CN104962638B - A kind of hypodontia alpine rush or palm-bark rain cape moss ISSR PCR molecule labelling methods - Google Patents
A kind of hypodontia alpine rush or palm-bark rain cape moss ISSR PCR molecule labelling methods Download PDFInfo
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Abstract
The present invention relates to a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR PCR molecule labelling methods, comprise the following steps：1) genomic DNA of extraction hypodontia alpine rush or palm-bark rain cape moss, 2) carry out ISSR PCR amplifications；Step 2) carry out ISSR PCR amplifications reaction system be：Contain 10 × LAPCR buffer solutions 3 μ L, template DNA 50ng, Mg in 30 μ L reaction systems2+For 1.5mmol/L, dNTP 0.25mmol/L, Taq enzyme 1.5U, primer is 0.5 μm of ol/L.The condition of the ISSR amplifications is by taking primer UBC834 as an example：94 DEG C of annealing 4min, then 94 DEG C of 45S, 53 DEG C of 45S, 72 DEG C of 1min45 circulations, last 72 DEG C of extensions 10min.Compared with prior art, the band stability that the hypodontia alpine rush or palm-bark rain cape moss ISSR PCR reaction systems that the present invention is established amplify is high, and definition is high, has the characteristics of very high polymorphism.The domestic deficiency to hypodontia alpine rush or palm-bark rain cape moss genetic diversity Journal of Sex Research is compensate for, the achievement of invention has very big scientific value and application value available for the research in terms of hypodontia alpine rush or palm-bark rain cape moss genetic affinity, molecular labeling and biogeography.
Description
Technical field
The present invention relates to biology field, more particularly, to a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule labelling methods.
Background technology
Orthotrichaceae (Orthotrichaceae) alpine rush or palm-bark rain cape Rhodobryum (Macromitrium Bridel) is the class of general tropical distribution
Group.The alpine rush or palm-bark rain cape Rhodobryum floristics of China is very abundant, and hypodontia alpine rush or palm-bark rain cape moss (Macromitrium gymnostomum) is therein one
Big species, because capsule cap it is anodontia it is therefore named be hypodontia alpine rush or palm-bark rain cape moss, this kind is mainly characterized by stem and crawls to overgrow, dense rufous rhizoid；Branch
Bar is upright, single or have several small brachyplasts, up to 5.0mm.Middle part of blade cell is transparent, rectangle, heavy wall, smoothly；Top is thin
Born of the same parents are slightly opaque, circular, have unconspicuous wart.Branches and leaves crimp when drying, and linear during moistening, wire lanceolar or ovate drape over one's shoulders pin
Shape, sporangium is upright, ellipticity cylinder, has shrinkage, peristomal teeth missing.Capsule balaclava shape, long 1.7-2.0mm are long only once in a while
1.5mm, base portion is very wide, how many divisions and tool gauffer, hairless.The strip gemmule being made up of 4-10 cell with brownish red
Born of the same parents.Sullivant, William Starling are in 1859 in Proceedings of the American Academy of
Arts and Sciences deliver the name article of this kind first.Bryophyte is one of pioneer of plant kingdom, in plant kingdom
There is special status during phylogeny.The bryophyte part important as the ecosystem, to rock decay, soil
Earth organic matter accumulation, forest swamping etc. have important Ecological Functions, and it leads in environmental protection, medicinal, gardening etc.
Domain and scientific research value in the subjects such as ecology, Phytochemistry, cytogenetics are also of increasing concern.With DNA points
This technology is widely used in identification, diversity analysis of species etc. by the development of sub- labelling technique, people.
DNA molecular marker technology has developed to more than ten and planted at present, and conventional means is including RAPD, ISSR, AFLP, SRAP etc..
ISSR (inter-simple sequence repeat, Inter-simple sequence repeat) is by Canada
The it is proposeds such as Zietkiewicz, it using the microsatellite of grappling is primer that its principle, which is, and augmental interval is not two very big SSR
Between sequence, then pass through gel electrophoresis strip analyze sample DNA polymorphism.The advantages of ISSR molecular engineerings is to be known a priori by
Any information of genome can be carried out marking, and can be detected in any stage of growth cycle, and design of primers
Easily, cost is low, and DNA dosages are few, therefore is considered as a kind of more satisfactory molecule labelling method.ISSR molecular marking techniques
Be widely used in the analysis of genetic diversity of many angiosperms, but the research for bryophyte be not it is a lot,
Rarely seen Skotnicki to cucumber silk moss, Liu Li etc. to the research of rat-tail moss and Li Qianying to the genetic diversity of Haplocladium,
For ISSR labelling methods using less, the country sees side moss, Bryaceae and the Wang Yingying big to narrow leaf such as Wang Chenying to Zhejiang thousand
The research of island lake big hypnum moss, Pogonatum inflexum (Lindb.) Lac genetic diversity.
Therefore, the research to hypodontia alpine rush or palm-bark rain cape moss and other species compare also gap, and establishing one relatively scientific and reasonable has
The ISSR-PCR reaction systems of effect, optimal processing combination is sought, good technology can be provided for follow-up scientific research and referred to
Lead and theories integration.
The content of the invention
The purpose of the present invention is just to provide for a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule labelling methods, to fill up existing skill
The blank of art.
The purpose of the present invention can be achieved through the following technical solutions：
A kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule labelling methods, comprise the following steps：
1) genomic DNA of hypodontia alpine rush or palm-bark rain cape moss is extracted；
2) ISSR-PCR amplifications are carried out.
Step 2) carry out ISSR-PCR amplifications reaction system be：Contain 10 × LA PCR buffer solutions in 30 μ L reaction systems
3 μ L, template DNA 50ng, Mg2+For 1.5mmol/L, dNTP 0.25mmol/L, Taq enzyme 1.5U, primer is 0.5 μm of ol/L.
Any bar of the described primer in SEQ ID No.1~10, shown in table specific as follows：
Step 2) carry out ISSR-PCR amplifications condition be：94 DEG C annealing 4min, then 94 DEG C of 45S, 53 DEG C of 45S, 72 DEG C
40 circulations of 1min, last 72 DEG C of extensions 10min.
The period that step 2) carries out ISSR-PCR amplifications is 25-45, preferably 40.
Compared with prior art, the band that the hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR reaction systems that the present invention is established amplify is stable
Property it is high, definition is high, has the characteristics of very high polymorphism.Compensate for the country to hypodontia alpine rush or palm-bark rain cape moss genetic diversity Journal of Sex Research not
Enough, the research in terms of the achievement of invention can be used for hypodontia alpine rush or palm-bark rain cape moss genetic affinity, molecular labeling and biogeography has very big
Scientific value and application value.
Brief description of the drawings
Fig. 1 is the amplification of ISSR-PCR exploration Orthogonal Experiment and Design reaction systems；
M is represented：DL2000Marker；Primer is UBC834；1-9 is that 1-9 is combined in the processing of table 3；
Fig. 2 is ISSR-PCR fine tuning orthogonal test figures；
M is represented：DL2000Marker；Primer UBC834；1-9 is that 1-9 is combined in the processing of table 3
Fig. 3 is the primer amplification of the suitable hypodontia alpine rush or palm-bark rain cape moss ISSR amplifications filtered out；
M is represented：DL2000Marker；1,2.UBC807；3,4.UBC810；5,6.UBC 811；7,8.UBC 808；9,
10.UBC 834；11,12.UBC 835；13,14.UBC889；15,16.UBC 880；17,18.UBC 890；19,20.UBC
826；21,22.UBC 842；23,24.UBC 856.
Fig. 4 is the amplification of different cycle-indexes in ISSR-PCR reaction systems；
1-4 is respectively number of cycles 25,30,35,40 and 45；M is represented：DL2000；Primer is UBC834.
Embodiment
The present invention is described in detail with specific embodiment below in conjunction with the accompanying drawings.
Embodiment 1
Extract hypodontia alpine rush or palm-bark rain cape moss genomic DNA.
Embodiment 2
The screening and optimization of amplification system
1st, the Orthogonal Experiment and Design of exploratory ISSR-PCR reaction systems
Orthogonal Optimum Design screening ISSR peak optimization reaction system (four factor three is horizontal), from L9(34) orthogonal arrage, design
The factor of each composition of PCR amplification system-horizontal quadrature contrived experiment table (scheme be shown in Table 2 and table 3).Using UBC834 as primer, profit
With 9 systems in table 3, ISSR amplifications are carried out to hypodontia alpine rush or palm-bark rain cape moss genomic DNA.Reaction system is 30 μ L, except in table it is listed because
Plain outer, often pipe contains 10 × LA PCR buffer 3 μ L, DNA profiling 50ng.Amplification program is：94 DEG C of annealing 4min, then 94
DEG C 45S, 53 DEG C of 45S, 72 DEG C of 1min40 circulations, last 72 DEG C of extensions 10min, 10 DEG C of ° of preservations.
The primer and sequence of the ISSR of table 1 amplifications
Orthogonal Experiment and Design [L9 (34)] unit of the exploratory ISSR-PCR reaction systems of table 2：μL
Exploratory orthogonal experiments are shown in Fig. 1, and No. 1, No. 8 do not amplify band, and No. 2, No. 3, No. 4, No. 5 processing have micro-
Weak band, No. 7 bars are not high with still definition, and how reproducible the bands of No. 6 and No. 9 processing are, illustrates away from optimal set
It is little to close deviation, as basic point, the concentration gradient for reducing each factor carries out fine tuning orthogonal test for we.
2nd, the foundation of fine tuning ISSR-PCR reaction systems
The Orthogonal Experiment and Design of fine tuning ISSR-PCR reaction systems is shown in Table 3.
The Orthogonal Experiment and Design of the fine tuning ISSR-PCR reaction systems of table 3
Fine tuning orthogonal experiments are shown in Fig. 2, and in addition to No. 5 and No. 6 are handled, others processing combination has all been run out of obvious
Band.No. 7, the band that No. 9 processing are run out of is few, illustrates that repeatability is not high.No. 1, No. 2, No. 8 have amplified band, but phase
One band has been lacked to No. 3 and No. 4, has illustrated that repeatability is bad, it is No. 4 processing to therefore deduce that best combination.Total coamplification
6 bands are gone out, and band is clear, is combined for optimal processing.
Using the system after optimization, using identical DNA as template, to the 100 of Canadian Columbia University (UBC) offer
Individual primer is screened, and finally screening draws UBC807, UBC810, UBC 811, UBC826, UBC834, UBC835, UBC
842, UBC 856, UBC 889, UBC 890 is primer, carries out ISSR amplifications, can amplify definition height, and stability is good
Band (Fig. 3 represents primer picture), illustrate that the reaction system is suitable for hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR reactions.
3rd, the determination of optimum cycle number
This experiment ISSR reaction primers use the sequence that Canadian Columbia University (UBC) is provided, according to orthogonal test
The result of design and the annealing temperature provided, carries out the determination of period.Period is followed successively by：25,30,35,40,45.Often
It is individual to be circularly set 2 repetitions.
According to orthogonal test and the result of annealing temperature, the detection of period is carried out, as can be seen from Figure 4：Work as circulation
Amplification does not almost have band when number is 25,30；Band is not apparent from when period is 35, and repeatability is not high；Period is 40
It is all higher than more visible repeatability with band when 45, to save time and experimentation cost, select period to compare conjunction for 40
It is suitable.
4th, conclusion
Using Orthogonal Experiment and Design, from Mg2+Concentration, dNTP concentration, Taq DNA enzymatics concentration and the water of 4 factor of primer concentration 3
It is flat, analysis is optimized to hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR reaction system, and PCR period is tried on this basis
Test.As a result show：In 30 μ L reaction systems, 10 × LA PCR buffer solutions 3 μ L, template DNA 50ng are contained in 30 μ L reaction systems,
Mg2+For 1.5mmol/L, dNTP 0.25mmol/L, Taq enzyme 1.5U, when primer is 0.5 μm of ol/L, and ISSR-PCR is most suitable
Period be 40 when, expanding effect is best.For hypodontia alpine rush or palm-bark rain cape moss genetic diversity further research established technical foundation and
Scientific basis.
The above-mentioned description to embodiment is understood that for ease of those skilled in the art and using invention.
Person skilled in the art obviously can easily make various modifications to these embodiments, and described herein general
Principle is applied in other embodiment without by performing creative labour.Therefore, the invention is not restricted to above-described embodiment, ability
Field technique personnel do not depart from improvement that scope made and modification all should be the present invention's according to the announcement of the present invention
Within protection domain.
Claims (1)
1. a kind of hypodontia alpine rush or palm-bark rain cape moss ISSR-PCR molecule labelling methods, it is characterised in that comprise the following steps：
1) genomic DNA of hypodontia alpine rush or palm-bark rain cape moss is extracted；
2) ISSR-PCR amplifications are carried out；
Step 2) carry out ISSR-PCR amplifications reaction system be：Contain the μ L of 10 × LA PCR buffer solutions 3 in 30 μ L reaction systems,
Template DNA 50ng, Mg2+For 1.5mmol/L, dNTP 0.25mmol/L, Taq enzyme 1.25U, primer is 0.5 μm of ol/L, described
Primer sequence as shown in SEQ ID No.5；
Carry out ISSR-PCR amplifications condition be：94 DEG C of annealing 4min, then 94 DEG C of 45s, 53 DEG C of 45s, 72 DEG C of 1min 40 are followed
Ring, last 72 DEG C of extensions 10min, the period for carrying out ISSR-PCR amplifications is 40.
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101451163A (en) * | 2008-12-22 | 2009-06-10 | 陈乃富 | Construction and identification method of molecular marking fingerprint of Dendrobium huoshanense and similitude species thereof |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN101451163A (en) * | 2008-12-22 | 2009-06-10 | 陈乃富 | Construction and identification method of molecular marking fingerprint of Dendrobium huoshanense and similitude species thereof |
Non-Patent Citations (2)
| Title |
|---|
| 水藓科植物ISSＲ－ PCＲ反应体系的优化;买买提明·苏来曼等;《生物技术》;20131231;第23卷;第42-46页 * |
| 真藓科植物ISSR-PCR 反应体系的优化及ISSR 指纹图谱的初步构建;汪琛颖等;《安徽农业科学》;20111231;第39卷(第27期);第16491页第1段、第16491页左栏第1-2段、第16492页第1-2段、表2 * |
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