CN100572560C - Plant monad high-throughput is collected and the PCR detection method - Google Patents

Abstract

The present invention discloses a kind of plant monad high-throughput and collects and the PCR detection method, comprises that step is: microscope separates pollen and broken cyst wall of pollen and dyeing; Packing alkaline bleach liquor cleavage liquid is in the PCR plate, and sealing and the centrifugal lysate that makes are sunken to the pipe end; Microscopically is selected painted monad grain with special tweezers and is put into PCR pipe lysate; Packing mineral oil is in the PCR plate, and is of short duration centrifugal; Place on the PCR instrument cracking under the 90-98 ℃ of temperature; In each pipe, add the neutralization of TE damping fluid, centrifugal, add the PCR reacted constituent; Of short duration centrifugal, place on the PCR instrument, reaction parameter is: 90-98 ℃ kept 3-8 minute, and thermal cycling is decided according to differential responses, and cycle number is 35-40, and last 72 ℃ were extended 5-10 minute, and remained under 4 ℃; The PCR product is taken pictures with electrophoretic separation and in the enterprising line scanning of imaging system.The present invention has the efficient advantages of higher, is specially adapted to the reproduction genetic statistics and analyzes.

Description

Plant monad high-throughput is collected and the PCR detection method

Affiliated technical field:

The invention belongs to a kind of plant monad high-throughput collects and the PCR detection method.

Background technology:

Monad PCR success on several species, but because single pollen separates and the cracked difficulty, monad PCR only limits on a small quantity (reaching 60 at most) pollen granule.This has limited the application of monad PCR in plant genetic analysis and karyomit(e) drawing.

Very little (the Pinar and Inceoglu 1999 of pollen; Su and Saunders 2003; Tel-Zur et al.2003; Wen and Nowicke 1999), several minutes (the Fritz andLukaszewski 1989 of after the pollen sac cracking, can only surviving under the natural condition; Khatun and Flowers 1995; Luna et al.2001).Therefore based on the research of pollen activity, the efficient of sampling and genetic analysis seems very important.PCR method is at unicellular and heritable variation (Azizand Sauve 2003 monad; Li et al.1990; Matsunaga et al.1999; Parducci et al.2005; Petersenet al.1996; Zhang et al.1992), genetic expression (Shoemaker et al.2005) and genotype (Aziz etal.2005; Li and Yeung 2002) success in the analysis.Yet, in these researchs, only use a spot of monad grain, have only an example to reach 60 (Aziz et al.2005) at most.Pollen is one group of haploid set with different genotype, and each monad has only obtained the part (Copenhaver et al.2000) of the whole nuclear gene group of its donor.From the angle of statistics, a large amount of pollen analyses is most important for genetic research.In general, pollen granule is very little and have a thick pollen wall (Pinar and Inceoglu 1999; Su and Saunders 2003; Tel-Zur et al.2003; Wen and Nowicke 1999), obtain enough pollen granules and DNA wherein to be discharged and do not suppress that PCR reacts be two bottleneck places of monad PCR method.

Unicellular, can utilize flow velocity cell counter (Li et al.1990 as human spermoblast; Zhang et al.1992), micromanipulator (Shoemaker et al.2005) separates with kapillary (Li and Yeung 2002); The useful separation by laser instrument of single pollen granule (Matsunaga et al.1999), micromanipulator (Aziz and Sauve 2003; Aziz etal.2005; Haliyo and Regnier 2003) separates with special glass microtubule (Parducci et al.2005).Micromanipulator is the key instrument of the single pollen of current separation, but because the glass microtubule frangibility of using can not directly pollen granule be put in the PCR pipe, so efficient is very low the highest 60 (the Aziz etal.2005) that do not surpass of the pollen granule number of being tried.The separation by laser instrument can the single pollen granule of high efficiency separation, but very expensive.The wall that the mature pollen grain is not only thick but also hard (Parre and Geitmann 2005; Santos and Mariath 1999) with and size (0.005 to 0.25mm in diameter) suit very much directly to separate with meticulous tweezers at microscopically.On the other hand, it is more much more difficult than zooblast that the cracking pollen granule discharges nucleic acid.Once attempt several different methods for this reason, comprised laser beam cutting (Matsunaga et al.1999), damping fluid cracking (Matsunaga et al.1999), pre-(the Aziz and Sauve 2003 that germinates that cultivates; Azizet al.2005), or with pipette crushing (Parducci et al.2005) etc.Yet most laboratory there is not ability to purchase expensive laser beam cutting unit, and chemical substance (the Aziz andSauve 2003 of many inhibition PCR reaction is introduced in substratum presprouting meeting; Aziz et al.2005), pipette crushing method is only applicable to the big fossil pollen grain of crushing.Some studies show that pollen wall can be by some chemical agent dissolves (Loewus et al.1985; Southworth 1974).Recently, Xin etc. (2003) has reported a kind of fast method that does not influence the PCR reaction, is applicable to extracting various plants DNA.

Summary of the invention:

The object of the present invention is to provide a kind of plant monad high-throughput to collect and the PCR detection method, this method can be applied to the ability that monad PCR was collected and carried out to monad in enormous quantities, for plant genetic analysis and karyomit(e) drawing provide a kind of new method.

The object of the present invention is achieved like this, and described plant monad high-throughput is collected and the PCR detection method, comprises the steps: in regular turn

1) microscope separates complete, clean not cracking pollen and broken cyst wall of pollen and dyeing, with the careful emptying of staining fluid; The pollen granule that stays cleans, relief pollen granule complete drying;

2) suit μ l lysate in the PCR plate in suitable number hole with the liquid getting device packing, sealing and the centrifugal lysate that makes are sunken to the pipe end;

3) select painted monad grain and painted monad grain is put into the PCR pipe lysate at the end with tweezers at microscopically;

4) packing suits μ l mineral oil in the PCR plate in suitable number hole, and is of short duration centrifugal;

5) the PCR plate in the suitable number hole of step 4) under 90-98 ℃ of temperature, keeping 16 minutes and-18 minutes and 30 seconds on the PCR instrument;

6) the TE damping fluid of the suitable μ l of adding in each pipe of step 5) is centrifugal, adds PCR reacted constituent mixed solution then and (generally contains 1 * PCR reaction buffer, sufficient quantity dATP, dTTP, dGTP and dCTP, MgCl ₂, primer, template DNA, Taq archaeal dna polymerase, H ₂O), adjusting final volume is 20 μ l;

7) of short duration centrifugal, place on the PCR instrument, the PCR reaction parameter is: 90-98 ℃ keeps carrying out in 3-8 minute pre-sex change, the corresponding PCR program setting that thermal circulation parameters carries out according to plan, cycle number of the present invention is set at 35-40, last 72 ℃ were extended 5-10 minute, obtained the PCR product, remained under 4 ℃ of temperature;

8) the PCR product of step 7) is taken pictures with electrophoretic separation and in the enterprising line scanning of imaging system.

It is to separate complete, clean one by one not cracking pollen sac at microscopically with tweezers to put on the slide glass with painted step that the described microscope of step 1) separates complete, clean pollen and the broken cyst wall of pollen of not ftractureing, dripping staining fluid and needling cyst wall overflows pollen, slide glass is put into culture dish with cover is 23-30 ℃ in temperature and cultivated 20-40 minute down, the back take out slide glass with tweezers with the careful emptying of staining fluid; The pollen granule that stays is adjusted density in the visual field simultaneously with sterilization sucrose liquid repeated washing; Relief pollen granule complete drying.

It is above-mentioned that slide glass is put into culture dish with cover is to be 28 ℃ in preferred temperature to cultivate 20-40 minute down in the present invention.

The used staining fluid of step 1) is to be made into by 0.7mM aniline Secondary ammonium phosphate indigo plant and 30mM sucrose.

Step 2) preparation method of described lysate is:: draw an amount of sterilization distilled water, and an amount of 1M NaOH or 1M KOH and an amount of 20%Tween-20, place the 2.0ml centrifuge tube, vortex is promptly made lysate for a moment, described lysate volume can according to how many increases and decreases of reaction number, but the final concentration of NaOH or KOH is 0.1M in the lysate, and the Tween-20 final concentration is 2%; The concrete preparation method of lysate of the present invention is: draw 1600 μ l sterilization distilled water, 200 μ l 1M NaOH or 1M KOH and 200 μ l 20%Tween-20 place the 2.0ml centrifuge tube, and vortex is promptly made lysate for a moment.

Described step 2) liquid getting device in is 12 road liquid getting devices, adopts 12 road liquid getting device packing, 1 μ l lysate in 96 hole PCR plates, is sunken to the pipe end with tinfoil paper rubber belt sealing and the centrifugal lysate that makes; Be that branch is equipped with 5 μ l mineral oil in the 96 hole PCR plates of described step 4); Described step 6) be to add 1 μ l TE damping fluid in each pipe.

The tip diameter of the tweezers that step 1) is used is smaller or equal to 0.01mm, and separating pollen is to finish at 63 times microscopically.

Step 4) described of short duration centrifugal be to refer to that certainly at centrifugal force be under the 1479g centrifugal 1 minute.

PCR plate described in the step of the present invention is to be placed in the PCR instrument and the preferred temperature in the PCR instrument is controlled to be 95 ℃, and the PCR plate is generally selected 96 hole PCR plates for use, and the PCR plate of step 5) is preferably 17 minutes and 30 seconds 95 ℃ of following hold-times of temperature on the PCR instrument; Step 7) is of short duration centrifugal, places on the PCR instrument, and be to carry out pre-sex change in 95 ℃ of hold-time 3-8 minutes in preferred temperature.

The PCR reacted constituent mixed solution that step 6) added is generally by 1 * PCR reaction buffer, sufficient quantity dATP, dTTP, dGTP and dCTP, MgCl2, primer, template DNA, Taq archaeal dna polymerase and H ₂O forms, and concrete preferred content can be used the described content of embodiment.

The PCR product that step 7) obtains carries out electrophoretic separation with 1.5% agarose that contains 0.5 μ g/ml bromination second pyridine and takes pictures in the enterprising line scanning of imaging system.

The characteristics that the present invention has are: present method is used a kind of special tweezers and is separated single pollen at microscopically, neutralizes with TE buffered soln after the cracking in alkali and surfactant soln.The cracking mixed solution is directly worked in 5S rrna internal transcribed spacer district (5S rDNA-ITS), randomly amplified polymorphic DNA (RAPD) and little satellite PCR (SSR-PCR) method as template, and amplification PCR reaction parameter is in advance optimized.Use this method, the people through training can carry out 288 monad PCR reactions every day.This method is that material is set up with the sugarcane monad, is to verify on corn, spot thatch, green soya bean, Chinese sorghum and the tomato other the five kinds plants that can collect pollen, and is respond well, and collection and PCR that this inventive method also can be used for other pollen detect.This method is collected in enormous quantities and the ability of carrying out monad PCR provides a kind of new method for plant genetic analysis and karyomit(e) drawing, and makes that the genetic analysis condition is more controlled, expense reduces significantly.This method has special meaning for the species of using classical genetic analysis method difficulty.

The advantage that the present invention has is:

1) efficient height: up to the present, owing to the loaded down with trivial details operating process restriction of micromanipulator itself, the pollen granule number that single test is tried does not surpass 60 at most, and separation monad grain efficient is very low; And using method of the present invention, a people through training can carry out the separation and the PCR reaction of 288 monads every day.And, make a plate pollen granule slide glass and can save steps such as other method pollen germination cultivation and preservation for repeatedly direct sampling use; Moreover the pollen lysate can directly use as the PCR reaction template, without precipitate and separate DNA, has further improved efficient.

2) highly versatile: the inventive method can be applicable to the monad pcr analysis of various plants, comprises the tomato that the pollen diameter is very little; Can be applicable to multiple PCR response procedures, as special primer PCR, arbitrarily primed PCR and little satellite pcr analysis.

3) cost is low, and simple and easy to do: do not need expensive instrument, required reagent also is all conventional reagent, and specimen preparation is simple, does not need complex DNA extracting and pre-amplification; Operating process is simple and easy to do, does not have molecular biology experiment operation steps complicated and that requirement is harsh.

4) stability is strong: guarantee that according to pollen activity dyeing authentication method selected pollen granule all contains dna profiling; Pollen granule separated and collected means are reliable; Pollen granule cracking released dna effect waits other method better than germinateing.

5) practicality and workable: according to the inventive method, general laboratory all possesses the condition of carrying out correlative study; Ordinary person through training all can carry out test operation, thereby the researchist is freed from the molecular biology experiment operation of complexity, is absorbed in data analysis.

6) being specially adapted to the reproduction genetic statistics analyzes: molecular biology steps into the genome times afterwards comprehensively, and the clone enters into a plurality of gene synergism researchs by individual gene.And present method can high-throughput be collected the parental gene group genetic information that monoploid pollen that plant produces through reduction division by diploid is obtained, can obtain most important etap of plant-reproduction genetic development by analyzing, good and bad and improve breeding efficiency guidance is provided for estimating the parent.

Description of drawings:

Fig. 1 is pollen granule of the present invention and forceps tips diameter dimension comparison diagram.

Among the figure: (A) sugarcane; (B) corn; (C) green soya bean; (D) Caulis Miscanthis floriduli; (E) Chinese sorghum; (F) tomato.Measuring scale: adopt OLYMPUS Objective Micrometer OB M 1/100 (lattice are represented 0.01mm among the figure).

Fig. 2 is the electrophoretic examinations figure of sugarcane monad grain PI/PII PCR reaction product of the present invention.

Swimming lane definition: La:DNA standard molecular weight (Catalog#BN2050); Among the figure: swimming lane 1-36, following four the sugar cane breed monad PCR reaction product of optimal conditions are relatively; Swimming lane 37-48, dyeing (39 to 43) is compared with (44 to 48) monad grain PCR reaction product of being unstained.Swimming lane 1,10,19,28,37, negative contrast (H ₂O); Swimming lane 2,11,20,29,38 is over against shining (being followed successively by total DNA of sugar cane breed L03-378, HoCP03-718, HoCP02-618, TCP99-4474, L03-378); Swimming lane 3 to 9,39 to 48, L03-378 monad grain; 12 to 18, HoCP03-718 monad grain; 21 to 27, HoCP02-618 monad grain; 30 to 36, TCP99-4474 monad grain.

Fig. 3 is the electrophoretic examinations figure of five species monad grain PI/PII PCR reaction product of the present invention.

Swimming lane definition: La:DNA standard molecular weight (Catalog#BN2050); Among the figure: swimming lane 1,3,7,11,15,19, negative contrast (H ₂O); Swimming lane 2 is over against shining (the total DNA of L03-378); Swimming lane 4 to 6, corn; Swimming lane 8 to 10, Chinese sorghum; Swimming lane 12 to 14, Caulis Miscanthis floriduli; Swimming lane 16 to 18, green soya bean; Swimming lane 20 to 22, tomato.

Fig. 4 is the application drawing (monad grain with HoCP02-618 be sample) of high-throughput monad PCR method of the present invention in RAPD and SSR PCR response procedures.

Swimming lane definition: La:DNA standard molecular weight (Catalog#BN2050); Among the figure: swimming lane 1 to 16, the PCR reaction of using two different RAPD primers; Swimming lane 17 to 32, the RAPD-PCR reaction of using two kinds of different lysates; Swimming lane 33 to 44, the PCR reaction of using two pairs of different SSR primers; Swimming lane 1,9,17,25,33,39, negative contrast (H ₂O); Swimming lane 2,10,18,26,34,40 is over against shining (the total DNA of HoCP02-618); Swimming lane 1 to 8 and 17 to 32, primer are the RAPD-PCR of OPBE-04; Swimming lane 9 to 16, primer are the RAPD-PCR of OPA-17; Swimming lane 17 to 24 contains the lysate of KOH; Swimming lane 1 to 16 and 25 to 44 contains the lysate of NaOH; Swimming lane 33 to 38, primer are the SSR PCR of SMC336BS; Swimming lane 39 to 44, primer are the SSR PCR of SMC1604SA.

Embodiment:

The present invention is described in detail below in conjunction with embodiment and accompanying drawing:

The specific embodiment of the present invention is as follows:

(1) materials and methods

1) instrument and reagent

The instrument that this research is used comprises micromanipulator (Model the MN-153) (EastMeadow that a Narishige company produces, NY, USA), with Blang's microtubule drawing device (Sutter Instrument Company, Novato, CA, USA) the glass microtubule of Zhi Zuoing, Zeiss microscope (Carl Zeiss MicroImaging, Inc., Thornwood, NY, USA), tweezers (DUMONT No.11254-20) (Fine Science Tools USA, Inc., Foster City, CA, USA) and 0.5-10 μ l 12 road liquid getting devices (Thermo Electron Corporation, Milford, MA, USA).

The preparation following chemical solution agents useful for same available from Sigma-Aldrich company (Kansas, MO, USA): 1MNaOH; 1M KOH; 20%Tween-20 (tween 20); TE damping fluid (0.1M Tris-HCl, 2mM EDTA); 1% (W/V) bovine serum albumin (BSA); 10% (W/V) PVP-40 (the polyethylene pyrrole irons alkane ketone 40); Staining fluid (0.7mM aniline Secondary ammonium phosphate indigo plant, 30mM sucrose); The 30mM sucrose solution.The series lysate (0.025M, 0.05M, 0.1M, 0.15M, 0.20M NaOH or 0.025M, 0.05M, 0.1M, 0.15M, 0.20M KOH, 2%Tween-20 (tween 20) now joins with preceding.In addition, mineral oil (mineral oil), 10mM dNTPs (deoxyribonucleotide) mixture and agarose are also available from Sigma-Aldrich company.The DNA standard molecular weight available from Bionexus company (Oakland, CA, USA); The Taq archaeal dna polymerase, (the PCR reaction buffer generally contains 10-50mmol/L TrisCl (20 ℃ of following pH8.3-8.8), the Mg of 50mmol/L KCl and proper concn to 10 times of PCR buffer ²⁺, in addition, reaction solution can add the dithiothreitol (DTT) (DDT) of 5mmol/L or the bovine serum albumin (BSA) of 100 μ g/ml) and the 25mM magnesium chloride solution available from Roche diagnostic reagent company (Indianapolis, IN, USA).

2) plant and pollen material

It (is L03-378 that used colored fringe picks up from four good sugar cane breeds respectively, HoCP03-718, HoCP02-618 and TCP99-4474), Hawaii super-sweet corn No. 10 (Zea mays L.), M 81-E Chinese sorghum (Sorghum bicolor L.), Derby (Derby) mung bean (Phaseolus vulgaris L.) and No. 91 tomatoes of Florida (Lycopersiconesculentum L.).The positive control that the DNA that extracts from the leaf of four sugar cane breeds according to the method for (2000) such as Pan reacts as PCR.

3) pollen cracking and monad PCR

Single pollen granule is positioned in the single PCR pipe or 96 hole PCR plates that 1 μ l lysate is housed, and the pollen post precipitation adds 5 μ l mineral oil.The secondary contrast of two covers is set, and a sleeve pipe adds 1 μ l lysate, does not add pollen granule; Another set of 1 μ l water and pollen granule of adding.Be incubated 15,17.5 and 20 minutes with the cracking pollen granule at 95 ℃ respectively behind the sample of short duration centrifugal (1479g 1 minute).Afterwards, add TE buffer (TE damping fluid, TE damping fluid composition is generally 0.1M Tris-HCl, the 2mM EDTA) neutralization of equimolar amount, of short duration centrifugal.

Utilize five kinds of dna primers to carry out the PCR reaction.(5 ' to 3 ') is as follows for the primer base sequence: 5S rDNA-ITS (ribosomal intergenic spacer) primer PI[TGGGAAGTCCT (C/T) GTGTTGCA] and PII[(T/G) T (A/C) G (T/C) GCTGGTATGATCGCA] (Cox etc. 1992); Two RAPD (randomly amplifiedpolymorphic DNA) primer OPBE-04 (CCCAAGCGAA) and OPA-17 (GACCGCTTGT); And two pairs of sugarcane micro-satellite primers (SSR) SMC336BS (upstream ATTCTAGTGCCAATCCATCTCA and downstream CATGCCAACTTCCAAACAGAC) and SMC1604SA (upstream AGGGAAAAGGTAGCCTTGG and downstream TTCCAACAGACTTGGGTGG).Wherein PI/PII PCR reaction volume is 20 μ l, contains 1 μ l lysate, single pollen granule, equimolar TE damping fluid, 50mM KCl, 10mM Tris.HCl (pH 8.3), 3.0mM MgCl ₂, 0.2mM the TaqDNA polysaccharase of dATP, dTTP, dGTP and dCTP (four kinds of deoxyribonucleotides), 0.25 μ M primer, 0.1% (W/V) BSA (bovine serum albumin) and 1 unit.The PI/PII-PCR response procedures is 95 ℃ 5 minutes (extend to 10 minutes in optimization Test, 15 minutes), thermal circulation parameters be 93 ℃ 55 seconds, 60 ℃ 10 seconds, 72 ℃ 40 seconds, 30 circulations (extending to 40 or 50 circulations in optimization Test), last 72 ℃ were extended 10 minutes.The RAPD-PCR reaction volume is 20 μ l, except that dATP, dTTP, dGTP and dCTP concentration are that 0.25mM, primer are the 0.5 μ M, and the same PI/PII-PCR of other composition; The RAPD-PCR program is 95 ℃ and kept 5 minutes, thermal circulation parameters be 93 ℃ 40 seconds, 40 ℃ 30 seconds, 72 ℃ 60 seconds, 40 circulations, last 72 ℃ were extended 5 minutes.The SSR-PCR reaction volume also is 20 μ l, removes MgCl ₂For 2.75mM, dATP, dTTP, dGTP and dCTP concentration are that 0.25mM, primer are outside the 0.21 μ M, the same PI/PII-PCR of other composition; The SSR-PCR response procedures is 95 ℃ and kept 5 minutes, thermal circulation parameters be 94 ℃ 30 seconds, 58 ℃ 30 seconds, 72 ℃ 30 seconds, 40 circulations, last 72 ℃ were extended 10 minutes.

The pcr amplification instrument is PCR System 9700 (Applied Biosystems, Inc., Foster CityCA, USA) the .PCR product carries out electrophoretic separation with 1.5% agarose that contains 0.5 μ g/ml bromination second pyridine and at GEL Logic200Imaging System (New Haven, CT takes pictures on USA).

(2) result and discussion

1) slide preparation and monad are collected

Alcohol wipe tweezers, stainless steel needle and slide glass with 75% (75 * 25mm, Clay Adams ^TM), the 50ml sterilization beaker that lysate is housed with clean and the wiping tweezers after aseptic paper is used to separate pollen.On slide glass, drip staining fluid, under 63 power microscopes, separate a not cracking pollen sac excellent, that outer wall WUHUAFEN grain is infected with and put into staining fluid, needle cyst wall pollen is overflowed with tweezers.Slide glass is put into culture dish with cover cultivated 20-40 minute in 28 ℃, the back take out slide glass with tweezers with the careful emptying of staining fluid.The pollen granule that stays is adjusted pollen granule density in the visual field simultaneously with 15 sterilization sucrose liquid repeated washing, relief pollen granule complete drying.

The micromanipulator that uses is three-dimensional tunable arrangement, and the glass microtubule of drawing is used this device separating the monad grain and putting into the process frangibility of PCR pipe, and separation difficulty and speed are low.On the contrary, DUMONT No.11254-20 type forceps tips diameter reaches 0.01mm, can directly separate a plurality of species monad grains (Fig. 1) of diameter greater than 0.01mm.Amplifying under 63 power microscopes, the gatherer can know the single pollen granule of seeing on the slide glass, separate rapid and reliable, by folding tweezers 2-3 time, pollen granule is easy to enter into the PCR pipe lysate at the end, before getting next pollen granule at test under microscope tweezers point to guarantee that a pollen granule is arranged in each PCR pipe.Generally speaking, everyone per hour on average can collect 60 pollen granules.

Separating pollen granule with this tweezers has an incomparable advantage, be exactly can the perception pollen granule certain physical characteristics such as hardness and full degree, and these characteristics and pollen vigor are closely related, to the success of pollen PCR very important (seeing below).When the pollen granule diameter near or during less than the forceps tips diameter, velocity of separation can reduce, as the pollen granule (seeing Fig. 1 F) of tomato. in addition, pollen granule should complete drying, otherwise although the tweezers point has certain pliability, pollen granule also can be out of shape.

2) lysate concentration is optimized

PCR in all NaOH that tried and KOH lysate (0.025M, 0.05M, 0.1M, 0.15M 0.20M) all can work.Yet, when NaOH and KOH concentration are 0.1M and 0.15M, the clear and noiseless band of PCR band, this result of study with Xin etc. (2003) is consistent.For verifying this result, get the lysate that contains 0.1M NaOH all four the monad grains that tried sugar cane breed are tested.Through 17 minutes 30 seconds heat pre-treatment, operation PI/PII-PCR program, all are tried pollen granule PCR and have all been produced purpose band (seeing Fig. 2,1 to 36 swimming lane).Then do not produce any PCR band (picture does not show) with 1 μ l water as the contrast of lysate, contain 1 μ l lysate and the contrast of WUHUAFEN grain does not produce any PCR band (sees Fig. 2, swimming lane 1,10,19,28 and 37 yet; Fig. 3, swimming lane 1,3,7,11,15 and 19).Obviously, cracking has vital role to this lysate for pollen granule, and cracking is not a varietY specificity.The lysate that contains KOH then provides new selection for similar PCR reaction.

3) added ingredients of monad PCR and influence thereof

For avoiding the loss of lysate volume in the heat-processed, in each PCR pipe, add 5.0 μ l mineral oil again behind adding 1.0 μ l lysates and the monad grain, contrast does not then add mineral oil.Operation PI/PII-PCR program, the reaction mixture electrophoretic separation.As a result, add the PCR reaction band clear and stable (seeing Fig. 2, swimming lane 1 to 36) of mineral oil, the PCR reaction or the band of no mineral oil are fuzzy, or do not have band (picture does not show).The material that some materials such as NaOH, Tween-20 is arranged and discharge from pollen wall can suppress the PCR reaction.When dna profiling purity was not high, the stablizer of BSA Chang Zuowei Taq enzyme used (Al-Soud and

2000; Xin et al.2003).Another kind of polymer P VP can eliminate the interference (Xin etc. 2003) of complex compound to the Taq enzyme according to report in rough DNA sample.This test adds 0.1%BSA and 1%PVP in the PCR reaction mixture, but PCR result is not obvious with the contrast difference that does not add these materials.Consider in the mixture of cracking pollen wall to contain the number of chemical material that suggestion adds BSA when carrying out pollen PCR reaction.

4) pre-sex change of PCR and heating pyrolyze time and cycle number are to the influence of PCR reaction

In the PI/PII-PCR response procedures, the pre-sex change time span of several differences is tested, the result shows: 5 minutes in monad PCR reaction effect best, PCR reaction band abundance is higher, and this is very important as RAPD-PCR for the PCR reaction that a plurality of bands are arranged.5 minutes pre-sex change time is different with the PCR program of (2003) such as Xin.The Taq enzymic activity descends with 95 ℃ of soaking times, and the transformation period of Roche company's T aq enzyme has only 40 minutes (Roche Applied Science2005/2006).Obviously, monad PCR is very sensitive to 95 ℃ of soaking times, because the monad pcr amplification needs more circulation and longer time.

Monad is to have heavy-walled monoploid, thus the time length of heating pyrolyze and PCR cycle number reaction has vital role to PCR.Utilize the PI/PII-PCR program, it is that 17 minutes 30 seconds and PCR cycle number are 40 o'clock (seeing Fig. 2, swimming lane 1 to 36) that best PCR reaction result comes across the heating pyrolyze time.These swimming lanes all present band clearly, although they are from the monad grain of different sugar cane breeds.By successful cracking, the dna profiling that discharges after the cracking of monad grain has been amplified a proper level to these monad grains of above presentation of results after 40 PCR circulations when the heating pyrolyze time is 17 minutes and 30 seconds.Other combination of heating pyrolyze time and PCR cycle number does not all produce ideal effect.

5) the pollen granule activity is to the influence of PCR reaction

Some pollen granule dyeing backs show blue, other the still displaing yellow that then can not dye.Since great-hearted pollen can absorb staining fluid, and unvital pollen can not (Pline et al.2002), we have just selected some blue pollen granules specially, and these pollen granules are normally circular; On the contrary, the xanchromatic pollen granule looks like trilateral and become thin slice easily with the tweezers gripping time.With the amplification of PI/PII-PCR program, blue pollen granule has produced stable PCR band (seeing Fig. 2, swimming lane 1 to 36), xanchromatic pollen granule then widely different (seeing Fig. 2, swimming lane 44 to 48).Above result clearly illustrates that with molecular method great-hearted pollen granule includes DNA, and the pollen vigor is most important for the success of PCR.For preventing pollen germination, the aniline blue (0.7mM) of having selected high density is to reach the purpose of rapid dyeing.

6) application of high-throughput monad PCR method in other species and PCR program

In order to test the versatility of this high-throughput monad PCR method, we use the PI/PII-PCR program is that corn, Caulis Miscanthis floriduli, green soya bean, Chinese sorghum and tomato have carried out the monad pcr amplification to other the five kinds plants that can collect pollen, and the pcr amplification condition is with above-described sugar cane breed pcr amplification condition.From the monad of all these species all by cracking successfully and obtained specific amplified purpose band (see figure 3).Monad grain with sugar cane breed HoCP02-618 is a template, carry out the PCR reaction with RAPD-PCR and SSR-PCR program, except that the corresponding PCR program setting that thermal circulation parameters carries out according to plan, other amplification condition is with above-described sugar cane breed pcr amplification condition, and effect is (see figure 4) equally well.RAPD-PCR has disclosed the pollen that derives from this donor and has had genetic diversity widely.According to the study known to the person, this is to use RAPD-PCR method proof to derive from the different RAPD molecule marker of pollen granule heredity of same donor first, and some pollen granule carries identical RAPD molecule marker and (sees Fig. 4, swimming lane 7 and 21; Swimming lane 13 and 14).This has also disclosed the potentiality aspect this method derives from same donor pollen granule in mensuration the proportion of composing of different genotype.We have also tested simultaneously and have contained the influence to RAPD-PCR of NaOH and KOH lysate, and found that does not have difference (seeing Fig. 4, swimming lane 17 to 32).

Generally speaking, the monad high-throughput PCR detection method that we set up comprises that at all species that tried sugarcane all uses well, this mainly should give the credit to this method in the cracking of monad separation efficiency and pollen and the distinct advantages aspect the PCR stability, and has also saved the step that the pollen granule that contains trace amount DNA is increased in advance.Can use multiple molecule marking method to pollen granule genomic heritable variation carry out high throughput analysis, and expense reduces significantly.If a certain research need be carried out multiple molecular marker analysis to the heritable variation with a collection of pollen granule, the method that can use full genome duplication before monad PCR is expanded to doubly (Blanco et al.1989 of 400-500 with the trace amount DNA of pollen granule; Esteban et al.1993; Zhang et al.1992).

(3) conclusion of high-throughput monad PCR detection method

Other organ of pollen and plant is compared, and all exists aspect many remarkable different at size, structure and vigor etc.So one efficiently the pollen PCR method should carry out multifactor optimization, comprise pollen staining, cracked solution, PCR reacted constituent, pollen cracking time and template sex change time etc.

In sum, the present invention's high-throughput PCR detection method of studying foundation specifically can be summarized as follows:

1, separates complete, a clean not cracking pollen sac at microscopically (63 times) with tweezers and put into staining fluid (0.7mM aniline Secondary ammonium phosphate indigo plant on the slide glass, 30mM sucrose) in and needle cyst wall pollen is overflowed, slide glass is put into culture dish with cover cultivated 20-40 minute in 28 ℃, the back take out slide glass with tweezers with the careful emptying of staining fluid.The pollen granule that stays is adjusted density in the visual field simultaneously with 15 sterilization sucrose liquid repeated washing.Relief pollen granule complete drying;

2, draw 1600 μ l sterilization distilled water, 200 μ l 1M NaOH (perhaps 1M KOH) and 200 μ l Tween-20, place the 2.0ml centrifuge tube, vortex is promptly made lysate for a moment, the lysate volume can according to how many increases and decreases of reaction number, but the final concentration of NaOH or KOH is 0.1M in the lysate, and the Tween-20 final concentration is 2%;

3, with 12 road liquid getting device packing, 1 μ l lysate in 96 hole PCR plates, with the tinfoil paper rubber belt sealing and centrifugal make lysate be sunken to the pipe end;

4, select painted monad grain with tweezers (DUMONT No.11254-20) down at microscope (63 times) and put into the PCR pipe lysate at the end, folding tweezers 2-3 time;

Whether have monad adhere to, the tweezers tip is immersed in the 50ml beaker that clean lysate is housed shaken cleaning subsequently, and then separate another monad grain if 5, tweezers being put back to test under microscope;

6, the monad grain separate finish after, packing 5 μ l mineral oil in 96 hole PCR plates, of short duration centrifugal (centrifugal 1 minute of 1479g);

7, putting 96 hole PCR plates kept 17 minutes and 30 seconds at 95 ℃ on the PCR instrument;

8, in each pipe, add 1 μ l TE damping fluid, centrifugal, add PCR reacted constituent mixed solution then, adjusting final volume is 20 μ l;

9, of short duration centrifugal, place on the PCR instrument, the PCR reaction parameter is: 95 ℃ keep carrying out pre-sex change in 5 minutes, the corresponding PCR program setting that thermal circulation parameters carries out according to plan, the PCR cycle number is set at 40, and last 72 ℃ were extended 10 minutes, and remained in 4 ℃;

10, the PCR product carries out electrophoretic separation with 1.5% agarose that contains 0.5 μ g/ml bromination second pyridine and takes pictures in the enterprising line scanning of imaging system.

According to this schedule of operation, generally speaking, everyone uses can finish the i.e. collections of 288 monad grains of three the 96 hole PCR plates performing PCR amplified reaction of going forward side by side in the PCR instrument 8 times each workday.

Claims (10)

1, a kind of plant monad high-throughput is collected and the PCR detection method, comprises the steps: in regular turn

1) microscope separates complete, clean not cracking pollen and broken cyst wall of pollen and dyeing, with the careful emptying of staining fluid; The pollen granule that stays cleans, relief pollen granule complete drying;

2) suit μ l lysate in the PCR plate in suitable number hole with the liquid getting device packing, sealing and the centrifugal lysate that makes are sunken to the pipe end;

3) select painted monad grain and painted monad grain is put into the PCR pipe lysate at the end with tweezers at microscopically;

4) packing suits μ l mineral oil in the PCR plate in suitable number hole, and is of short duration centrifugal;

5) the PCR plate in the suitable number hole of step 4) under 90-98 ℃ of temperature, keeping 16 minutes and-18 minutes and 30 seconds on the PCR instrument;

6) the TE damping fluid neutralization of the suitable μ l of adding in each pipe of step 5), centrifugal, add PCR reacted constituent mixed solution then, adjusting final volume is 20 μ l;

7) of short duration centrifugal, place on the PCR instrument, the PCR reaction parameter is: 90-98 ℃ keeps carrying out pre-sex change in 3-8 minute, and the cycle number of thermal cycling is set at 35-40, and last 72 ℃ were extended 5-10 minute, obtained the PCR product, remained under 4 ℃ of temperature;

8) the PCR product of step 7) is taken pictures with electrophoretic separation and in the enterprising line scanning of imaging system.

2, plant monad high-throughput according to claim 1 is collected and the PCR detection method, it is characterized in that it is to separate complete, clean one by one not cracking pollen sac at microscopically with tweezers to put on the slide glass with painted step that the described microscope of step 1) separates complete, clean pollen and the broken cyst wall of pollen of not ftractureing, dripping staining fluid and needling cyst wall overflows pollen, slide glass is put into culture dish with cover is 23-30 ℃ in temperature and cultivated 20-40 minute down, the back take out slide glass with tweezers with the careful emptying of staining fluid; The pollen granule that stays is adjusted density in the visual field simultaneously with sterilization sucrose liquid repeated washing; Relief pollen granule complete drying.

3, plant monad high-throughput according to claim 1 is collected and the PCR detection method, it is characterized in that described staining fluid is made into by 0.7mM aniline Secondary ammonium phosphate indigo plant and 30mM sucrose.

4, plant monad high-throughput according to claim 1 is collected and the PCR detection method, the preparation method who it is characterized in that described lysate is: draw an amount of sterilization distilled water, and an amount of 1M NaOH or 1M KOH and 20% an amount of tween 20, place the 2.0ml centrifuge tube, vortex is promptly made lysate for a moment, the final concentration of NaOH or KOH is 0.1M in the described lysate of making, and the tween 20 final concentration is 2%.

5, plant monad high-throughput according to claim 1 is collected and the PCR detection method, and the tip diameter that it is characterized in that described tweezers is smaller or equal to 0.01mm, and separating pollen is to finish at 63 times microscopically.

6, a kind of plant monad high-throughput according to claim 1 is collected and the PCR detection method, it is characterized in that step 4) is described of short durationly centrifugally to be meant that at centrifugal force be under the 1479g centrifugal 1 minute.

7, plant monad high-throughput according to claim 1 is collected and the PCR detection method, it is characterized in that described PCR plate is to be placed in the PCR instrument and the temperature in the PCR instrument is controlled to be 95 ℃, the PCR plate adopts 96 hole PCR plates, and the PCR plate of step 5) is 17 minutes and 30 seconds 95 ℃ of following hold-times of temperature on the PCR instrument; Step 7) is of short duration centrifugal, places on the PCR instrument, and be to carry out pre-sex change in 95 ℃ of hold-time 3-8 minutes in temperature.

8, plant monad high-throughput according to claim 1 is collected and the PCR detection method, it is characterized in that the PCR product carries out electrophoretic separation with 1.5% agarose that contains 0.5 μ g/ml bromination second pyridine and takes pictures in the enterprising line scanning of imaging system.

9, plant monad high-throughput according to claim 1 is collected and the PCR detection method, it is characterized in that described step 2) in liquid getting device be 12 road liquid getting devices, adopt 12 road liquid getting device packing, 1 μ l lysate in the PCR plate of suitable number hole, be sunken to the pipe end with tinfoil paper rubber belt sealing and the centrifugal lysate that makes; Divide in the suitable number hole PCR plate of described step 4) 5 μ l mineral oil are housed; Add 1 μ lTE damping fluid in each pipe of described step 6).

10, plant monad high-throughput according to claim 2 is collected and the PCR detection method, it is characterized in that described slide glass is put into culture dish with cover is to be 28 ℃ in temperature to cultivate 20-40 minute down.

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