermostat water bath (HHS type, the medical treatment of Shanghai Bo Xun cause Co., Ltd
Instrument factory), anatomical lens (Beijing Fu Kai Instrument Ltd.), body formula fluorescence DIC imaging system (NikonAZ100), electrophoresis apparatus
(DYY-10C type), electrophoresis tank (DY CP-31DN type Beijing Liuyi Instrument Factory), plastic plate and comb, PCR pipe
(Genexbeta), liquid-transfering gun (10 μ L, 200 μ L, 1000 μ L), eight unions, graduated cylinder (100mL), conical flask (100mL) gel slab,
Tweezers, volumetric flask, beaker, 1.5mL centrifuge tube, dissecting needle, blotting paper, culture dish, glass slide, coverslip etc..
1.2.2 drug and reagent
QIAGENDNeasy organize & blood DNA extracts kit (include BufferAL, BufferAW1, BufferAW2,
BufferAE, BufferATL, DNeasyMini filter column, 2mL collecting pipe, protein kinase K), 95% ethyl alcohol, 85% ethyl alcohol, 75%
Ethyl alcohol, 50% ethyl alcohol, Nei Shi liquid, Euparal natural gum, the raw work PCR kit (SterilizedddH in Shanghai2O, dNTP, MgCl2
(25mmol/L), 10 × TaqBuffer, 10 × TaqBuffer, TaqDNApolymerase, primer solution COI (on), COI
(under), disodium ethylene diamine tetraacetate, agar powder, DNAMarker (10kb), 6 × LoadingBuffer, EB dye liquor, pure water, surpass
Pure water, nail polish.
1.3 experimental methods and process
1.3.1 the production of midge slide
(1) with tweezers by the midge being immersed in 85% ethyl alcohol take out, under anatomical lens, by the ipsilateral foot of midge, feeler,
Wing is removed, and is placed on glass slide, and extra ethyl alcohol is sucked with blotting paper, drips upper Arabic gum covered respectively；
(2) the foot separation of remaining chest and side is put into the centrifuge tube equipped with 85% ethyl alcohol and is saved, and post mark
Label；
(3) head and abdomen are put into the centrifuge tube equipped with Nei Shi liquid, and the rear taking-up of 80 DEG C of heating water bath 10min or so,
It is adhered to respectively on the glass slide in (1) with Euparal natural gum, covered is labelled.
(4) ready-made slide is put into baking oven drying (55 DEG C or so, 15d), later with transparent nail sheet for oil seal, then put
Enter baking oven drying.
(5) slide of drying is placed under body formula fluorescence DIC imaging system, is taken pictures with suitable amplification factor, weight
Point claps the abdomen of midge, and carries out relevant record.
(6) it according to photo, selects suitable midge and is tested.
1.3.2 the gene of midge is extracted
(1) foot of the chest that saves when making sample and side is taken out, be respectively put into the 1.5mL of the sterilizing marked from
In heart pipe, 2h is impregnated with the pure water after sterilizing, changes a water per hour；It is abandoned finally, the water in centrifuge tube is sucked out with liquid-transfering gun
Fall, 100 μ lBufferATL are added, add 40 μ l protein kinase Ks, plays mixing with pipette tips suction, be put in 56 DEG C of thermostat water baths
Overnight, about 14h.
(2) centrifuge tube in (1) is taken out, it is slight to shake 15S or more, the chest of midge and foot are chosen with the pipette tips of sterilizing
Out, it is put into the original centrifuge tube equipped with ethyl alcohol, into remaining liquid plus 200 μ lBufferAL, oscillation are mixed, added
200 μ l dehydrated alcohols, shaken well.
(3) will in (2) mixture move into filter column in, be centrifuged 2min under the conditions of 6000 × g (8000rpm), discard filtrate and
Centrifuge tube.
(4) filter column is put into new 2mL collecting pipe, 500 μ lBufferAW1, >=6000 × g is added and are centrifuged 2min, so
After discard filter liquor and collecting pipe.
(5) filter column is put into new collecting pipe, 500 μ lBufferAW2,20000 × g (14000rpm) centrifugation is added
Then 3min discards filter liquor and collecting pipe.
(6) filter column is moved to new 1.5mL centrifuge tube, 100 μ lBufferAE are added into filter column, takes DNA for eluting,
It is incubated for after five minutes under room temperature (15 DEG C -25 DEG C), 6000 × g (8000rpm) is centrifuged 1min；Again 6000 × g (8000rpm) from
Heart 2min.
(7) filter liquor (DNA profiling) is marked, is put into 4 DEG C and temporarily saves backup.
1.3.3 target gene carries out PCR amplification
Using 16SrDNA as primer, PCR amplification is carried out to target gene, is matched with reference to the raw work PCR kit specification in Shanghai
Amplification system is set, specific amplification system is shown in Table 1.2 to table 1.4, and amplification condition is shown in Table 1.5 to table 1.6.
1.2 PCR amplification system 1 of table
Tab1.2 PCRamplificationsystem(25μl)
1.3 PCR amplification system 1 of table
Tab1.3 PCRamplificationsystem(25μl)
1.4 PCR amplification system 1 of table
Tab1.4 PCRamplificationsystem(25μl)
1.5 PCR amplification condition 1 of table
Tab1.5 PCRamplificationconditions
1.6 PCR amplification condition 1 of table
Tab1.6 PCRamplificationconditions
1.3.4PCR amplified production detects
The present invention has chosen 16SrDNA genetic fragment, and 2 pairs of primer amplification gene orders are respectively adopted.The primer is upper
The universal primer of Hai Shenggong synthesis according to the TE buffer that specification is added after sterilizing is 10 μ l strength solutions when use,
And 20 DEG C save backup.The primer information is shown in Table 1.7.
The primer sequence of 1.7 midge gene 16SrDNA segment of table
Tab3.8 PrimersequenceofthegenefragmentoftheChironomusnucleus
(1) preparation of TBE solution: 18.612g disodium ethylene diamine tetraacetate is weighed, is placed in the beaker of 100mL.Take constant volume
Good solution 100mL uses ddH2O (ultrapure water) constant volume to 1000mL again, repeatedly once, TBE solution is made, with stand-by.
(2) Ago-Gel that compound concentration is 1%: weighing a certain amount of agar Icing Sugar in conical flask, is added corresponding
The TBE solution of amount makes the concentration 1% of agarose in solution, is heated to agar Icing Sugar and is completely dissolved, then solution is poured on glue
On plate, after waiting solution to solidify, comb is taken out.
(3) point sample: in second hole, the 2.5 μ lmarker of gel, the hole below sequentially adds 5 μ lPCR amplified productions
With the mixture of 6 × loadingBuffer.First is not added any substance with the last one hole.
(4) gel after point sample electrophoresis: is put into PCR amplification instrument, 120V electrophoresis 40min；After the completion of electrophoresis, it will coagulate
Glue is put into EB solution and dyes 10min.
(5) it observes gel-tape: after dyeing, placing the gel in gel imaging system ImageLab4.0 and see whether
It expands successfully, such as expands successfully, then save the picture of imaging.
1.3.5 sequencing
The PCR product of Successful amplification chosen in gel electrophoresis imaging to band is bright, clearly sample is numbered, is sent to
Wuhan six directions Hua Da Gene science limited liability company is sequenced, such as Fig. 2.
1.3.6 sequence alignment
Sequence is converted into meg. format, imports in MEGA7.0 software, is compared completely, then comparison result is saved
Get off, carries out other analyses as basic data.
1.3.7 the base composition of analytical sequence
Various analyses are carried out to the base of sequence.Need the conserved positions of the sequence of calculation, variant sites, brief information bit
Point.And the case where composition of analytical sequence base, the composition of base-pair and codon use.
1.3.8 sequence replacement saturability analysis
MEGA7.0 software, calculate separately out sequence conversion genetic distance (S), transversion genetic distance (V) correction heredity away from
It from (D), is then mapped in Excel using resulting value, if done figure is linear, it was demonstrated that be unsaturated between sequence
, it is unsaturated between sequence if gained figure line sexual intercourse.
1.3.9 genetic distance and phylogenetic tree construction are calculated
Using MEGA7.0 software, genetic distance between the sequence of calculation, and phylogenetic tree construction.Select p-
Distance, Kimura2-parameter Model Reconstruction Phylogenetic Relationships.
2, result and analysis
2.1 Morphological Identifications are as a result, as shown in table 2.1:
2.1 Morphological Identification result of table
2.2 16SrDNA gene order Phylogenetic Analysis
2.2.1 16SrDNA sequence composition analysis
The sequence measured to stick arteries and veins midge is compared through Clustal software, after being cut off to height variant sites,
16SrDNA gene order total length is 440bp, wherein conserved positions C, variant sites V, brief information bit Pi, from descendants site S,
0-foldDegeneratesites-272,2-foldDegeneratesites-66,4-foldDegene ratesites-294
Data see Table 2 for details .2.
C, V, Pi, S etc. of 2.2 16SrDNA gene of table
Tab2.2 The valueC、V、Pi、S of 16SrDNA gene
It can be concluded that in the midge 16SrDNA gene order measured, the total length of sequence is by table 2.2
440bp, wherein conserved positions 272, variant sites 155, brief informative site 109, from 44, descendants site.16SrDNA
The variability of gene order is 35.2%, and brief informative site accounts for 4.8% in gene order, accounts for 10% from descendants site.
2.2.2 16SrDNA sequence different loci base frequency
2.3 different loci base frequency of table
Tab2.3 The base freqency of the different sites
By table 2.3 can be concluded that the average content of T, C, A, G be respectively 43.3%, 5.8%, 38.6%,
12.3%.The content that the content that wherein content of A+T is 82%, G+C is 18%, A+T is apparently higher than G+C.Codon site contains
Amount is respectively 83.2%, 85%, 77.5%.Thus illustrate, 16SrDNA gene has apparent composition skewed popularity.
2.2.316SrDNA the base replacement analysis of gene nucleotide
By the analysis of table 2.4, R value average value is 0.3, and the ratio of base transition and transversion frequency is R value, then proves: from
On the whole, the transversion of base is greater than conversion.Base transition (transition) rate value is 10%, transversion
(transversion) rate is 37%.Third site R value then shows that conversion wants low compared with transversion less than 1.0,
There may be replacement saturability problems for 16SrDNA genetic fragment, so having before carrying out Phylogenetic Analysis to genetic fragment must
It is replaced saturability analysis.Speculate that transversion number is higher than the reason of conversion number and A+T content is higher in 16SrDNA sequence has
It closes, high A+T content may increase the probability of A-T transversion.
The base of 2.4 16SrDNA gene nucleotide of table is replaced
Tab 2.4 The base substitution of 16SrDNA gene
2.2.4 based on the replacement saturability analysis of 16SrDNA genetic fragment base
Using MEGA7.0 software, " pairs of sequence genetic distance calculates " is carried out, XY scatter plot is done, sees Fig. 3.Analyze result
Display: conversion number and transversion number partially overlap, and are linearly distributed, increase with the increase of p-distance, but the two does not have
Apparent saturation trend, so the sequence can be used in searching system development relationship.
2.2.5 genetic distance
16SrDNA gene order is imported in ClustalX software, alignment is then carried out, saves as .meg. format.
The calculating of genetic distance, single genetic distance are shown in Table 2.5 between carrying out sequence using MEGA7.0 software.Between not of the same race, they
Genetic distance between 0.07-0.76, inbred genetic distance wants small relative to Genetic distance, thus this experiment tentatively demonstrate,prove
The more close then genetic distance of bright affiliation is small, the big rule of the more remote then genetic distance of affiliation.
2.5 genetic distance of table
Tab 2.5 Genetic distance
2.2.6 it is assessed based on 16SrDNA genetic fragment ABGD
It such as Fig. 4, is assessed according to ABGD, 6 molecular classification units is divided into when P value is 0.0050-0.264, when P value is
When 0.0514-0.0368, taxon is 2, close to the morphologic species classification studied.
2.2.7 it is contribute based on 16SrDNA segment
Based on NJ tree such as Fig. 5 constructed by adjacent method, can be obtained by phylogenetic tree: Corynoneura,
Thienemanniella two belong to and separating, in form it is identical it is of the same race between to gather be one, it is of the same race between the value of the confidence all very
Height, therefore show: Molecular Identification result and Morphological Identification result are coincide substantially,
Fig. 6 is that the present invention implements the stick arteries and veins midge gonosomite outline drawing provided.
The foregoing is merely illustrative of the preferred embodiments of the present invention, is not intended to limit the invention, all in essence of the invention
Made any modifications, equivalent replacements, and improvements etc., should all be included in the protection scope of the present invention within mind and principle.
Claims (4)
1. the analysis method that a kind of pair of stick arteries and veins midge gene 16SrDNA fragment sequence combines its form, which is characterized in that described right
Stick arteries and veins midge gene 16SrDNA fragment sequence combine its form analysis method the following steps are included:
Step 1, the production of midge slide；
Step 2, typoiogical classification；
Step 3 extracts the gene of midge；
Step 4, target gene carry out PCR amplification；
Step 5, pcr amplification product detection；
Step 6, sequencing；
The PCR product of Successful amplification chosen in gel electrophoresis imaging to band is bright, clearly sample is numbered, is surveyed
Sequence；
Step 7, sequence alignment；
Sequence is converted into meg. format, imports in MEGA7.0 software, is compared completely, then comparison result is preserved,
Other analyses are carried out as basic data；
Step 8, the base composition of analytical sequence；
Various analyses are carried out to the base of sequence；Need the conserved positions of the sequence of calculation, variant sites, brief informative site.And
The case where composition of analytical sequence base, the composition of base-pair and codon use；
Step 9, sequence replace saturability analysis；
With MEGA7.0 software, conversion genetic distance (S), transversion genetic distance (V) the correction genetic distance of sequence are calculated separately out
(D), then mapped in Excel using resulting value, if done figure is linear, it was demonstrated that be between sequence it is unsaturated,
It is unsaturated between sequence if gained figure line sexual intercourse；
Step 10 calculates genetic distance and phylogenetic tree construction；
Using MEGA7.0 software, genetic distance between the sequence of calculation, and phylogenetic tree construction；Selection p-distance,
Kimura2-parameter Model Reconstruction Phylogenetic Relationships.
Step 11 is combined with typoiogical classification and is analyzed.
2. the analysis method of its form is combined to stick arteries and veins midge gene 16SrDNA fragment sequence as described in claim 1, it is special
Sign is that the production of midge slide specifically includes:
(1) midge being immersed in 85% ethyl alcohol is taken out with tweezers, under anatomical lens, by the ipsilateral foot of midge, feeler, wing
It removes, is placed on glass slide, extra ethyl alcohol is sucked with blotting paper, drip upper Arabic gum covered respectively；
(2) the foot separation of remaining chest and side is put into the centrifuge tube equipped with 85% ethyl alcohol and is saved, and post label；
(3) head and abdomen are put into the centrifuge tube equipped with Nei Shi liquid, and the rear taking-up of 80 DEG C of heating water bath 10min or so, are used
Euparal natural gum adheres to it on glass slide in (1) respectively, and covered is labelled；
(4) ready-made slide is put into baking oven and dries 55 DEG C, 15d, later with transparent nail sheet for oil seal, place into baking oven baking
It is dry；
(5) slide of drying is placed under body formula fluorescence DIC imaging system, is taken pictures with suitable amplification factor, emphasis is clapped
The abdomen of midge, and carry out relevant record；
(6) midge is selected to be tested.
3. the analysis method of its form is combined to stick arteries and veins midge gene 16SrDNA fragment sequence as described in claim 1, it is special
Sign is that the gene for extracting midge specifically includes:
(1) foot of the chest saved when making sample and side is taken out, is respectively put into the 1.5mL centrifuge tube for the sterilizing marked
In, 2h is impregnated with the pure water after sterilizing, changes a water per hour；Finally, being discarded the water suction in centrifuge tube with liquid-transfering gun, add
Enter 100 μ lBufferATL, add 40 μ l protein kinase Ks, plays mixing with pipette tips suction, be put in 56 DEG C of thermostat water baths overnight；
(2) centrifuge tube in (1) is taken out, it is slight to shake 15S or more, the chest of midge and foot are chosen with the pipette tips of sterilizing, put
Enter in the original centrifuge tube equipped with ethyl alcohol, into remaining liquid plus 200 μ lBufferAL, oscillation mix, add 200 μ l without
Water-ethanol, shaken well；
(3) mixture in (2) is moved into filter column, is centrifuged 2min under the conditions of 6000 × g (8000rpm), discards filtrate and centrifugation
Pipe；
(4) filter column is put into new 2mL collecting pipe, 500 μ lBufferAW1, >=6000 × g centrifugation 2min is added, then abandon
Remove filter liquor and collecting pipe；
(5) filter column is put into new collecting pipe, 500 μ lBufferAW2,20000 × g (14000rpm) is added and are centrifuged 3min,
Then filter liquor and collecting pipe are discarded；
(6) filter column is moved to new 1.5mL centrifuge tube, 100 μ lBufferAE are added into filter column, DNA is taken for eluting, in room
It is incubated for after five minutes under warm (15 DEG C -25 DEG C), 6000 × g (8000rpm) is centrifuged 1min；6000 × g (8000rpm) is centrifuged again
2min；
(7) filter liquor (DNA profiling) is marked, is put into 4 DEG C and temporarily saves backup.
4. the analysis method of its form is combined to stick arteries and veins midge gene 16SrDNA fragment sequence as described in claim 1, it is special
Sign is that pcr amplification product detection specifically includes:
(1) preparation of TBE solution: 18.612g disodium ethylene diamine tetraacetate is weighed, is placed in the beaker of 100mL；Take constant volume good
Solution 100mL uses ddH2O constant volume to 1000mL again, repeatedly once, TBE solution is made, with stand-by；
(2) Ago-Gel that compound concentration is 1%: a certain amount of agar Icing Sugar is weighed in conical flask, corresponding amount is added
TBE solution makes the concentration 1% of agarose in solution, is heated to agar Icing Sugar and is completely dissolved, then solution is poured on plastic plate
On, after waiting solution to solidify, comb is taken out；
(3) point sample: in second hole, the 2.5 μ lmarker of gel, the hole below sequentially add 5 μ lPCR amplified productions and 6 ×
The mixture of loadingBuffer；First is not added any substance with the last one hole；
(4) gel after point sample electrophoresis: is put into PCR amplification instrument, 120V electrophoresis 40min；After the completion of electrophoresis, gel is put
Enter in EB solution and dyes 10min；
(5) it observes gel-tape: after dyeing, placing the gel in gel imaging system ImageLab4.0 and see whether to expand
Success such as expands successfully, then saves the picture of imaging.
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