CN102660648B - Chromosomal localization for FSML (female-specific marker of Laminaria japonica Aresch)-1488 - Google Patents
Chromosomal localization for FSML (female-specific marker of Laminaria japonica Aresch)-1488 Download PDFInfo
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Abstract
The invention relates to a chromosomal localization method for FSML (female-specific marker of Laminaria japonica Aresch)-1488. The method comprises the steps of preparation of DNA (deoxyribonucleic acid) with target fragment FSML-1488 plasmids, preparation of gametophyte chromosomes of the Laminaria japonica Aresch, preparation of probes, and fluorescence in situ hybridization (FISH). The method has the advantages of preparing the high-quality chromosomes on the basis of obtaining protoplast of antiphyte and gametophyte of the Laminaria japonica Aresch by the optimal coordination of various tool enzymes such as macerozyme, abalone enzyme, cellulose and pectinase, breaking through the technology bottleneck of the FISH technology in the research of algology, and successfully locating the FSML-1488 with the length being 1488bp on the chromosomes.
Description
Technical field
The present invention relates to genetically engineered field, specifically, is specificity FSML-1488(female-specific marker of of relevant Female Gametophytes of Laminaria Japonica
laminaria japonicaaresch) location of molecule marker on karyomit(e).
Background technology
Sea-tangle (
saccharinajaponica(Areschoug) C. E. Lane, C. Mayes, Druehl et G. W. Saunders, comb. nov.) or (
laminaria japonicaaresch.) be subordinate to one of economical alga important in phaeophyta (Phaeophyta), Laminariales (Laminariales) ，Shi China mariculture industry.Sea-tangle has obvious heterogeny the life history.When sporophyte (being the common edible major portions of people) is ripe, on strap, the sporocyte of unilocular sporangium, through reduction division and repeatedly division, produces 32 amphitrichous homotype zoospores of haploidy adnation; They are attached to soon in matrix after release, when growing environment is suitable, develop into respectively female, microgametophyte, and sex ratio is 1:1(Schreiber, and 1930).This sex ratio result is indicating that sea-tangle may exist the such sex determination system of similar higher plant unisexual flower.
Evans(1963,1965) to palmate sea-tangle (
l.
digitata) etc. several species carry out chromosome research, in megagametophyte, find two less and larger karyomit(e)s, and infer that larger karyomit(e) may be their sex chromosome.In addition, and a large amount of apomictic experimental results (Dai Jixun and side ancestor are prosperous, and 1976; Fang Zongxi etc., 1978; Motomura, 1991; Dai Jixun etc., 1992; Lewis
et al., 1993) confirm, the spore physical efficiency that Female Gametophytes of Laminaria Japonica produces through amixis monogenesis forms sporocyst and zoospore, and these zoospore energy normal developments all become megagametophyte, illustrates that sea-tangle ubiquity " parthenogenesis " phenomenon.According to these results of study, Fang Zongxi etc. (1978) infer that sea-tangle exists similar X/Y sex determination system, and wherein the sporophyte of diploidy is XY type, and female, the microgametophyte of haploidy are respectively X-type and Y type.But regrettably, Dai Jixun and side ancestor prosperous (1977), Yabu; Yasui(1991) and Liu Yu etc. (2012) when utilizing cytology method to observe sea-tangle karyomit(e), though confirmation sea-tangle karyomit(e) is little and the large a little microgametophyte of megagametophyte (size is between 0.78-2.61 μ m) (size is between 0.57-2.17 μ m), they are rod-short or point-like more, but these reports are all presented at and in megagametophyte, are not all found to significant large-scale sex chromosome.Can infer accordingly, sea-tangle may existence karyomit(e), but it and euchromosome do not have obvious difference in form.
Molecule marker has been widely used in sex identification (the Jiang & of higher plant; Sink, 1997; Gunter<i TranNum="78">et al</i>., 2003a; 2003b; Stehlik & Blattner, 2004; Xu et al., 2004; Yakubov<i TranNum="79">et al</i>., 2005; Chaves-Bedoya & Nu ez, 2007; Li<i TranNum="80">et al</i>., 2010; Samantaray<i TranNum="81">et al</i>., 2010).But the report that rarely has so far algae Sexual-related molecule marker.Sim<i TranNum="82">et al</i>.(2007) think randomly amplified polymorphic DNA (random amplified polymorphic DNA, RAPD) technology can be used for Zhang Shi fragrant plant mentioned in ancient texts (<i TranNum="83">gracilaria changii</i>) evaluation of female, staminiferous plant and sporophyte.Martinez<i TranNum="84">et al</i>.(1999) utilize RAPD technology from thin fragrant plant mentioned in ancient texts (<i TranNum="85">gracilaria gracilis</i>) in obtain respectively 1 female relevant PCR mark and 2 male related molecular markers.Recently, by micro-satellite primers, screen Shan & Pang(2010) from wakame (<i TranNum="86">undaria pinnatifida</i>) molecule marker relevant to megagametophyte of middle acquisition.This seminar institute according to marchantia (<i TranNum="87">marchantia polymorpha</i>l.) Y chromosome distinguished sequence (GenBank accession number AB069714) design primer amplifies a differential fragment from Female Gametophytes of Laminaria Japonica, and the SCAR mark (Liu of the called after FRML-494 that takes the lead in the world this fragment to be successfully converted into<i TranNum="88">et al</i>., 2009); But can these Sexual-related molecule markers be positioned on karyomit(e) specifically, by directly having influence on these, be marked at the grow seedlings evaluation of female, microgametophyte relevant to breeding aspect and in the further investigation of the basic theories such as kelp gametophyte sexual differentiation, sex karyomit(e) of kelp clone.
Fluorescence in situ hybridization (fluorescence<i TranNum="90">in situ</i>hybridization, FISH) technology be based upon in situ hybridization (<i TranNum="91">in situ</i>hybridization) (Gall and Pardue, 1969 on the principle of technology and working method basis; John<i TranNum="92">et al</i>., 1969), first in mammiferous research, be developed (Pinkel<i TranNum="93">et al</i>., 1986), it is to utilize DNA that fluorescent substance mark is good or the nucleic acid of rna probe and cell chromosome to hybridize, and detects the latter in the method for intracellular existence and quantity and structural modification.Since Le<i TranNum="94">et al</i>.(1989) and Schwarzacher<i TranNum="95">et al</i>.(1989), in plant after successful Application FISH technology, this powerful means have been widely used in higher plant gene group analysis (referring to summary Lavania, 1998; Jiang & Gill, 2006; Schwarzacher & Heslop-Harrison, 2011), because it can make target dna sequence no matter in mitotic division or on maiotic karyomit(e), can carry out physics drawing.Thereby can be used for location (the Ueda & of higher plant Sexual-related gene or DNA marker; Tanaka, 1995; Mariotti<i TranNum="96">et al</i>., 2009) and heterosomal evaluation (Shibata<i TranNum="97">et al</i>., 1999; 2000; Hobza<i TranNum="98">et al</i>., 2004; Lan<i TranNum="99">et al</i>., 2006) etc. research.But FISH technology is the location on algae karyomit(e) for target dna sequence not also so far.
Chinese patent literature CN101280339B discloses the SCAR molecule marker that can distinguish Female Gametophytes of Laminaria Japonica, can identify fast and effectively kelp gametophyte sex, also can identify unisexuality sea tangle sporophyte parental source and offspring's thereof sex.Chinese patent literature CN101280340B discloses and can distinguish that sea-tangle is female, the specific molecular marker of microgametophyte, can identify kelp gametophyte sex, monogenesis sea tangle sporophyte parental source and offspring's sex thereof, also can on nucleus molecular level, to sea-tangle sexual differentiation, study.
Summary of the invention
The object of the invention is for deficiency of the prior art, the chromosome localization method of a Female Gametophytes of Laminaria Japonica specific molecular marker FSML-1488 is provided.
For achieving the above object, the technical scheme that the present invention takes is: the chromosome localization method of a Female Gametophytes of Laminaria Japonica specific molecular marker FSML-1488, comprises the following steps:
(1) with the preparation of target fragment FSML-1488 plasmid DNA: extract after kelp gametophyte DNA by CTAB method, utilizing the round pcr length that increases is the fragment of the megagametophyte specificity FSML-1488 of 1488 bp, then after TA clone, be converted in intestinal bacteria, finally extract plasmid and obtain target fragment plasmid DNA, the sequence of described target fragment FSML-1488 is SEQ ID NO.5;
(2) the chromosomal preparation of kelp gametophyte: utilize mixed enzyme solution, cellulase, polygalacturonase, macerozyme and abalone enzyme, process kelp gametophyte cell, with DAPI dyeing, obtains division and compares good high quality stains body;
(3) preparation of probe: the plasmid extracting is obtained to target fragment FSML-1488 plasmid DNA and utilize the flat method of otch to carry out green bio element label probe;
(4) fluorescence in situ hybridization: the chromosomal localization that carries out megagametophyte specificity FSML-1488 molecule marker according to the ordinary method of fluorescence in situ hybridization.
Mixed enzyme solution in described step (2) is the mixed enzyme solution of cellulase, polygalacturonase, macerozyme and abalone enzyme, and the ratio of its cellulase, polygalacturonase, macerozyme and abalone enzyme is 2:1:1:1.5, and the concentration of mixed enzyme solution is 10 mg/mL.
The sequence of the described primer pair for the Female Gametophytes of Laminaria Japonica genomic dna that increases: forward primer SEQ ID NO.3 and reverse primer SEQ ID NO.4.
The invention has the advantages that:
The present invention utilizes various tool enzyme, as the optimization of macerozyme, abalone enzyme, cellulase, polygalacturonase etc. coordinates to obtain on the basis of sea tangle sporophyte and gametophyte protoplastis, prepare high-quality karyomit(e), break through the technology " bottleneck " of FISH technology in phycology research, successfully by one long be that the Female Gametophytes of Laminaria Japonica specificity FSML-1488 molecule marker of 1488 bp is positioned on karyomit(e).
Accompanying drawing explanation
Fig. 1. the electrophoretogram of Female Gametophytes of Laminaria Japonica ISSR difference molecule marker.1-5 swimming lane: the microgametophyte of Different Individual; 6-10 swimming lane: the megagametophyte of Different Individual; 11 swimming lanes: blank; 12 swimming lanes: GeneRuler
tM100 bp DNA Marker Plus.
Fig. 2. the electrophoretogram of Female Gametophytes of Laminaria Japonica FSML-1488 molecule marker.1-10 swimming lane: the microgametophyte of Different Individual; 12-21 swimming lane: the megagametophyte of Different Individual; 22 swimming lanes: blank; The DNA standard of 11 and 23 swimming lane: D2000.
Fig. 3. kelp gametophyte DNA restriction enzyme digestion and electrophoresis collection of illustrative plates (left side) and Southern hybridization collection of illustrative plates (right side).The DNA standard of M1:D2000; The DNA standard of M2:1 kb; 1 ♂ and 1 ♀ are used
pstthe genomic dna that I enzyme is cut; 2 ♂ and 2 ♀ are used
xhoi and
notthe genomic dna of I double digestion.
Fig. 4 A. length is the megagametophyte specificity FSML-1488 molecule marker of the 1488 bp in situ hybridization collection of illustrative plates on Female Gametophytes of Laminaria Japonica metaphase nucleus, karyomit(e) DAPI negative staining.Arrow indication is green hybridization signal.
Fig. 4 B. length is the megagametophyte specificity FSML-1488 molecule marker of the 1488 bp in situ hybridization collection of illustrative plates on Female Gametophytes of Laminaria Japonica interphasic nucleus, interphasic nucleus DAPI negative staining.Green area is hybridization signal.
Fig. 4 C. length is the megagametophyte specificity FSML-1488 molecule marker of the 1488 bp in situ hybridization collection of illustrative plates on sea-tangle microgametophyte metaphase nucleus, karyomit(e) DAPI negative staining.
Fig. 4 D. length is the megagametophyte specificity FSML-1488 molecule marker of the 1488 bp in situ hybridization collection of illustrative plates on sea-tangle microgametophyte interphasic nucleus, interphasic nucleus DAPI negative staining.
Embodiment
Below in conjunction with accompanying drawing, embodiment provided by the invention is elaborated.
extraction and the evaluation of embodiment 1 specific molecular marker FSML-1488
1) get the kelp gametophyte clone that fresh weight is 0.1 g.
2) in liquid nitrogen, lightly gametophyte clone is pulverized last, be transferred to immediately in 0.6 mL CTAB Extraction buffer of preheating, it is the mercaptoethanol that 3% CTAB (cetyl trimethylammonium bromide), 1.4 M NaCl, 20 mM EDTA, the 10 mM pH Tris-HCl that is 7.8 and weightmeasurement ratio are 2% that this damping fluid contains weightmeasurement ratio, mixes 1 min on vortex blending instrument.
3) 65 ℃ of temperature are bathed 30 ~ 60 min, and every 10 min put upside down and shake 1 time.
4) adding isopyknic volume ratio is the chloroform of 24:1: primary isoamyl alcohol mixed solution, and put upside down gently and mix 3-5 min, till there is emulsion layer.
5) 12 000 rpm(rev/min under room temperature) centrifugal 10 min, supernatant liquor is transferred in the centrifuge tube of another sterilizing.
6) to the Virahol that adds ℃ precooling of 2/3 volume-20 in centrifuge tube, put upside down and mix, till there is precipitation.
7) at-20 ℃, place behind 15 min left and right centrifugal 10 min of 8 000 rpm.Be deposited in 70% washing with alcohol and after air seasoning, be dissolved in the ddH of 200 μ L sterilizings in aseptic operating platform
2in O.
8) add until completely dissolved the ammonium acetate of 7.5 M and the dehydrated alcohol of 2 times of volume-20 ℃ precoolings of 1/3 volume.Put upside down and mix, till there is precipitation.
9), more than placing 15 min at-20 ℃, after centrifugal 10 min of 12 000 rpm, then precipitate by 70% washing with alcohol.Finally will be precipitated and dissolved in the sterilizing ddH of 40 ~ 100 μ L left and right
2o ,-20 ℃ save backup.
10) the primer of purchasing from Canadian British Columbia university, select 10 primers, by optimize PCR reaction conditions, finally determine that response procedures is: first carry out taking turns 94 ℃ of sex change 3 min; Then 45 circulations comprise 94 ℃ of sex change 45s, 50 ~ 54 ℃ of annealing 1.5 min, and 72 ℃ are extended 1min; Finally at 72 ℃ of downward-extension 10 min.The PCR reaction system of 25 μ L: 2.5 μ L 10 * PCR damping fluids [50 mM KCl, 10 mM Tris-Cl (pH 8.3), 1.5 ~ 2.5 mM Mg
2+, 100 μ M dNTP], the 0.2 μ M ISSR primer Taq of ，1.1 unit archaeal dna polymerase, 30 ngDNA templates.2.0% agarose gel electrophoresis for PCR product, ethidium bromide staining, gel imaging system is observed and is taken pictures.In 10 pairs of primers, find primer UBC809(SEQ ID NO.1) can in megagametophyte, amplify a size at the differential band (Fig. 1) of 1500 bp left and right.
11) use the DNA glue of Beijing Ai Delai company to reclaim test kit, with reference to businessman's experimental procedure description operation.Sea-tangle is female, microgametophyte genomic dna amplified production carries out electrophoresis on 2% sepharose, and female, male differential fragment and other amplified bands are separated as far as possible, then with clean blade, cuts off differential band, puts into 2.0 mL centrifuge tubes.
12) every 100 mg gels add 500 μ L colloidal sol damping fluids, and 55 ℃ of temperature are bathed 10 min, and gel is thoroughly melted.
13) liquid of thawing is poured in adsorption column, standing 1 min, centrifugal 30 s of 12000 rpm, abandon liquid in collection tube.
14) add 700 μ L rinsing liquids, centrifugal 30 s of 12000 rpm room temperature.Abandon collection tube liquid.Repeat this step once.
15) outwell the waste liquid in collection tube, adsorption column is put into same collection tube, centrifugal 2 min of 12 000 rpm room temperature.
16) adsorption column post is put into the centrifuge tube of 1.5 new mL, the air-dry adsorption column EC of Bechtop.
17) to adsorption column film central authorities, add 40 μ L elutriants, room temperature is placed 2 min, centrifugal 2 min of 12 000 rpm room temperature, the liquid in centrifuge tube is the DNA segment of recovery, be stored in-20 ℃ standby.
18) in Eppendorf tube, add 1 μ L pMD19-T plasmid vector, 0.1 ~ 0.3 pmol is inserted into DNA fragmentation, then adds ddH
2o to 5 μ L.
19) add the connection mixed solution of 5 μ L, 16 ℃ of reactions are spent the night.
20) above-mentioned 10 μ L reaction solutions are added in the escherichia coli jm109 competent cell of 100 μ L, shake gently centrifuge tube 2 ~ 3 times, fully mix.In ice, place 30 min.
21) after ice bath, centrifuge tube is proceeded to 42 ℃ of water-bath 90 s, immediately ice bath 3 ~ 5 min.
22) add the LB liquid nutrient medium of 890 μ L, the velocity fluctuation of 150 rpm is cultivated 60 min at 37 ℃.(every liter of LB liquid nutrient medium contains 10 g Tryptoness, 5 g yeast extracts, 10 g NaCl, finally regulate after pH to 7.0 with the NaOH of 5 M, are settled to one liter, sterilizing with distilled water).
23) get 0.1 mL and contain the bacterium liquid that connects product and coat on LB Agar Plating and cultivate, 37 ℃ of overnight incubation, form single bacterium colony.LB agar plate preparation: add 4 g agar powders in every 100 mL LB liquid nutrient mediums, nutrient agar subject to sterilization adds the bromo-4-of 5-chloro-3-indoles-beta galactose glycosides (X-Gal) of 4 mg, the isopropylthio-β-D-galactoside (IPTG) of 2.4 mg and the penbritin (Amp of 10 mg while being cooled to 50 ℃ of left and right
+), be made into X-Gal, IPTG, Amp
+plate culture medium, is down flat plate after shaking up.
24) select white colony on LB solid medium, be inoculated into containing in the LB liquid nutrient medium of penbritin of 2 mL.
25) speed of 200 rpm concussion overnight incubation at 37 ℃.
26) get 1 μ L bacterium liquid as template DNA, carry out PCR reaction.25 μ L reaction systems containing 12.5 μ L days root 2 * Taq PCR MasterMix [the 0.1 Taq Polymerase/ μ L of unit, 500 μ M dNTP, 20 mM Tris-HCl(pH 8.3), 100 mM KCl, 3 mM MgCl
2], each 0.5 μ L of the upstream and downstream primer of 10 μ M, 1 μ L bacterium liquid template DNA.Amplification program comprises 94 ℃ of denaturation 3 min, and then according to 94 ℃ of sex change 45 s, 52 ℃ of annealing 45 s, 72 ℃ of programs of extending 1.5 min, carries out 30 circulations, and last 72 ℃ are extended 10 min.After PCR reaction finishes, get product 6 μ L at 1.0% agarose gel electrophoresis.
27) the bacterium liquid that contains object fragment being delivered to Shanghai bio-engineering corporation checks order.
28) according to sequencing result SEQ ID NO.2, forward primer is extended to respectively to 32 and 29 bases to 3 '-end and reverse primer to 5 '-end, obtain the sequence of FSML-1488 special primer: forward primer SEQ ID NO.3 and reverse primer SEQ ID NO.4.
29), microgametophyte genomic dna female to sea-tangle carries out pcr amplification.25 μ L reaction systems containing 12.5 μ L days root 2 * Taq PCR MasterMix [the 0.1 Taq Polymerase/ μ L of unit, 500 μ M dNTP, 20 mM Tris-HCl(pH 8.3), 100 mM KCl, 3 mM MgCl
2], each 0.5 μ L of the upstream and downstream primer of 10 μ M, 1 μ L bacterium liquid template DNA.Amplification program comprises 94 ℃ of denaturation 3 min, and then according to 94 ℃ of sex change 45 s, 65 ℃ of annealing 45 s, 72 ℃ of programs of extending 1.5 min, carries out 30 circulations, and last 72 ℃ are extended 10 min.Amplification carries out electrophoresis on 1.0% sepharose, and gel imaging system is observed and taken pictures.
30) according to 11)-27) step is carried out the recovery of DNA glue, connect to be transformed, the confirmation of white colony screening, Insert Fragment etc., finally the bacterium liquid that contains object fragment being delivered to Shanghai bio-engineering corporation checks order, acquisition can be identified a FSML-1488 molecule marker of Female Gametophytes of Laminaria Japonica, and sequence is referring to SEQ ID NO.5.
31) Southern hybridization confirms the specificity of FSML-1488 molecule marker: result as shown in Figure 3.Concrete Southern hybridization step is as follows:
I) according to the North2South hybridization kit process specifications label probe of Thermo company
By the biotin labeled N of 20 μ L
4-dCTP(Biotin-dUTP) with 5 * dNTP mix(1 mM dATP of 80 μ L, 1 mM dTTP, 1 mM dGTP, 1 mM dCTP) abundant mixing for standby use.
1. the PCR purified product of 100 ng object fragments is diluted to 24 μ L in centrifuge tube, as template DNA;
2. add 10 μ L Heptanucleotide mix primers in above-mentioned centrifuge tube, boil 5 min with sex change, then at mixture of ice and water, place 5 min with annealing rapidly;
3. add 10 μ L N
4-dCTP and 5 * dNTP mix(1 mM dATP, 1 mM dTTP, 1 mM dGTP, 1 mM dCTP) mixture, 5 μ L reaction buffers, 1 μ L Klenow enzyme (10U/ μ L) mixes;
4. 37 ℃ of incubation 60min;
5. the EDTA liquid (500 nM, pH 8.0) that adds 2 μ L makes Klenow enzyme deactivation.
II) genomic dna enzyme is cut
To extract the kelp gametophyte genomic dna of purifying, and select not have in molecule marker the restriction endonuclease of restriction endonuclease sites to carry out enzyme and cut.This research is determined and is used one group after preliminary experiment
psti single endonuclease digestion and one group by
xhoi and
notthe double digestion that I forms.
III) transferring film
1. genomic dna enzyme is cut rear electrophoresis, observes and takes a picture, and writes down the region of disperse DNA;
2. the HCl that gel is placed in to 0.2 M processes 10 min;
3. discard HCl solution, use rinsed with deionized water 2 times, subsequently gel is soaked in the sex change liquid (0.5 M NaOH, 1.5 M NaCl) of 20 mL volumes, after 30 min, again gel is soaked to 30 min in neutralizer;
4. get a porcelain dish, add and shift liquid (20 * SSC contains 175.3 g NaCl, 88.2 g Trisodium Citrates, 800 mL water, adjusts pH 7, is settled to 1 L), on put a sheet glass as platform, glass pane surface paving one deck filter paper, the both sides of filter paper are immersed in shifts in liquid, displaces the bubble between sheet glass and filter paper;
5. according to submergence 5 min in shifting liquid of 2 elder generations of the equirotal filter paper of the big or small cutting of glue, then be layered on sheet glass, gel is put on filter paper, on gel, place again nylon membrane (size the same as glue in advance submergence 10 min in deionized water, after in shifting liquid submergence 5 min), on nylon membrane, put into again 2 metafiltration paper and drive bubble;
6. on filter paper, place the thieving paper that 15 cm are high, use sheet glass to flatten, and on sheet glass, place the weight of 0.5 kg.Transferring film 10 h, during the thieving paper that more renews at interval of 2 h;
7. transferring film is complete, removes thieving paper, and film is placed in 6 * SSC solution and soaks 5 min, with filter paper, blots liquid, is placed on new filter paper 80 ℃ of roasting film 45 min.
IV) hybridization
1. prehybridization: the film taking a turn for the better is placed in to hybrid pipe, and point sample faces up, and adds hybridization solution (at least 0.1 mL/cm
2) and guarantee that film can be by hybridization solution uniform fold, prehybridization 30 min under 80 rpm room temperatures;
2. thermally denature probe: boil probe 10 min and make its sex change, be then placed in rapidly mixture of ice and water 10 min that anneal;
3. hybridization: in hybridization solution, add the DNA probe of sex change, make its concentration reach 30 ng/mL, fully mix, 80 rpm, 55 ℃ of hybridization are spent the night.
V) the rigorous film of washing
1. in advance the North2South of refrigeration is hybridized to rigorous film washing liquid and be slowly warming up to room temperature, add isopyknic deionized water;
2. film is transferred in new hybrid pipe and used the 1. rigorous film washing liquid of step to wash film 3 times, with 20 mL film washing liquids, wash film 15 min at every turn.
VI) chemoluminescence method detects hybridization signal
In advance confining liquid, 4 * rinsing damping fluid and the substrate balance liquid of refrigeration are slowly warming up to room temperature, and configure 1 * rinsing damping fluid.
1. sealing: abandon rigorous film washing liquid, add 15 mL confining liquids, membrane closure 15 min are washed in 42 ℃ of vibrations;
2. the HRP that configures the avidin mark that 1:300(66.7 μ L is stable joins in the confining liquid of 20 mL) antibody diluent;
3. antibody incubation: abandon confining liquid, add antibody diluent.Room temperature 15 min that vibrate gently;
4. wash film: with 1 * rinsing damping fluid drip washing film of 20 mL, then use vibrate soft rinsing film 4 times of 1 * rinsing damping fluid room temperature of 20 mL, each 5 min;
5. balance: Hybond membrane is moved in new culture dish, use the substrate balance liquid of 30 mL, 5 min that vibrate gently;
6. the configuration of substrate working fluid (operation of the inside, darkroom): 1:1 equal-volume mixing North2South
tMluminol/Enhanser solution and North2South
tMperoxidase stabilizing solution is as the luminous working fluid of substrate;
7. moistening film is put into new culture dish, add 6. substrate working fluid (operating in darkroom) 5 min of step, guarantee that working fluid floods Hybond membrane;
8. film is taken out from culture dish, filter paper blots film surface liquid.With preservative film, wrap Hybond membrane;
9. the Hybond membrane of wrapping is placed in magazine, puts into Kodak's X-ray film, 2 min that expose, (dark room operation);
10. from magazine, take out Kodak's X-ray, develop and photographic fixing.The slice result of X-ray as shown in Figure 3.
the chromosomal localization of embodiment 2 specific molecular marker FSML-1488
The step of the chromosomal localization of Female Gametophytes of Laminaria Japonica specific molecular marker FSML-1488 comprises:
(1) with the preparation of target fragment (SEQ ID NO.5) plasmid DNA
By CTAB method, extract after kelp gametophyte DNA; utilizing the round pcr length that increases is the fragment of the megagametophyte specificity FSML-1488 of 1488 bp; then after TA clone, be converted in intestinal bacteria, finally extract plasmid and obtain target fragment (SEQ ID NO.5) plasmid DNA.
(2) the chromosomal preparation of kelp gametophyte
Utilize cellulase, polygalacturonase, macerozyme and abalone enzyme optimum combination to process kelp gametophyte cell, with DAPI dyeing, obtain division and compare good high quality stains body.
(3) preparation of probe
The plasmid extracting is obtained to target fragment (SEQ ID NO.5) plasmid DNA utilizes the flat method of otch to carry out green bio element label probe.
(4) fluorescence in situ hybridization
According to the ordinary method of fluorescence in situ hybridization, carry out the chromosomal localization of megagametophyte specificity FSML-1488 molecule marker.
preparation with target fragment (SEQ ID NO.5) plasmid DNA
the extraction of 1.1 kelp gametophyte DNA
1) get the kelp gametophyte clone that fresh weight is 0.1 g.
2) in liquid nitrogen, lightly gametophyte clone is pulverized lastly, be transferred to immediately in 0.6 mL CTAB Extraction buffer of preheating.It is 3% CTAB, 1.4 M NaCl, the Tris-HCl(pH 7.8 of 20 mM EDTA, 10 mM that this damping fluid contains weightmeasurement ratio) and the weightmeasurement ratio mercaptoethanol that is 2%.On vortex blending instrument, mix 1 min.
3) 65 ℃ of temperature are bathed 45 min, and every 10 min put upside down and shake 1 time.
4) adding isopyknic volume ratio is the chloroform of 24:1: primary isoamyl alcohol mixed solution, and put upside down gently and mix 3-5 min, till there is emulsion layer.
5) centrifugal 10 min of 12 000 rpm under room temperature, supernatant liquor is transferred in the centrifuge tube of another sterilizing.
6) to the Virahol that adds ℃ precooling of 2/3 volume-20 in centrifuge tube, put upside down and mix, till there is precipitation.
7) at-20 ℃, place behind 15 min left and right centrifugal 10 min of 8 000 rpm.Be deposited in 70% washing with alcohol and after air seasoning, be dissolved in the ddH of 200 μ L sterilizings in aseptic operating platform
2in O.
8) add until completely dissolved the ammonium acetate of 7.5 M and the dehydrated alcohol of 2 times of volume-20 ℃ precoolings of 1/3 volume.Put upside down and mix, till there is precipitation.
9), more than placing 15 min at-20 ℃, after centrifugal 10 min of 12 000 rpm, then precipitate by 70% washing with alcohol.Finally will be precipitated and dissolved in the sterilizing ddH of 40 ~ 100 μ L left and right
2o ,-20 ℃ save backup.
the pcr amplification of target fragment
Use a pair of forward primer and reverse primer (forward primer SEQ ID NO.3 and reverse primer SEQ ID NO.4), kelp gametophyte genomic dna is carried out to pcr amplification.25 μ L reaction systems containing 12.5 μ L days root 2 Taq PCR MasterMix [the 0.1 Taq Polymerase/ μ L of unit, 500 μ M dNTP, 20 mM Tris-HCl(pH 8.3), 100 mM KCl, 3 mM MgCl
2], each 0.5 μ L of the upstream and downstream primer of 10 μ M, 1 μ L bacterium liquid template DNA.Amplification program comprises 94 ℃ of denaturation 3 min, and then according to 94 ℃ of sex change 45 s, 65 ℃ of annealing 45 s, 72 ℃ of programs of extending 1.5 min, carries out 30 circulations, and last 72 ℃ are extended 10 min.Amplification carries out electrophoresis on 1.0% sepharose, and gel imaging system is observed and taken pictures.
clone and intestinal bacteria transform
Use the DNA glue of Beijing Ai Delai company to reclaim test kit, with reference to businessman's experimental procedure description operation.Sea-tangle is female, microgametophyte genomic dna amplified production carries out electrophoresis on 2% sepharose, and female, male differential fragment and other amplified bands are separated as far as possible, then with clean blade, cuts off differential band, puts into 2.0 mL centrifuge tubes.
1) every 100 mg gels add 500 μ L colloidal sol damping fluids, and 55 ℃ of temperature are bathed 10 min, and gel is thoroughly melted.
2) liquid of thawing is poured in adsorption column, standing 1 min, centrifugal 30 s of 12 000 rpm, abandon liquid in collection tube.
3) add 700 μ L rinsing liquids, centrifugal 30 s of 12 000 rpm room temperature.Abandon collection tube liquid.Repeat this step once.
4) outwell the waste liquid in collection tube, adsorption column is put into same collection tube, centrifugal 2 min of 12 000 rpm room temperature.
5) adsorption column post is put into the centrifuge tube of 1.5 new mL, the air-dry adsorption column EC of Bechtop.
6) to adsorption column film central authorities, add 40 μ L elutriants, room temperature is placed 2 min, centrifugal 2 min of 12 000 rpm room temperature, the liquid in centrifuge tube is the DNA segment of recovery, be stored in-20 ℃ standby.
7) in Eppendorf tube, add 1 μ L pMD19-T plasmid vector, 0.1-0.3 pmol is inserted into DNA fragmentation, then adds ddH
2o to 5 μ L.
8) add the connection mixed solution of 5 μ L, 16 ℃ of reactions are spent the night.
9) above-mentioned 10 μ L reaction solutions are added in the escherichia coli jm109 competent cell of 100 μ L, shake centrifuge tube 2-3 time gently, fully mix.In ice, place 30 min.
10) after ice bath, centrifuge tube is proceeded to 42 ℃ of water-bath 90 s, immediately ice bath 3-5 min.
11) add the LB liquid nutrient medium of 890 μ L, the velocity fluctuation of 150 rpm is cultivated 60 min at 37 ℃.(every liter of LB liquid nutrient medium contains 10 g Tryptoness, 5 g yeast extracts, 10 g NaCl, finally regulate after pH to 7.0 with the NaOH of 5 M, with distilled water, are settled to 1 L, sterilizing)
12) get 0.1 mL and contain the bacterium liquid that connects product and coat on LB Agar Plating and cultivate, 37 ℃ of overnight incubation, form single bacterium colony.LB agar plate preparation: add 4 g agar powders in every 100 mL LB liquid nutrient mediums, nutrient agar subject to sterilization adds the bromo-4-of 5-chloro-3-indoles-beta galactose glycosides (X-Gal) of 4 mg, the isopropylthio-β-D-galactoside (IPTG) of 2.4 mg and the penbritin (Amp of 10 mg while being cooled to 50 ℃ of left and right
+), be made into X-Gal, IPTG, Amp
+plate culture medium, is down flat plate after shaking up.
13) select white colony on LB solid medium, be inoculated into containing in the LB liquid nutrient medium of penbritin of 2 mL.
14) speed of 200 rpm concussion overnight incubation at 37 ℃.
15) get 1 μ L bacterium liquid as template DNA, carry out PCR reaction.25 μ L reaction systems containing 12.5 μ L days root 2 Taq PCR MasterMix [0.1 U Taq Polymerase/ μ L, 500 μ M dNTP, 20 mM Tris-HCl(pH 8.3), 100 mM KCl, 3 mM MgCl
2], each 0.5 μ L of the upstream and downstream primer of 10 μ M, 1 μ L bacterium liquid template DNA.Amplification program comprises 94 ℃ of denaturation 3 min, and then according to 94 ℃ of sex change 45 s, 52 ℃ of annealing 45 s, 72 ℃ of programs of extending 1.5 min, carries out 30 circulations, and last 72 ℃ are extended 10 min.After PCR reaction finishes, get product 6 μ L at 1.0% agarose gel electrophoresis.
16) the bacterium liquid that contains object fragment being delivered to Shanghai bio-engineering corporation checks order.
the extraction of plasmid
Use the plasmid extraction kit of Beijing Ai Delai company, according to its operation steps explanation, carry out plasmid extraction.
1) inoculate bacterium liquid that 100 μ L contain object fragment to 5 mL containing in the LB liquid nutrient medium of penbritin, incubated overnight, extracts plasmid.
2) get the bacterium liquid of 5 mL, the centrifugal supernatant of abandoning.
3) with the solution p1 resuspension bacteria liquid of 250 μ L.
3) add gentle the upset 3 times of solution p2 of 350 μ L, room temperature is placed 4 min.
4) add the p3 of 350 μ L, gentle upset at once 5 times, standing 4 min in iceberg, centrifugal 10 min of 13 000 rpm, carefully get supernatant.
5) supernatant of previous step is carefully moved into adsorption column (adsorption column will be put into collection tube), 13 000 rpm, 30 s, abandon liquid in collection tube.
6) add 700 μ L rinsing liquid WB, 12000 rpm, 30 s, abandon waste liquid in collection tube.
7) again add the rinsing liquid WB of 500 μ L, centrifugal 30 s of 12000 rpm.
8) abandon waste liquid in collection tube, adsorption column is put into centrifugal 2 min of collection tube.
9) abandon collection tube, adsorption column is put into new centrifuge tube, in Bechtop, blow the ethanol in 5 min volatilization rinsing liquids.
10) to the elutriant that adds 60 μ L on the adsorption film of adsorption column central authorities.
the chromosomal preparation of kelp gametophyte
Get the centrifugal removal excessive moisture of the good kelp gametophyte of appropriate cultivation conditions, with sterilizing seawater, clean 2-3 time, prepare stand-by.Add the vibration of 0.02% colchicine to shake up, process 6-12 h for 4 ℃, centrifugal gametophyte precipitation.Add Kano stationary liquid (Glacial acetic acid: ethanol=1:3) after vibration shakes up, more than 4 ℃ of processing 24 h.Product after distilled water cleaning is fixing 3-5 time, each 2 min, centrifuging and taking precipitation frond, put into 2 mL centrifuge tubes and add appropriate mixed enzyme solution (cellulase: polygalacturonase: macerozyme: abalone enzyme=2:1:1:1.5 (10 mg/mL)), on 37 ℃ of shaking tables in 200 r/min enzymolysis 18 h.Ethanol enzymolysis reaction with 70% and with distilled water cleaning of enzyme hydrolysis products 3-4 time to remove ethanol, add the Glacial acetic acid of appropriate 4 ℃ of preservations, vibration mixes, whether observation enzymolysis product even, the enzymolysis product that takes a morsel drips sheet, naturally dries.Get and carry the chromosomal slide glass that naturally dries, under phase microscope, carry out microscopy.Then choose to have and disperse good karyomit(e) division phase, add 15 μ L DAPI(Vector Laboratories, USA) to dye, after 15 min, in the lower observation of Olympus BX61 fluorescent microscope (Japan), take pictures and amplify, measure,, microgametophyte chromosomal relative length female to calculate.
the preparation of probe
Utilize Chroma Tide Alexa Fluor 488-5-dUTP(green bio element mark for nick translation test kit (ADVANCE Nick Translation Kit, Sigma-Aldrich)) (Invitrogen, USA) label probe.The whole process of probe of carrying out fluorescence in situ hybridization all requires to carry out on ice.In order to avoid other reagent such as enzyme are because temperature variation inactivation or do not have good effect.
Nick Translation (plasmid DNA 200 ng/ μ L) 10 μ L
10×Nick Translation Buffer 2 μL
Labeled dNTP (1 mM) 0.5 μL
Non-labeled dNTP (2 mM each mixed) 2 μL
DNA Polymerase I (10 U/μL) 4 μL
DNase I (100 mU/μL) 0.4 μL
Cumulative volume 20 μ L
Add 1.5 mL eppendorf pipes to mix above reagent, put into metal bath, 15 ℃ of constant temperature 2 h.Add afterwards 140 μ L ssDNA(Sigma-Aldrich, USA), the mixed solution of 90% ethanol-10% acetic acid of 400 μ L ,-20 ℃ are spent the night.At 4 ℃, centrifugal 30 min of 13 000 rpm, remove supernatant, 70% washing with alcohol.Unglazed, air-dry.Air-dry rear use 20 μ L 2 * SSC[(contain 17.53 g NaCl, 8.82 g Trisodium Citrates, 800 mL water, adjust pH 7, are settled to 1 L), 1 * TE(10 mM Tris-HCl, 1 mM EDTA, pH 8.0)] solution dissolving ,-20 ℃ of storages.
fluorescence in situ hybridization
1) utilize fluorescence microscope division to compare better kelp gametophyte karyomit(e), take out several slice, thin piece marker annotations that do not have DAPI to dye, female, microgametophyte are separated.
2) under UV-crosslinked instrument, carry out UV-crosslinked, 2 times.
3) hybridization solution of 8 μ L [1.5 μ L+6.5 μ L (2 * SSC, 1 * TE)] being dropped in to karyomit(e) division goes up mutually.
4) cover plastic coverslip, 100 ℃, heat denatured dna 5 min.
5) the hybridization sheet after heating is put in plastics receiver, put into the hybridization of spending the night of 55 ℃ of constant incubators.
6) slice, thin piece of having hybridized washes away cover glass with 2 * SSC elutriant.Air-dry in dark.
7) in karyomit(e) hybridization region, drip 15 μ L DAPI staining fluids, after 5 min, carry out fluorescent microscope microscopy.
Please refer to accompanying drawing 4, Fig. 4 A is that length is the megagametophyte specificity FSML-1488 molecule marker of the 1488 bp in situ hybridization collection of illustrative plates on Female Gametophytes of Laminaria Japonica metaphase nucleus, karyomit(e) DAPI negative staining.Arrow indication is green hybridization signal.
Fig. 4 B is that length is the megagametophyte specificity FSML-1488 molecule marker of the 1488 bp in situ hybridization collection of illustrative plates on Female Gametophytes of Laminaria Japonica interphasic nucleus, interphasic nucleus DAPI negative staining.Green area is hybridization signal.
Fig. 4 C is that length is the megagametophyte specificity FSML-1488 molecule marker of the 1488 bp in situ hybridization collection of illustrative plates on sea-tangle microgametophyte metaphase nucleus, karyomit(e) DAPI negative staining.
Fig. 4 D is that length is the megagametophyte specificity FSML-1488 molecule marker of the 1488 bp in situ hybridization collection of illustrative plates on sea-tangle microgametophyte interphasic nucleus, interphasic nucleus DAPI negative staining.
The above is only the preferred embodiment of the present invention; it should be pointed out that for those skilled in the art, do not departing under the prerequisite of the inventive method; can also make some improvement and supplement, these improvement and supplement and also should be considered as protection scope of the present invention.
SEQUENCE LISTING
<110> Shanghai Ocean University
<120>chromosomal localization of a Female Gametophytes of Laminaria Japonica specific molecular marker FSML-1488
<130> /
<160> 5
<170> PatentIn version 3.3
<210> 1
<211> 17
<212> DNA
<213>artificial sequence
<400> 1
agagagagag agagagg 17
<210> 2
<211> 1488
<212> DNA
<213>sea-tangle (Laminaria japonica Aresch)
<400> 2
agagagagag agagaggatg cctgcttgtt gtgccgcata ttagtggaag cgaaacgata 60
tcgcgcacag tcgtgaaatt attcattgca cacgttagtc gacaatatac tggatgaatt 120
aggagtaaaa accgagcatt tgatttcccc cgtggtgtca tgtagtcgta ctactgtagg 180
aatagcacac tcaaccatcc cttttatttg cttcgttctc tgctgtattg tcgtgagcag 240
cactgctaca gacatcggca tcgacccaga cgttacggat ggcacgtgcg gtgagactac 300
gtagcatagg tcgtcctttc atccacgctt gaagtgtgtc aacctgctca tttagcttgg 360
tgcttgcaac acaagcttgt tgtaaccgac gcgcgcaaat ggcgcatgtt gcttgaggag 420
gagctagtat ggtatcagac ttgtcgcttc acgcttcgcc ccgaaggatt atccagaccc 480
gcaaatgact ggttggaacg gcctgtccct tcgcttcggc gtattatgtc gcgcagagtg 540
tactcccgtt gatgccgtct tctgcgactt tgggctatcc tgtcagagtg gaatgactgg 600
ctacacttgc ggtgagtttc cggtggggcg tttggttttc ctgtcgtttc tcaatgggtg 660
cacgacatca catgattgag caccctcccg atataccgag cgttgggctg ttgacgatgt 720
tggtcgccat tcgtccccat gcggcgccat cacccgggcg atatactact cctagcatgc 780
cccgtaacca catgtttggg gaaaaactac ttgaaattag tgtgatatat ttgtttcagt 840
tcggaatggg ttaacacatt tgtgtttttg agtggcgccg ttgaccaatg aaatgtatag 900
tacggtgtta tacggcgtct acgtgccttg taaggcaggt gatggagtgg cacaggccac 960
ctcgcgtcca gcattcgagt tgaggcagta cgttgtcaca accccgattt tgtttatgtg 1020
actaacccta atcatacgtt gtcgttcaac gtttgttttt cacagtcgcc taacagtaca 1080
tcaattcggc ggtatgagag cttactcaat cctgggggtc acagcccatc gctgtgcatt 1140
gctgtgggta tctcacgtgg agtgtctggc acatatgggc agatggctcg atgactctct 1200
tgcccgagca catggcgatg gaccaggctg cgaggacaaa tacgcttgag cgtgtcgggc 1260
gcgcacgtag caccggcgta atctattgtc ggtataactc gtgttcgacg atgacctgcc 1320
atggtgcaac tagcttgccc tcagggcccg ggcaaggaca taatgtctgc tcaaacacgg 1380
gggttcaaca ccctcccccc tgctgtggaa tattctgaat agtgtttctg tgcaatagta 1440
cacaaccatt acctactgta ggaaatggcg ccctctctct ctctctct 1488
<210> 3
<211> 32
<212> DNA
<213>artificial sequence
<400> 3
agagagagag agagaggatg cctgcttgtt gt 32
<210> 4
<211> 29
<212> DNA
<213>artificial sequence
<400> 4
agagagagag agagagggcg ccatttcct 29
<210> 5
<211> 1488
<212> DNA
<213>sea-tangle (Laminaria japonica Aresch)
<400> 5
agagagagag agagaggatg cctgcttgtt gtgccgcata ttagtggaag cgaaacgata 60
tcgcgcacag tcgtgaaatt attcattgca cacgttagtc gacaatatac tggatgaatt 120
aggagtaaaa accgagcatt tgatttcccc cgtggtgtca tgtagtcgta ctactgtagg 180
aatagcacac tcaaccatcc cttttatttg cttcgttctc tgctgtattg tcgtgagcag 240
cactgctaca gacatcggca tcgacccaga cgttacggat ggcacgtgcg gtgagactac 300
gtagcatagg tcgtcctttc atccacgctt gaagtgtgtc aacctgctca tttagcttgg 360
tgcttgcaac acaagcttgt tgtaaccgac gcgcgcaaat ggcgcatgtt gcttgaggag 420
gagctagtat ggtatcagac ttgtcgcttc acgcttcgcc ccgaaggatt atccagaccc 480
gcaaatgact ggttggaacg gcctgtccct tcgcttcggc gtattatgtc gcgcagagtg 540
tactcccgtt gatgccgtct tctgcgactt tgggctatcc tgtcagagtg gaatgactgg 600
ctacacttgc ggtgagtttc cggtggggcg tttggttttc ctgtcgtttc tcaatgggtg 660
cacgacatca catgattgag caccctcccg atataccgag cgttgggctg ttgacgatgt 720
tggtcgccat tcgtccccat gcggcgccat cacccgggcg atatactact cctagcatgc 780
cccgtaacca catgtttggg gaaaaactac ttgaaattag tgtgatatat ttgtttcagt 840
tcggaatggg ttaacacatt tgtgtttttg agtggcgccg ttgaccaatg aaatgtatag 900
tacggtgtta tacggcgtct acgtgccttg taaggcaggt gatggagtgg cacaggccac 960
ctcgcgtcca gcattcgagt tgaggcagta cgttgtcaca accccgattt tgtttatgtg 1020
actaacccta atcatacgtt gtcgttcaac gtttgttttt cacagtcgcc taacagtaca 1080
tcaattcggc ggtatgagag cttactcaat cctgggggtc acagcccatc gctgtgcatt 1140
gctgtgggta tctcacgtgg agtgtctggc acatatgggc agatggctcg atgactctct 1200
tgcccgagca catggcgatg gaccaggctg cgaggacaaa tacgcttgag cgtgtcgggc 1260
gcgcacgtag caccggcgta atctattgtc ggtataactc gtgttcgacg atgacctgcc 1320
atggtgcaac tagcttgccc tcagggcccg ggcaaggaca taatgtctgc tcaaacacgg 1380
gggttcaaca ccctcccccc tgctgtggaa tattctgaat agtgtttctg tgcaatagta 1440
cacaaccatt acctactgta ggaaatggcg ccctctctct ctctctct 1488
Claims (3)
1. the chromosome localization method of a Female Gametophytes of Laminaria Japonica specific molecular marker FSML-1488, is characterized in that, comprises the following steps:
(1) with the preparation of target fragment FSML-1488 plasmid DNA: extract after kelp gametophyte DNA by CTAB method, utilizing the round pcr length that increases is the fragment of the megagametophyte specificity FSML-1488 of 1488 bp, then after TA clone, be converted in intestinal bacteria, finally extract plasmid and obtain target fragment plasmid DNA, the sequence of described target fragment FSML-1488 is as shown in SEQ ID NO.5;
(2) the chromosomal preparation of kelp gametophyte: utilize mixed enzyme solution, cellulase, polygalacturonase, macerozyme and abalone enzyme, process kelp gametophyte cell, with DAPI dyeing, obtains division and compares good high quality stains body;
(3) preparation of probe: utilize nick-translation method to carry out green bio element label probe to the target fragment FSML-1488 plasmid DNA of extracting;
(4) fluorescence in situ hybridization: the chromosomal localization that carries out megagametophyte specificity FSML-1488 molecule marker according to the ordinary method of fluorescence in situ hybridization.
2. the chromosome localization method of FSML-1488 according to claim 1, it is characterized in that, mixed enzyme solution in described step (2) is the mixed enzyme solution of cellulase, polygalacturonase, macerozyme and abalone enzyme, the ratio of its cellulase, polygalacturonase, macerozyme and abalone enzyme is 2:1:1:1.5, and the concentration of mixed enzyme solution is 10 mg/mL.
3. the chromosome localization method of FSML-1488 according to claim 1, it is characterized in that, for the sequence of the primer pair of the Female Gametophytes of Laminaria Japonica genomic dna that increases: the sequence of forward primer as shown in SEQ ID NO.3 and the sequence of reverse primer as shown in SEQ ID NO.4.
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| CN101280340A (en) * | 2008-05-29 | 2008-10-08 | 上海海洋大学 | Specific molecular marker capable of identifying sea-tangle female and male gametophytes |
| CN101280339A (en) * | 2008-05-29 | 2008-10-08 | 上海海洋大学 | SCAR molecular marker capable of identifying sea-tangle female gametophyte |
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| CN101280339A (en) * | 2008-05-29 | 2008-10-08 | 上海海洋大学 | SCAR molecular marker capable of identifying sea-tangle female gametophyte |
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