CN108411030A - The method of primer pair and the kit comprising it, purposes and the detection M. truncatula ecotype A17 and R108 - Google Patents
The method of primer pair and the kit comprising it, purposes and the detection M. truncatula ecotype A17 and R108 Download PDFInfo
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Abstract
The present invention provides a kind of primer pair and the methods of the kit comprising it, purposes and the detection M. truncatula ecotype A17 and R108, are related to biotechnology.The primer pair includes primer NST480 F and primer NST480 R, it is respectively provided with the sequence as shown in SEQ ID NO.1 and SEQ ID NO.2, the DNA fragmentation of M. truncatula ecotype A17 and R108 different fragments length can be amplified simultaneously, therefore primary amplification can tell in sample to be tested whether contain the M. truncatula ecotype A17 and R108.Use the above-mentioned primer pair detection M. truncatula ecotype A17 and R108, the problem of having the characteristics that sensitivity is strong, accuracy rate is high, repeated strong and rapid and convenient, alleviating a kind of quickly and accurately method detection of shortages existing in the prior art and discriminating M. truncatula ecotype A17 and R108.
Description
Technical field
The present invention relates to biotechnologies, more particularly, to a kind of primer pair and include its kit, purposes and inspection
The method for surveying the M. truncatula ecotype A17 and R108.
Background technology
M. truncatula (Medicago truncatula) is used as model legume, has drought-resistant, cold-hot, high yield
With high-quality feature, while soil can be improved again, also have that growth cycle is short, genome is small, genetic transformation rate is high and is awarded from flower
The advantages of powder.M. truncatula, most of legume including alfalfa (M.sativa) are similar genetically having
Property.The information obtained from M. truncatula can be used for reference to other legumes.Therefore, in molecular biology of plants and gene
Group is learned M. truncatula in research and is concerned.
With the development of animal husbandry, the demand of herbage is increasing, and safety requirements is also higher and higher.Molecular biosciences learns to do
Section, the effect on herbage is more and more significant, and leguminous forage provides as one of most important Liang Ge sections in herbage for domestic animal
A large amount of protein resource, thus be the important topic in herbage field to the improvement of genes of leguminous forage.
For legume molecular biology field, model plant of the M. truncatula as leguminous forage is to study other
Whether excellent gene expresses in legume provides excellent experiment material.The accurately M. truncatula seed of high quality
It is the guarantee carried out to experiment safety, thus accurately identifies M. truncatula different ecological type rapidly, for improves China's pulse family
The molecular biology research of forage grass has a very important significance.
M. truncatula different ecological type A17 and R108 is very common under lab, but both M. truncatulas are ecological
Type laboratory purposes and differ.A17 M. truncatula genome sequences are that gained is sequenced by single seed progeny, are usually used in T-
DNA is inserted into analysis；The M. truncatula R108 ecotypes are because with that genome is small, seed production is big and the seed maturity time is short etc. is special
Point, so being commonly used for studying relevant molecular genetic process.
And both ecotypic seeds are extremely similar on mode of appearance, resolution is very difficult, once mix, it will be to puncture
Lamb's-quarters alfalfa gene functional study causes irremediable loss, and now both at home and abroad not yet one it is unified, effective
Discrimination method.It can only rely on and visually observe, so being as a result inaccurate.
Therefore a kind of quickly and accurately method detection highly desirable at present and differentiate M. truncatula ecotype A17 and
R108。
In view of this, special propose the present invention.
Invention content
The first object of the present invention is to provide a kind of primer pair of the detection M. truncatula ecotype A17 and R108, alleviate
The technical issues of detecting M. truncatula ecotype A17 and R108 technical deficiency in the prior art, which can amplify
The DNA fragmentation of environmental A17 and ecotype R108, and the fragment length of amplified production is different, therefore can detect to wait for simultaneously
Whether contain the M. truncatula ecotype A17 and R108 in sample.
The second object of the present invention is to provide a kind of kit including above-mentioned primer pair, which coordinates other points
Common agents tool has been widely used in sub- biological experiment.
The third object of the present invention is to provide a kind of above-mentioned primer pair or mentioned reagent box in detection M. truncatula ecology
Purposes in type A17 and R108, the purposes is very extensive, such as can be applied in detection sample to be tested whether contain puncture vine lucerne
The ingredient of the Mu ecotype A17 and/or R108, such as can also be applied to differentiate the M. truncatula ecotype A17 and R108；And have
There is detection or differentiates accurately, quickly, high-throughput advantage, and above-mentioned primer pair or kit apply also for detecting or differentiate
The mutant strain of the M. truncatula ecotype A17 and R108, therefore purposes is very extensive.
The fourth object of the present invention is to provide a kind of method of the detection M. truncatula ecotype A17 and R108, alleviate
It is existing in the prior art to lack a kind of quick and accurate method detection and differentiate asking for the M. truncatula ecotype A17 and R108
Topic.
In order to solve the above technical problems, spy of the present invention adopts the following technical scheme that：
A kind of primer pair of detection M. truncatula ecotype A17 and R108, the primer pair include primer NST480-F and
Primer NST480-R；
Wherein, there is the primer NST480-F sequence as shown in SEQ ID NO.1, the primer NST480-R to have such as
Sequence shown in SEQ ID NO.2.
Further, primer NST480-F and primer NST480-R are by modification；
Further, described to modify the 5 ' ends for being included in primer and 3 ' addition markers；
Further, the marker includes fluorescent marker, biotin labeling or digoxigenin labeled.
The present invention also provides a kind of kits including above-mentioned primer pair.
Further, the kit further includes Mg2+, dNTP, archaeal dna polymerase and PCR Buffer.
The present invention also provides a kind of above-mentioned primer pairs or mentioned reagent box in detection the M. truncatula ecotype A17 and R108
In purposes.
The present invention also provides a kind of method of the detection M. truncatula ecotype A17 and R108, this method includes following step
Suddenly：PCR amplification is carried out to the DNA of sample to be tested, by detect in amplified production whether comprising M. truncatula ecotype A17 and/
Or the DNA fragmentation of R108, judge the ingredient for whether including the M. truncatula ecotype A17 and/or R108 in sample to be tested；Wherein,
The primer used when the PCR amplification includes above-mentioned primer pair.
Further, the sample to be tested is seed.
Further, the genomic DNA of seed is extracted after first sprouting seed again.
Further, under the conditions of 22-27 DEG C, seed cdna group is extracted again after seed is sprouted 60-84h on filter paper
DNA。
Further, the condition of the PCR amplification is a) 94 DEG C of pre-degenerations 4 minutes；B) it is denaturalized 30 seconds for 94 DEG C；c)50-56
DEG C annealing 30 seconds；D) extend 30 seconds for 72 DEG C；Step b)-d) it recycles 25-40 times；E) extend 7 minutes for 72 DEG C；
Wherein, step b)-d) preferred cycle 30-38 times；More preferably cycle 32-36 times；
Annealing temperature is preferably 51-55 DEG C；More preferably 51-53 DEG C.
Compared with prior art, the present invention has the advantages that：
The primer pair of the detection M. truncatula ecotype A17 and R108 provided by the invention, can amplify puncture vine lucerne simultaneously
The DNA fragmentation of the Mu ecotype A17 and R108, and it expands the ecotypic amplified production length of two kinds of M. truncatula and differs,
Therefore primary amplification sample to be tested can tell whether sample to be tested includes ecotype A17 and R108, avoid separated amplification
When the error that is operated due to the difference of amplification efficiency or personnel caused by false positive or false negative, and above-mentioned primer pair may be used also
To expand the mutant strain of M. truncatula ecotype A17 and R108.
The kit provided by the invention for including above-mentioned primer pair, the kit coordinate in other molecular biology experiments often
Can have with reagent and have been widely used.
The use of above-mentioned primer pair provided by the invention or mentioned reagent box in detecting the M. truncatula ecotype A17 and R108
On the way, above-mentioned primer pair can not only detect wherein whether contain M. truncatula ecotype A17 and/or puncture vine from sample to be tested
The ingredient of clover ecotype R108, it is often more important that above-mentioned primer pair can differentiate M. truncatula ecotype A17 and M. truncatula
The seed of the seed of environmental R108, the ecotypic M. truncatula of above two is extremely similar on mode of appearance, differentiates very
It is difficult.But the ecotypic M. truncatula of above two has the function of different, the ecotype in molecular biology research field
A17 is usually used in T-DNA and is inserted into analysis；Environmental R108 is because with genome, small, seed production is greatly and the seed maturity time is short
The features such as, it is commonly used for studying relevant molecular genetic process.So if having obscured the kind of environmental A17 and ecotype R108
Son will cause irremediable loss to M. truncatula gene functional research.Therefore differentiate the ecotypic seed of above two very
There are application value and meaning.
The method of the detection M. truncatula ecotype A17 and R108 provided by the invention, it is time saving and energy saving, it is easy to operate, in reality
It tests under the conditions of room and can be good at completing in the short period, have that sensitivity is strong, accuracy rate is high, repeated strong and rapid and convenient etc.
Feature.This method is applied to differentiate the seed of the M. truncatula ecotype A17 and R108, contributes to Seed Inspection personnel to A17
With the differentiation of R108 seeds, it is legume molecular studies service to make M. truncatula preferably, and the method has sensitivity strong, accurate
The features such as true rate is high, repeatability is strong and rapid and convenient, can carry out the detection of the high-volume M. truncatula seed true and false, to ensure puncture
Lamb's-quarters alfalfa seed purity detecting provides feasible foundation.
Description of the drawings
It, below will be to specific in order to illustrate more clearly of the specific embodiment of the invention or technical solution in the prior art
Embodiment or attached drawing needed to be used in the description of the prior art are briefly described, it should be apparent that, in being described below
Attached drawing is some embodiments of the present invention, for those of ordinary skill in the art, before not making the creative labor
It puts, other drawings may also be obtained based on these drawings.
Fig. 1 is PCR product gel electrophoresis figure in the embodiment of the present invention 4；
Fig. 2 is PCR product gel electrophoresis figure in the embodiment of the present invention 4.
Specific implementation mode
Technical scheme of the present invention is clearly and completely described below in conjunction with attached drawing, it is clear that described implementation
Example is a part of the embodiment of the present invention, instead of all the embodiments.Based on the embodiments of the present invention, ordinary skill
The every other embodiment that personnel are obtained without making creative work, shall fall within the protection scope of the present invention.
The person that is not specified actual conditions in embodiment, carries out according to conventional conditions or manufacturer's recommended conditions.Agents useful for same or instrument are not
Production firm person is indicated, is the conventional products that can be obtained by commercially available purchase.
The present invention provides a kind of primer pairs of the detection M. truncatula ecotype A17 and R108, and the primer pair includes to draw
Object NST480-F and primer NST480-R；Wherein, the primer NST480-F has the sequence as shown in SEQ ID NO.1, described
Primer NST480-R has the sequence as shown in SEQ ID NO.2.
It is understood that when containing only environmental A17 in sample to be tested, ecology can be amplified using above-mentioned primer pair
The DNA fragmentation of type A17；When containing only environmental R108 in sample to be tested, the ecotype can be amplified using above-mentioned primer pair
The DNA fragmentation of R108；When containing ecotype A17 and ecotype R108 simultaneously in sample to be tested, above-mentioned primer pair can be simultaneously
Amplify the DNA fragmentation of environmental A17 and ecotype R108, and the piece of the amplified production of ecotype A17 and ecotype R108
Duan Butong can tell two kinds of products that above-mentioned primer pair amplifies go out after amplification, therefore can be from the amplification of above-mentioned primer pair
Known in product in sample to be tested while comprising ecotype A17 and the ecotype two kinds of ingredients of R108.
Primer NST480-F and primer NST480-R can amplify the DNA of M. truncatula ecotype A17 and R108 simultaneously
Segment, and it expands the ecotypic amplified production length of two kinds of M. truncatula and differs, therefore primary amplification sample to be tested is
It is distinguishable go out sample to be tested in whether include ecotype A17 and/or R108, due to the difference of amplification efficiency when avoiding separated amplification
False positive or false negative caused by the error of different or personnel operation, and above-mentioned primer pair can also expand M. truncatula ecology
The mutant strain of type A17 and R108.
In a kind of amplified production of the primer pair of the present invention, primer NST480-F and primer NST480-R amplify puncture vine
The DNA fragmentation of clover ecotype A17 has the sequence as shown in SEQ ID NO.3, which is 176bp, complementary
Sequence is as shown in SEQ ID NO.4；Primer NST480-F and primer NST480-R amplifies the DNA of M. truncatula ecotype R108
It is 167bp, complementary series such as SEQ IDNO.6 institutes that segment, which has the sequence as shown in SEQ ID NO.5, the sequence fragment length,
Show.
In some alternative embodiments, primer NST480-F and primer NST480-R are by modification；The modification example
Such as can be but be not limited to phosphorylation, biotin labeling, digoxigenin labeled, fluorophor label, thio-modification, deoxidation urine it is phonetic
Pyridine modification or deoxyinosine modification.As long as it can be understood that meeting the routine experiment principle of molecular biology experiment, originally
Invention does not limit the modification mode of primer.In some preferred embodiments, the primer is modified using fluorescent marker, through glimmering
The primer of signal modification can be used for quantitative fluorescent PCR.
The present invention also provides a kind of kits including above-mentioned primer pair.Above-mentioned primer pair is previously prepared as reagent
Conventional efficient can be improved in box, which coordinates in other molecular biology experiments common agents that can have to have been widely used.
Such as the kit optionally includes Mg2+, dNTP, the PCR reagents such as archaeal dna polymerase when can be used as PCR amplification kit, when into
One step can be used as PCR kit for fluorescence quantitative when further including fluorescent dye.It is understood that the kit can be used for examining
It is whether independent comprising M. truncatula ecotype A17 or M. truncatula ecotype R108 in survey sample to be tested, it can be used for examining
Whether survey simultaneously includes M. truncatula ecotype A17 or M. truncatula ecotype R108；When the kit also includes to detect other
When the primer of species, it can also be used to while detecting the sample of a variety of species.
The present invention also provides a kind of above-mentioned primer pairs or mentioned reagent box in detection the M. truncatula ecotype A17 and R108
In purposes.Above-mentioned primer pair can not only be detected from sample to be tested wherein whether containing M. truncatula ecotype A17 and/
Or the ingredient of M. truncatula ecotype R108, it is often more important that above-mentioned primer pair can differentiate M. truncatula ecotype A17 and puncture
The seed of lamb's-quarters clover ecotype R108, the seed of the ecotypic M. truncatula of above two is extremely similar on mode of appearance, point
Distinguish very difficult.But the ecotypic M. truncatula of above two have the function of in molecular biology research field it is different,
Environmental A17 is usually used in T-DNA and is inserted into analysis；Genome is small, seed production is big and seed maturity because having by environmental R108
The features such as time is short is commonly used for studying relevant molecular genetic process.So if having obscured environmental A17 and the ecotype
The seed of R108 will cause irremediable loss to M. truncatula gene functional research.Therefore differentiate that above two is ecotypic
Seed extremely has application value and meaning.
The present invention also provides a kind of method of the detection M. truncatula ecotype A17 and R108, this method includes following step
Suddenly：PCR amplification is carried out to the DNA of sample to be tested, by detect in amplified production whether comprising M. truncatula ecotype A17 and/
Or the DNA fragmentation of R108 judges whether include the ingredient of the M. truncatula ecotype A17 and/or R108 in sample to be tested；Wherein, institute
It includes above-mentioned primer pair to state the primer used when PCR amplification, both primer NST480-F and primer NST480-R.It is appreciated that
It is that the primer that when PCR amplification uses can also include other primer pairs in addition to primer NST480-F and primer NST480-R,
To achieve the purpose that while expand other purposes segment.Such as when need to detect in M. truncatula ecotype A17 and R108 whether
When adulterating other species and other ecotypes, corresponding primer can be added, composite PCR is carried out, the present invention does not limit addition
Other primer pairs, it is to be understood that the primer pair separately added should not influence the amplification efficiency of primer pair of the present invention.It can manage
Solution, above-mentioned PCR for example can be but be not limited to regular-PCR, quantitative fluorescent PCR or high-resolution solubility curve
(HRM) etc., the present invention does not limit the type of PCR, as long as it is anti-that primer pair provided by the invention can be used to carry out chain polymerization enzyme
It should；The present invention can also further be improved to enhance the reaction efficiency of PCR the system of PCR and the reagent used,
But what these improvement were completed when being all based on amplification using primer pair provided by the invention.
The method of the detection M. truncatula ecotype A17 and R108 provided by the invention, it is time saving and energy saving, it is easy to operate, in reality
It tests under the conditions of room and can be good at completing in the short period, have that sensitivity is strong, accuracy rate is high, repeated strong and rapid and convenient etc.
Feature.
In some preferred embodiments, the sample to be tested is the seed of M. truncatula.This method can be effective
The seed for identifying M. truncatula different ecological type A17 and R108, contributes to area of the Seed Inspection personnel to A17 and R108 seeds
Point, it is legume molecular studies service to make M. truncatula preferably, and the method is with sensitivity is strong, accuracy rate is high, repeated
The features such as strong and rapid and convenient, additionally it is possible to the discriminating for carrying out the high-volume M. truncatula seed true and false, to ensure M. truncatula seed
Purity detecting provides feasible foundation.
In some preferred embodiments, the genomic DNA of seed is extracted after first sprouting seed again.It is optional at some
In ground embodiment, under the conditions of 22-27 DEG C, seed cdna group DNA is extracted again after seed is sprouted 60-84h on filter paper.It can
Selection of land, the temperature that seed is sprouted for example can be but be not limited to 22 DEG C, 23 DEG C, 24 DEG C, 25 DEG C, 26 DEG C or 27 DEG C, preferably 25
℃；Optionally, seed sprout time for example can be but be not limited to 60h, 62h, 65h, 70h, 72h, 75h, 78h, 80h or
84h, preferably 72h；It is preferred that sprouting method germination seed using double-layer filter paper.Since kind of skin is thicker non-breakable, first seed is sprouted
Convenient for subsequent extracted seed DNA, it is preferred to use the method extraction seed DNA of SDS.
In some alternative embodiments, the system of the PCR amplification be 20 μ l systems, including TaKaRa 2 ×
The primer NST480-F 1 μ l of PCR TaqMix 10 μ l, a concentration of 50ng/ μ l DNA profilings 1 μ l, a concentration of 10nM, it is a concentration of
Primer NST480-R 1 the μ l and ddH of 10nM2O7μl；In the pcr amplification reaction for detection, 20 μ l systems can meet
The needs of the dosage of product detection after amplification.It is understood that above-mentioned PCR system can be according to actual experiment and detection feelings
Condition is adjusted and optimizes.
In some alternative embodiments, the condition of the PCR amplification is a) 94 DEG C of pre-degenerations 4 minutes；B) 94 DEG C of changes
Property 30 seconds；C) it anneals 30 seconds for 50-56 DEG C；D) extend 30 seconds for 72 DEG C；E) extend 7 minutes for 72 DEG C；Wherein, step b)-d) cycle 25-
40 times.Optionally, above-mentioned annealing temperature for example can be but be not limited to 50 DEG C, 51 DEG C, 52 DEG C, 53 DEG C, 54 DEG C, 55 DEG C or 56
DEG C, preferably 50-56 DEG C, more preferable 52 DEG C；Optionally, step b)-d) cycle-index for example can be but be not limited to 25,28,
30,32,35,38 or 40 times, preferably 32-36 times.Above-mentioned PCR amplification condition can be adjusted according to the change of PCR system
And optimization.
In some alternative embodiments, such as it can be but be not limited to using polyacrylamide gel electrophoresis, agar
The methods of sugared gel electrophoresis or capillary gel electrophoresis detect PCR amplification product, can also by PCR product direct Sequencing, or
Person can also select not make when that can show the method for product while PCR amplification using quantitative fluorescent PCR or HRM etc.
The amplified production that PCR is detected with additional technological means, it will therefore be appreciated that the present invention does not limit the detection side of PCR product
Method.In an embodiment preferably, using polyacrylamide gel electrophoresis method detect amplified production, this method at
This is low, and speed is fast, does not need additional Mdification primer；It is preferable to use the PAGE glues that gel is 6%-10%, such as can be but not
It is limited to the PAGE glue of a concentration of 6%, 7%, 8%, 9% or 10%, the PAGE glue that preferred concentration is 8% can be by adjusting excellent
Changing the condition of electrophoresis keeps the band of electrophoresis relatively sharp.
The advantageous effect further illustrated the present invention with reference to embodiment.
A kind of primer pair of detection M. truncatula ecotype A17 and R108 of embodiment 1
A kind of detection the M. truncatula ecotype A17 and R108 are present embodiments provided, which includes primer NST480-
F and primer NST480-R, wherein there is primer NST480-F the sequence as shown in SEQ ID NO.1, primer NST480-R to have such as
Sequence shown in SEQ ID NO.2.
NST480-F(5′-3′)：GGAGAACAATGAGAACAACAATG
NST480-R(5′-3′)：GACCACCATCACCACATGC
A kind of kit of detection M. truncatula ecotype A17 and R108 of embodiment 2
A kind of kit of the detection M. truncatula ecotype A17 and R108 is present embodiments provided, which includes such as
Lower component：Primer NST480-F and primer NST480-R, 2 × PCR Master and ddH2O；
Wherein 2 × PCR Master include 3mM MgCl2, 0.2mM dNTP, 0.1U/ μ l Taq and 2 × PCR Buffer.
A kind of method for the seed differentiating purple the M. truncatula ecotype A17 and R108 of embodiment 3
Present embodiments provide a kind of method for the seed differentiating purple the M. truncatula ecotype A17 and R108, this method packet
Containing following steps：
A) collect M. truncatula ecotype A17 and R108 difference saltant types seed, using double-layer filter paper sprout method, 25 DEG C
It sprouts 72 hours, it is spare that SDS methods extract germination seed genomic DNA；
B) use primer NST480-F and primer NST480-R to step A) in extract genomic DNA carry out PCR amplification；
PCR amplification system is 20 μ l systems, including 2 × PCR of TaKaRa TaqMix 10 μ l, a concentration of 50ng/ μ l
Primer NST480-R 1 the μ l and ddH of primer NST480-F 1 the μ l, a concentration of 10nM of DNA profiling 1 μ l, a concentration of 10nM2O 7
μl；
PCR amplification program：A) 94 DEG C of pre-degenerations 4 minutes；B) it is denaturalized 30 seconds for 94 DEG C；C) it anneals 30 seconds for 52 DEG C；D) prolong for 72 DEG C
It stretches 30 seconds；Step b)-d) it recycles 35 times；E) extend 7 minutes for 72 DEG C；
C) PCR product detects：It is detected with 8% PAGE glue, 100ml systems, 53.0ml H20,26.3ml poly- third
Alkene, 20ml 35% ammonium persulfates of 5 × TBE, 700 μ l, 70 μ l TEMED solidify 50min, in riser electrophoresis apparatus, 200V electricity
Piezoelectricity swimming 1h40min, uses nucleic acid staining dye, is placed in gel imager and takes a picture later；Judge kind according to amplified production segment
Son is environmental A17 or ecotype R108.
4 primer NST480-F of embodiment and primer NST480-R amplification M. truncatula seeds DNA
Take 30 kinds of different mutant strain seeds, seed information in M. truncatula ecotype A17 as shown in table 1；Take puncture vine lucerne
30 kinds of different mutant strain seeds, seed information are as shown in table 2 in Mu ecotype R108；All seeds are sprouted using double-layer filter paper
Hair method, 25 DEG C are sprouted 72 hours, and SDS methods extract germination seed genomic DNA, are that 50ng/ μ l make by genomic DNA concentration dilution
For template, the system of PCR amplification, the method for reaction condition and amplified production electrophoresis is the same as embodiment 3；As a result such as Fig. 1 and Fig. 2 institutes
Show, wherein the sample that each swimming lane represents is as shown in Table 1 and Table 2, swimming lane M represents Marker；It can be seen that from Fig. 1 and Fig. 2 with puncture
The seed cdna group DNA of the lamb's-quarters clover ecotype A17 and R108 are that the electrophoretic band of template clearly becomes clear, with the M. truncatula ecotype
Band is 176bp when the genomic DNA of A17 seeds is template, using the genomic DNA of M. truncatula ecotype R108 seeds as mould
The electrophoretic band of plate is 167bp, and primer NST480-F and primer NST480-R can amplify the DNA of various mutations body.
1 M. truncatula ecotype A17 mutant information of table
1 M. truncatula ecotype R108 mutant information of table
Embodiment 5 primer NST480-F and primer NST480-R
Further to verify the validity of primer NST480-F and primer NST480-R, to M. truncatula ecotype A17 and
The DNA fragmentation of the amplified production of R108 is sequenced.The amplification production of the purple M. truncatula ecotype A17 and R108 of its result display
All include primer NST480-F and primer NST480-R in object segment.
Using 50ng/ μ l M. truncatula ecotype A17 genomic DNAs template, using primer NST480-F and NST480-R
Primer carries out PCR amplification.PCR system and method are the same as embodiment 3.PCR product in 2% agarose through detecting, and recycling length is about
The DNA fragmentation of 170bp obtains pGEM-T-NST480-A17 recombinant plasmids after connecting pGEM-T carriers, which is converted large intestine
Bacillus DH5 α competent cells.The Escherichia coli of the plasmid containing pGEM-T-NST480-A17 are applied to containing Amp, IPTG and X-Gal
On LB culture mediums, 37 DEG C are incubated overnight, and 3 positive colony bacterial plaques of picking are shaken for 37 DEG C overnight in the LB liquid medium containing Amp
Bacterium is cultivated.With it is expected that stripe size is consistent after bacterium solution detection, serves Hai Shenggong bioengineering Co., Ltd and be sequenced, select 3
A positive colony bacterial strain, sends to sequencing.The results show that primer NST480-F and primer NST480-R can be well to puncture vine lucerne
The DNA fragmentation of Mu ecotype A17 is expanded, and the special-purpose gene segment of 176bp has been amplified.The M. truncatula wherein amplified
It is 176bp that the segment of environmental A17, which has the sequence as shown in SEQ ID NO.3, the sequence fragment length, and complementary series is such as
Shown in SEQ ID NO.4.
Using 50ng/ μ l M. truncatula ecotype R108 genomic DNAs template, using primer NST480-F and NST480-R
Primer carries out PCR amplification.PCR system and method are the same as embodiment 3.PCR product is through 2% agar sugar detection, recycling length about 167bp
DNA fragmentation, connect pGEM-T carriers after obtain pGEM-T-NST480-R108 recombinant plasmids, by the plasmid convert Escherichia coli
DH5 α competent cells.The Escherichia coli of the plasmid containing pGEM-T-NST480-R108 are applied to the LB containing Amp, IPTG and X-Gal
On culture medium, 37 DEG C are incubated overnight, and 3 positive colony bacterial plaques of picking shake bacterium for 37 DEG C overnight in the LB liquid medium containing Amp
Culture.With it is expected that stripe size is consistent after bacterium solution detection, serves Hai Shenggong bioengineering Co., Ltd and be sequenced, select 3
Positive colony bacterial strain sends to sequencing.The results show that primer NST480-F and primer NST480-R can be well to M. truncatula
The DNA fragmentation of environmental R108 is expanded, and the special-purpose gene segment of 167bp has been amplified.The M. truncatula wherein amplified
It is 167bp that the segment of environmental R108, which has the sequence as shown in SEQ ID NO.5, the sequence fragment length, and complementary series is such as
Shown in SEQ ID NO.6.
Finally it should be noted that：The above embodiments are only used to illustrate the technical solution of the present invention., rather than its limitations；To the greatest extent
Present invention has been described in detail with reference to the aforementioned embodiments for pipe, it will be understood by those of ordinary skill in the art that：Its according to
So can with technical scheme described in the above embodiments is modified, either to which part or all technical features into
Row equivalent replacement；And these modifications or replacements, various embodiments of the present invention technology that it does not separate the essence of the corresponding technical solution
The range of scheme.
SEQUENCE LISTING
<110>Lanzhou University
<120>The method of primer pair and the kit comprising it, purposes and the detection M. truncatula ecotype A17 and R108
<160> 6
<170> PatentIn version 3.5
<210> 1
<211> 23
<212> DNA
<213>Artificial sequence
<400> 1
ggagaacaat gagaacaaca atg 23
<210> 2
<211> 19
<212> DNA
<213>Artificial sequence
<400> 2
gaccaccatc accacatgc 19
<210> 3
<211> 176
<212> DNA
<213>M. truncatula（Medicago truncatula）
<400> 3
ggagaacaat gagaacaaca atgatgggta tcatgcaatt attcaagtag atatggctaa 60
tgaaaatgaa gggtcattct ctgatcatca tcatcatcat catcataaca acatggttaa 120
taatccatta gaggcatcat catcatcaat ggtgatagca tgtggtgatg gtggtc 176
<210> 4
<211> 176
<212> DNA
<213>M. truncatula（Medicago truncatula）
<400> 4
cctcttgtta ctcttgttgt tactacccat agtacgttaa taagttcatc tataccgatt 60
acttttactt cccagtaaga gactagtagt agtagtagta gtagtattgt tgtaccaatt 120
attaggtaat ctccgtagta gtagtagtta ccactatcgt acaccactac caccag 176
<210> 5
<211> 167
<212> DNA
<213>M. truncatula（Medicago truncatula）
<400> 5
ggagaacaat gagaacaaca atgatgggta tcatgcaatt attcaagtag atatggctaa 60
tgaaaatgaa ggatcattct ctgatcatca tcatcataac aacatggtta ataatccatt 120
agaggcatca tcatcatcaa tggtgatagc atgtggtgat ggtggtc 167
<210> 6
<211> 167
<212> DNA
<213>M. truncatula（Medicago truncatula）
<400> 6
cctcttgtta ctcttgttgt tactacccat agtacgttaa taagttcatc tataccgatt 60
acttttactt cctagtaaga gactagtagt agtagtattg ttgtaccaat tattaggtaa 120
tctccgtagt agtagtagtt accactatcg tacaccacta ccaccag 167
Claims (10)
1. a kind of primer pair of the detection M. truncatula ecotype A17 and R108, which is characterized in that the primer pair includes primer
NST480-F and primer NST480-R；
There is the primer NST480-F sequence as shown in SEQ ID NO.1, the primer NST480-R to have such as SEQ ID
Sequence shown in NO.2.
2. primer pair according to claim 1, which is characterized in that primer NST480-F and primer NST480-R are by repairing
Decorations；
Preferably, described to modify the 5 ' ends for being included in primer and 3 ' addition markers；
Preferably, the marker includes fluorescent marker, biotin labeling or digoxigenin labeled.
3. a kind of kit including primer pair as claimed in claim 1 or 2.
4. kit according to claim 3, which is characterized in that the kit further includes Mg2+, dNTP, archaeal dna polymerase
With PCR Buffer.
5. the kit described in a kind of primer pair as claimed in claim 1 or 2 or claim 3 or 4 is in detection M. truncatula life
Purposes in state type A17 and R108.
6. a kind of method of the detection M. truncatula ecotype A17 and R108, which is characterized in that include the following steps：Treat test sample
The DNA of product carries out PCR amplification, whether includes the DNA of the M. truncatula ecotype A17 and/or R108 in amplified production by detecting
Segment judges the ingredient for whether including the M. truncatula ecotype A17 and/or R108 in sample to be tested；
Wherein, the primer used when the PCR amplification includes primer pair as claimed in claim 1 or 2.
7. according to the method described in claim 6, it is characterized in that, the sample to be tested is seed.
8. the method according to the description of claim 7 is characterized in that the genomic DNA of seed is extracted after first sprouting seed again.
9. according to the method described in claim 8, it is characterized in that, under the conditions of 22-27 DEG C, seed is sprouted on filter paper
Seed cdna group DNA is extracted after 60-84h again.
10. according to the method described in claim 8, it is characterized in that, the condition of the PCR amplification is that a) 94 DEG C of pre-degenerations 4 are divided
Clock；B) it is denaturalized 30 seconds for 94 DEG C；C) it anneals 30 seconds for 50-56 DEG C；D) extend 30 seconds for 72 DEG C；Step b)-d) it recycles 25-40 times；e)72
DEG C extend 7 minutes；
Wherein, step b)-d) preferred cycle 30-38 times；More preferably cycle 32-36 times；
Annealing temperature is preferably 51-55 DEG C；More preferably 51-53 DEG C.
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| CN108411030B (en) | 2021-05-14 |
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