CN103103278B - Molecule specificity marking primer and method for distinguishing Chinese bolete and North American bolete - Google Patents

Abstract

The invention provides a molecule specificity marking primer for distinguishing Chinese boletus roseoflavus and North American bolete B. speciousus and a method for quickly distinguishing the Chinese boletus roseoflavus and the North American bolete B. speciousus by using the primer. The molecule specificity marking primer has the following nucleotide sequence that an upstream primer is 5'-ATTGAAGACCGTCCGGGATG-3' and a downstream primer is 5'-AGTTAAAAGTGACCAAGCC-3'. The invention has the beneficial effects that the molecule specificity marking primer can perform quick molecule distinguishing on the Chinese boletus roseoflavus and the North American bolete B. speciousus; the method is simple, quick and accurate; and the molecule specificity marking primer is an effective assistance for distinguishing two types of bolete according to morphological characteristics.

Description

Differentiate molecular specificity labeled primers and the method for China and North America bolete

(1) technical field

The present invention relates to a kind of molecular specificity labeled primers of differentiating Chinese bolete Boletus roseoflavus and North America bolete B.speciosus, and one utilizes this primer pair China to distinguish fast method for distinguishing with North America bolete sample.

(2) background technology

Boletus roseoflavus is subordinate to the Appendiculati Konrad & Maubl.ex Lannoy & Estades monoid that under Boletales Boletales, Boletus belongs to, and only finds to be distributed at present China.The diagnostic characteristics of China B.roseoflavus is that cap is rose pink, red-purple, rose, and bacterial context is exposed to constant indigo plant in air, or slowly becomes blue, becomes at once blue after bacterium hole is injured, and the mean aspect ratio of sporidium is less than 3.In in the past a lot of year, the B.roseoflavus kind that is distributed in Yunnan always by the fungus scholar of China mistakenly titled with the kind name of North America B.speciosus.The diagnostic characteristics of North America B.speciosus is that cap color is scarlet in rose, bacterial context, the injured rear indigo plant that all becomes at once in bacterium hole.Due to B.roseoflavus and B.speciosus, the difference in morphological specificity, color and metachromasia is not remarkable, thereby the B.roseoflavus sample mistake of Chinese yunnan is accredited as to B.speciosus in the past always, domestic have lot of documents this to be had to record.In fact, B.roseoflavus and B.speciosus are not of the same race, not yet find that there is real B.speciosus so far exist in China, and this conclusion had also obtained the support of molecular data afterwards.

The principal character that B.roseoflavus and these two sibling species of B.speciosus are distinguished is upper and inapparent fruit-body color difference and metachromasia of macroscopic view.But because being subject to the impact of habitat, weather, soil, sporophore growth phase, physiological situation etc., color distortion usually causes the deviation in subjective differentiation, and for the exsiccata of long-term preservation, particularly the Study on Identification again to ancient sample, type specimen, is only difficult to precise Identification especially with color distortion.Therefore,, in macro fungi sort research, molecular engineering has become the important means to sample assistant identification.

The Internal Transcribed Spacer (the Internal Transcribed Spacer of the ribosomal gene rRNA of fungi, ITS) conservative property is relatively consistent in substantially showing as and planting, difference between species is obvious, this feature makes ITS section not only be suitable for belonging to the Phylogenetic Relationships analysis between interior species or between the obvious flora of intraspecies variation, and is very suitable for the Molecular Identification of fungal species.B.roseoflavus and B.speciosus ITS district sequencing result are shown, the ITS1-5.8S-ITS2 sequence length of B.roseoflavus and B.speciosus is respectively 690～706bp and 664bp, is difficult to utilize pcr amplification, the common electrophoresis of universal primer to (as ITS1/ITS4 or ITS5/ITS4) accurately to differentiate and come.There are some differences in the local sequence that the ITS sequence alignment of two sibling species is shown to the two, this is design ITS special primer, and the ITS specific fragment that increases pointedly, provides possibility to facilitate for the molecule assistant identification of these two sibling species.

(3) summary of the invention

The object of the invention is to provide a kind of molecular specificity labeled primers of differentiating Chinese bolete B.roseoflavus and North America bolete B.speciosus, and one utilizes this primer pair China and North America bolete to distinguish fast method for distinguishing.

The technical solution used in the present invention is:

A molecular specificity labeled primers of differentiating Chinese bolete B.roseoflavus and North America bolete B.speciosus, its nucleotide sequence is as follows:

Upstream primer: 5 '-ATTGAAGACCGTCCGGGATG-3 '

Downstream primer: 5 '-AGTTAAAAGTGACCAAGCC-3 '

This primer pair is to adopt round pcr, through the cloning and sequencing of the ribosomal gene ITS region sequence to Chinese bolete sample B.roseoflavus and North America bolete sample B.speciosus, carry out base difference comparison with the ITS sequence obtaining, and design ITS Auele Specific Primer pair with this.Carry out pcr amplification with this primer pair B.roseoflavus and B.speciosus, can from B.roseoflavus, obtain the specific fragment of 437bp size.

The invention still further relates to described molecular specificity labeled primers in the application of differentiating in Chinese bolete B.roseoflavus and North America bolete B.speciosus.

The invention still further relates to a kind of method of utilizing described molecular specificity labeled primers to differentiate Chinese bolete B.roseoflavus and North America bolete B.speciosus, described method comprises: extract bolete sample genomic dna to be measured as template, using described molecular specificity labeled primers as amplimer, carry out pcr amplification, amplified production is carried out to electrophoresis detection, if there is the DNA band of 437bp in electrophoresis result, bolete to be measured is B.roseoflavus, if the DNA band of 437bp does not appear in electrophoresis result, bolete to be measured is B.speciosus;

Described molecular specificity labeled primers sequence is:

Upstream primer: 5 '-ATTGAAGACCGTCCGGGATG-3 '

Downstream primer: 5 '-AGTTAAAAGTGACCAAGCC-3 '

Described method key is that selection, DNA extraction, PCR reaction system and the reaction conditions of amplimer is definite, and electrophoresis detection, all can carry out according to this area ordinary method.

It should be noted that, molecular specificity labeled primers of the present invention and detection method are only applicable to the discriminating of Chinese bolete B.roseoflavus and North America bolete B.speciosus, be that sample to be measured is to be defined as in the scope of Chinese bolete sample B.roseoflavus and North America bolete sample B.speciosus, those of ordinary skill in the art can first carry out preliminary judgement according to sporophore shape feature and color, then the bolete sample the inventive method that is judged as B.roseoflavus or B.speciosus is differentiated.

China bolete B.roseoflavus and the difference of North America bolete B.speciosus in morphological specificity, color and metachromasia are not remarkable, thereby have in the past a large amount of domestic literatures to record B.roseoflavus mistake is accredited as to B.speciosus.Main by a little difference on the two fruit-body color to distinguishing of B.roseoflavus and B.speciosus, but the differentiation of color distortion is had to very strong subjectivity, usually there is deviation, and for exsiccata, particularly ancient sample, type specimen have been difficult to only carry out precise Identification by color distortion.Employing the inventive method, can carry out Molecular Identification fast to sibling species B.roseoflavus and B.speciosus, and method is simple, quick, accurate, is that effective that morphological specificity is distinguished assists.

Described pcr amplification reaction composing system following (cumulative volume 25 μ L):

PCR reaction conditions is as follows:

After 94 DEG C of denaturation 6min; 94 DEG C of sex change 40s, 65 DEG C of annealing 40s, 72 DEG C are extended 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C.

Concrete, described method is as follows:

(1) get bolete sample sporophore 0.2g to be measured, add liquid nitrogen and thoroughly grind, extract the genomic dna of bolete to be measured with SDS-CTAB method;

(2) genomic dna extracting taking step (1), as template, using described molecular specificity labeled primers as amplimer, carries out pcr amplification:

Every 25 μ L are composed as follows for PCR reaction system:

PCR reaction conditions is as follows:

After 94 DEG C of denaturation 6min; 94 DEG C of sex change 40s, 65 DEG C of annealing 40s, 72 DEG C are extended 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C;

(3) get step (2) amplified production 5 μ L, mix with 1 μ L0.25% bromjophenol blue damping fluid, point sample is on 1.5% sepharose, electrophoresis in 1 × TAE damping fluid, under 5V/cm voltage, electrophoresis finishes rear EB dyeing, on automatic gel image analysis instrument, takes a picture, if there is the DNA band of 437bp in electrophoresis result, bolete to be measured is B.roseoflavus, if the DNA band of 437bp does not appear in electrophoresis result, bolete to be measured is B.speciosus.

PCR Buffer final concentration is 1 ×, refer to that in PCR Buffer, the concentration of each component in reaction system is identical with 1 × PCR Buffer, conventionally selecting volume is 10 × PCR Buffer of reaction system volume 1/10.10 × PCR Buffer composition is: 100mM Tris-HCl(pH8.5), 500mMKCl, 25mM MgCl ₂and 1.0%Triton-X-100, solvent is ddH ₂o.

Beneficial effect of the present invention is mainly reflected in: molecular specificity labeled primers of the present invention can carry out Molecular Identification fast to bolete sibling species B.roseoflavus and B.speciosus, method is simple, quick, accurate, is that morphological specificity is distinguished the effectively auxiliary of two kinds of boletes.

(4) brief description of the drawings

Fig. 1 is the result of B.roseoflavus and B.speciosus being carried out to pcr amplification; M:Marker, i.e. DNA molecular amount standard; C: negative control; Bs:B.speciosus; Bro:B.roseoflavus; Fig. 1 indication is that the B.speciosus sample that is numbered Bs does not amplify the special DNA band that molecular weight is 437bp, and the B.roseoflavus sample that is numbered Bro has amplified this band of arrow indication.

(5) embodiment

Below in conjunction with specific embodiment, the present invention is described further, but protection scope of the present invention is not limited in this:

Embodiment 1:

(1) extraction of bolete sample genomic dna: get bolete sample sporophore 0.2g to be measured, add liquid nitrogen and thoroughly grind, the extraction of genomic dna adopts SDS-CTAB method, through repeatedly extracting, extracts the genomic dna crude extract that obtains sample.DNA crude extract carries out purifying (rich day Bioer through Magabio nucleic acid purification test kit again, Hangzhou, China) after, the agarose gel electrophoresis by 1.5% and DNA/RNA ultraviolet spectrophotometer (GeneQuant Pro, GE Healthcare) detect integrity, purity and concentration.OD ₂₆₀/ OD ₂₈₀the DNA sample of >1.8 is for follow-up pcr amplification.DNA extraction thing is for subsequent use in-20 DEG C of refrigerator storages.

(2) design specific PCR amplimer, the sequence of primer pair is upstream primer 5 '-ATTGAAGACCGTCCGGGATG-3 ' and downstream primer 5 '-AGTTAAAAGTGACCAAGCC-3 ', synthetic by Shanghai biotechnology company limited.

(3) pcr amplification of ITS special primer:

PCR reaction system:

10 × PCR Buffer2.5 μ L, 10mmol/L dNTPs2 μ L, 25mmol/L MgCl ₂2 μ L, 5U/ μ L Taq DNA enzyme 0.3 μ L, the each 1 μ L of 10 μ mol/L upstream and downstream special primer, 17ng/ μ L template DNA 3 μ L, ddH ₂o14.2 μ L, reaction cumulative volume is 25 μ L.

Amplified reaction carries out on the TC-XP type amplification instrument of Hangzhou Bo company, reaction conditions: after 94 DEG C of denaturation 6min; 94 DEG C of sex change 40s, 65 DEG C of annealing 40s, 72 DEG C are extended 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C.

(4) electrophoresis detection: get step (3) pcr amplification product 5ul, mix with 1ul0.25% bromjophenol blue damping fluid, point sample is on 1.5% sepharose, in 1 × TAE damping fluid, electrophoresis under 5V/cm voltage, after electrophoresis finishes, dyeing 30 minutes containing in the aqueous solution of 0.5 μ g/m1EB, then train on the automatic gel image analysis instrument of clear JS-380A and take a picture in Shanghai.

According to the method described above, to B.roseoflavus and B.speciosus sample, (B.roseoflavus sample comes from sample shop, forest-science academy of Zhejiang Province (Herbarium of the Zhejiang Forestry Academy respectively, and Chinese Academy of Sciences's Kunming plant institute spore plant sample shop (Herbarium of Cryptogams HZFA), Kunming Institute of Botany, Chinese Academy of Sciences, HKAS); B.speciosus sample comes from University of Tennessee of the U.S. (University of Tennessee Herbarium, TENN)) detect, with the negative contrast of sterilized water, electrophoresis result is shown in Fig. 1.Wherein be numbered 7,8,9 B.speciosus sample and do not amplify the special DNA band that molecular weight is 437bp, amplified this band and be numbered 1,2,3,4,5,6 B.roseoflavus sample.

Claims (4)

1. differentiate the molecule spy of Chinese bolete B.roseoflavus and North America bolete B.speciosus for one kind

Opposite sex labeled primer, its nucleotide sequence is as follows:

Upstream primer: 5 '-ATTGAAGACCGTCCGGGATG-3 '

Downstream primer: 5 '-AGTTAAAAGTGACCAAGCC-3 '.

2. a method of utilizing molecular specificity labeled primers described in claim 1 to differentiate Chinese bolete B.roseoflavus and North America bolete B.speciosus, described method comprises: extract bolete sample genomic dna to be measured as template, using described molecular specificity labeled primers as amplimer, carry out pcr amplification, amplified production is carried out to electrophoresis detection, if there is the DNA band of 437bp in electrophoresis result, bolete to be measured is B.roseoflavus, if the DNA band of 437bp does not appear in electrophoresis result, bolete to be measured is B.speciosus;

Described molecular specificity labeled primers sequence is:

Upstream primer: 5 '-ATTGAAGACCGTCCGGGATG-3 '

Downstream primer: 5 '-AGTTAAAAGTGACCAAGCC-3 '.

3. method as claimed in claim 2, is characterized in that described pcr amplification condition is as follows: after 94 DEG C of denaturation 6min; 94 DEG C of sex change 40s, 65 DEG C of annealing 40s, 72 DEG C are extended 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C.

4. method as claimed in claim 2, is characterized in that method is as follows:

(1) get bolete sample sporophore to be measured, add liquid nitrogen and grind, extract the genomic dna of bolete to be measured with SDS-CTAB method;

(2) genomic dna extracting taking step (1), as template, using described molecular specificity labeled primers as amplimer, carries out pcr amplification:

10×PCR Buffer 2.5μL

10mM dNTPs 2μL

25mM MgCl ₂ 2μL

5U/ μ L Taq DNA enzyme 0.3 μ L

The each 1 μ L of 10 μ M upstream and downstream primer

17ng/ μ L template DNA 3 μ L

DdH ₂o complements to 25 μ L;

PCR reaction conditions is as follows:

After 94 DEG C of denaturation 6min; 94 DEG C of sex change 40s, 65 DEG C of annealing 40s, 72 DEG C are extended 2min, totally 30 circulations; Finally fill 7min in 72 DEG C, final temperature is 4 DEG C;

(3) get step (2) amplified production 5 μ L, mix with 1 μ L0.25% bromjophenol blue damping fluid, point sample is on 1.5% sepharose, electrophoresis in 1 × TAE damping fluid, under 5V/cm voltage, electrophoresis finishes rear EB dyeing, on automatic gel image analysis instrument, takes a picture, if there is the DNA band of 437bp in electrophoresis result, bolete to be measured is B.roseoflavus, if the DNA band of 437bp does not appear in electrophoresis result, bolete to be measured is B.speciosus.