CN1511948A - Method for constructing genome library - Google Patents
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Abstract
The process of constructing genome library includes preparing protoplast; swelling the protoplast with non-ionic osmosis pressure regulator solution of 0-0.1 M concentration to release cell nucleus; regulating the concentration of the non-ionic osmosis pressure regulator solution to 0.5-0.8 M; washing and enriching cell nucleus and extracting macromolecular nucleus DNA; partially enzyme incision of macromolecular nucleus DNA with limiting endoenzyme and recovering segments with proper size to constitute genome library. The process can obtain genome with high DNA quality, avoid the pollution to chlorophyll body and mitochondrial DNA, and reduce polysaccharide matter with inhibiting effect on jointing enzyme.
Description
Technical field
The present invention relates to a kind of method that makes up genomic library.
Technical background
Because individual gene only accounts for the extremely small ratio of chromosomal DNA molecule total amount, must just might separate obtaining containing the specific dna fragmentation of goal gene through amplification, need usually to make up gene library, or be called the DNA library
1The DNA material source that is used for gene clone mainly is that gene library can correspondingly be divided into genomic library and cDNA library two big classes from the chromosomal DNA of particular organization or organ extraction or the cDNA copy that the mRNA reverse transcription becomes.If the purpose of research is to study the proteinic aminoacid sequence of a certain coded by said gene, just can the construction cDNA library, derive out according to the nucleotide sequence of the cDNA molecule of being cloned; On the other hand, if research is the regulating and controlling sequence that controlling gene is expressed, or in mRNA non-existent particular sequence, just need to make up genomic library, the genomic dna sequence that the clone is relevant
2
The dna fragmentation of recombinating in genomic library is the chromogene group DNA that derives from certain particular organisms.The chromosomal DNA that extracts is handled through mechanical shearing or digestion with restriction enzyme, reclaim a certain size fragment, with these fragment clonings on appropriate carriers, import and make the recombinant dna fragment cloning in the suitable host cell, can obtain to comprise the genomic library of this biont genome full sequence information
1Genomic library is mainly used in the structure of physical map of genome, the analysis of genome sequence, and location and the genome structure analysis of gene on karyomit(e) is the basis of carrying out genomics research.In addition, genomic library also has irreplaceable purposes on clone and identified gene controlling element
2
The initial mean length of the genomic dna that extracts in order to make up genomic library should be more than 8 times of carrier capacity at least.The DNA of this length could guarantee that most of fragment of the partially digested generation of restriction enzyme is the interior segments that derives from high-molecular-weight DNA, have only these fragments just have can with vector arms complementary sticky end, thereby the efficient that guarantee to connect.Genomic library can be divided into plasmid library according to applied carrier difference, lambda particles phage library, cosmid phagemid library, types such as bacterial artificial chromosome (BAC) library and yeast artificial chromosome (YAC) library.Carrier difference, the ability of the foreign DNA of cloning are also different: for example to have a liking for clone's ability of thalline carrier be 15-23kb to λ, so make up the λ genomic library, the length of initiate dna should be greater than 200kb; Cosmid clone ability is 45-50kb, and initiate dna length should be more than 400kb; Clone's ability of BAC and yac vector should be greater than 1Mb so be used to build the genomic dna in storehouse all more than 100kb.This shows that the key that makes up the high quality genomic library is the integrity that keeps initial gene group DNA.
DNA in the vegetable cell mainly is present in the nucleus, claims nuclear DNA or chromosomal DNA; Also contain a spot of DNA and be called extranuclear DNA in tenuigenin, they mainly are distributed in chloroplast(id) and the plastosome, are called chloroplast DNA (ctDNA) and Mitochondrial DNA (mtDNA).As the initiate dna that makes up genomic library, should select nuclear DNA for use, if sneaked into chloroplast(id) and Mitochondrial DNA, usable storage is reduced greatly, also increased the workload of follow-up genomic library screening to a great extent.Extracted the method that nucleus is used liquid nitrogen grinding more in the past, it is freezing rapidly with liquid nitrogen to be about to material, acutely grinds to form subcellular structure in mortar, utilizes screen filtration or density gradient centrifugation that nucleus is separated again.In the process of liquid nitrogen grinding, be difficult to the degree that artificial control is ground: if grinding is insufficient, just can not break the hard cell walls of vegetable cell, nucleus is discharged; If that grinds is too fierce, will damage nuclear membrane, cause nucleus to break and the fracture of nuclear dna.Because the ununiformity of grinding condition, both of these case toward contact together with the time take place.Therefore, extract nucleus with liquid nitrogen flash freezer and abrasive method, efficient is very low and can't guarantee nuclear integrity.
When making up the Plant Genome library, the selection of plant parent material has significant effects to the selection of genome DNA extracting method and the DNA quality of being extracted in addition.A lot of vegetable materials contain secondary meta-bolitess such as a large amount of polysaccharide and polyphenol, and these materials are difficult to utilize conventional DNA purification process that they and genomic dna are separated because physical properties is close.The irreversible combination can take place with genomic dna in the product of polyphenols oxidation, makes between dna molecular to take place crosslinkedly, causes brownization of being extracted of genomic dna, is difficult to the digestion of being limited property restriction endonuclease.Polysaccharose substance is the inhibitor of restriction enzyme and T4 dna ligase, if the genomic dna that is extracted contains more polyose impurity, will suppress the restriction enzyme enzymic activity and reduce the joint efficiency that reclaims fragment and vector arms, finally cause constructed genomic library titre to reduce greatly.
Therefore not only step is loaded down with trivial details to utilize traditional method to extract genomic dna structure Plant Genome library, and success ratio is often very low.We are in the experiment that makes up genomic library; invent control and oozed method; promptly utilize the plasma membrane of the gentle cracking plant protoplast of hypotonic environment to discharge nucleus; utilize hypertonic solution to protect nuclear nuclear membrane again; utilize nucleus to extract genomic dna, improved the success ratio and the efficient of genomic library construction greatly.
Summary of the invention
The purpose of this invention is to provide a kind of method that makes up genomic library.
The invention provides a kind of method that makes up genomic library, this method may further comprise the steps:
(1) uses the plant explants evoked callus;
(2) prepare cellulase with washing lotion, polygalacturonase, the plant callus cell that the mixed enzyme solution digestion process of macerozyme obtains prepares complete protoplastis;
(3) utilizing concentration is greater than the non-ionic type osmotic pressure regulator solution swelling protoplastis of 0M less than 0.1M, discharges nucleus;
(4) concentration with the non-ionic type osmotic pressure regulator solution in the step (3) is adjusted to 0.5-0.8M, with the integrity of protection nuclear membrane;
(5) nucleus is washed and enrichment, and from nucleus, extract macromole nuclear DNA;
(6) with restriction enzyme high molecular nuclear DNA is carried out partially digestedly, reclaim suitable big or small fragment, make up genomic library.
In aforesaid method of the present invention, described non-ionic type osmotic pressure regulator is the solution of N.F,USP MANNITOL or sorbyl alcohol preferably, also can be sucrose or PEG, and its concentration is preferably 0.08-0.1M.Restriction enzyme in the step (6) is Sau3AI preferably, also can be isoschizomerss such as MobI, Bsp1431.
In aforesaid method of the present invention, use the plant explants evoked callus, be enlarged culturing by callus, obtain the vegetable cell of q.s.This can be undertaken 3 by method well known in the prior art.Use cellulase, mixed enzyme solution digestion process plant callus cells such as polygalacturonase and macerozyme, preparation protoplastis.This also can be undertaken 4,5,6 by method well known in the prior art.Can contain 10mM CaCl in the solution
2To improve the stability of prepared protoplastis.Can also contain 0.7mM KH
2PO
4, the pH value in order to stabilizing solution makes in the scope of its optimum pH that is in enzyme.
The method swelling protoplastis that method of the present invention utilizes Artificial Control born of the same parents exosmosis to press discharges nucleus, prevents that with hypertonic solution protection nuclear membrane nucleus from breaking again.Used non-ionic type osmotic pressure regulator has non-ionic type osmotic pressure regulators such as N.F,USP MANNITOL, sorbyl alcohol, because the salt ion of high density may produce toxic action to plant protoplast.When extraneous osmotic pressure regulator concentration during at 0.4-0.6M, plant protoplast outside and internal penetration are pressed relative equilibrium, and protoplastis can keep standard state; If be lower than this concentration, the osmotic pressure in the cytolemma is higher than outside the film, and water molecules enters in the cell under osmotic pressure drives, and protoplasma cognition expands; When extraneous osmotic pressure regulator concentration was lower than 0.1M, the cytolemma of protoplastis can be risen brokenly, discharged intracellular tenuigenin, and at this moment organoid and nucleus need osmotic pressure regulator concentration is returned to more than the 0.5M, prevent that nuclear membrane is damaged.The nucleus that obtains is washed and enrichment, extract macromole nuclear DNA at last from nucleus, this can be undertaken by known method
7,8Can carry out partially digestedly with restriction enzyme Sau3AI to high molecular nuclear DNA, reclaim suitable big or small fragment, with, for example, the λ vector arms links to each other.To connect product and carry out external packing, and make up genomic library, this also can be undertaken by known method
9
In order to make up the Radix Dauci Sativae genomic library, we attempted utilizing several different methods in the past, extracted genomic dna from different tissues, but the dna molecular amount that obtains is less relatively, and often contain inhibitory enzyme and cut secondary metabolite with ligation, the pollution of chloroplast(id) and Mitochondrial DNA is arranged again.Make up genomic library with such DNA, titre is very low, is difficult to cover fully the Radix Dauci Sativae genome.
We utilize control of the present invention to ooze the nucleus extraction genomic dna that legal system is equipped with carrot callus.The genomic dna quality of extracting is good, avoided again simultaneously chloroplast(id) and Mitochondrial DNA and pollution, having reduced again has inhibiting polysaccharose substance to ligase enzyme.
The control method of oozing makes up genomic library and can be applied to most of plant, is specially adapted to contain a large amount of polysaccharide, secondary meta-bolites such as polyphenol, and cause extracting the vegetable material of the htrb gene group DNA that is fit to build the storehouse with ordinary method.In addition for a lot of rare plants, the material of the q.s that can't obtain makes up genomic library, can utilize a spot of plant explants evoked callus, the method of the enlarged culturing by callus obtains the library material of building of capacity, extract nucleus by the control method of oozing again, make up genomic library.
Brief Description Of Drawings
Fig. 1 shows the carrot callus of suspension culture;
Fig. 2 shows the Radix Dauci Sativae protoplastis;
Fig. 3 is presented at expansible protoplastis under the hypotonic condition;
Fig. 4 shows the nucleus that discharges behind the protoplastis swelling;
Fig. 5 shows that different methods extracts genomic dna relatively, wherein, 1 expression λ Hind IIImarker, the genomic dna that 2 expression CTAB methods are extracted, the genomic dna that 3 expression SDS methods are extracted, 4-6 represents to control the nuclear DNA that the method for oozing is extracted;
Fig. 6 shows that genomic dna Sau 3AI gradient enzyme cuts the result, wherein, 1 expression Hind IIIMarker, 2-9 represents to cut genomic dna through the Sau of gradient dilution 3AI enzyme;
Fig. 7 shows the genomic library titer determination, wherein, and 1 expression, 1 μ l packing end reaction thing, the packing reactant of 2 expressions, 1 μ l dilution in 1: 10, the packing reactant of 3 expressions, 1 μ l dilution in 1: 100.
Embodiment: utilize the control method of oozing to make up the Radix Dauci Sativae genomic library
1. carrot callus inducing and cultivating
Our used vegetable material is the Radix Dauci Sativae commercial breed: Japan's branch greatly enhances genseng, the Radix Dauci Sativae seed is evenly sowed in soil surface, when sowing 12-13 days, the cotyledon of seedling launches and grows up, and true leaf does not grow as yet, and extract with root seedling this moment, water is rinsed well in super clean bench and is disinfected: soaked 1 second in 75% alcohol, use rinsed with sterile water immediately, again with 0.1% mercuric chloride sterilization 4 minutes, sterile water wash 5～7 times.Then cotyledon and hypocotyl are cut into the segment about 1 centimetre, be inoculated in respectively on the callus of induce substratum (the MS minimum medium adds 1.5mg/L 2,4-D, PH5.8).In illumination box, evoked callus under 28 ℃ of low light conditions of temperature.The callus that obtains every 20 days subcultures once, enlarged culturing in time discards the explant of brownization and pollution.
2. prepare protoplastis by callus
Get 50ml washing lotion (10mM CaCl
2, 0.7mM KH
2PO
4, 0.5M N.F,USP MANNITOL pH5.6) and adding 1.5% polygalacturonase, 2% cellulase and 0.2%Y23 enzyme.Be distributed into two bottles, add the 3g callus respectively.In 26 ℃ of shaking table overnight incubation (16 hours), microscopically detects, and finds that the carrot cell wall is digested, and at this moment protoplastis is ball-type, is evenly dispersed in the substratum.5000g collected protoplastis in centrifugal 10 minutes.
3. control is oozed legal system and is equipped with carrot callus's nucleus
With the resuspended protoplastis of 3ml washing lotion, getting 50 μ l is positioned on the slide glass, go into 50 μ l sterilization distilled water mixing, microscopically is observed, discovery protoplastis under hypotonic condition constantly expands, the cytolemma of most of protoplastis was risen brokenly when system osmotic pressure was reduced to 1/6 original (adding sterilized water 250 μ l), added the nuclear staining of a pinkish red dye liquor pair cell of improvement, found that nucleus still is kept perfectly.Therefore the gentle mixing of sterilized water that in the resuspended protoplastis of 3ml washing lotion, adds 5 times of volumes, the swelling situation of microscopy protoplastis and nuclear integrity.When the cytolemma of observing most of protoplastis begins to break (needing 2min usually), add isopyknic high washing lotion (10mM CaCl that oozes rapidly
2, 0.7mM KH
2PO
4, 1M N.F,USP MANNITOL pH5.6), slowly mix with the protection nucleus.Centrifugal 5 minutes precipitate nucleus of 2500g.
To be rich in nuclear precipitation and be suspended in 2ml 5% citric acid, open, be laid on clip rifle head in 30% sucrose solution of 5% citric acid preparation, usefulness horizontal rotor centrifugal 30 minutes with 5000g with clean writing brush brush.
Nucleus precipitation is resuspended with 1 * SSC, in 2500g centrifugal 2 minutes, the repeated washing nucleus up to supernatant liquor pH value greater than 7.
4. the extraction of nuclear gene group DNA:
With 10ml extraction buffer (100mM TrisHClPH8.0; 100mM EDTA; 250mM NaCl; The 100ug/ml Proteinase K) resuspended carrot cell nuclear, adding sodium lauroyl sareosine (sarkosine) to final concentration behind the mixing is 1%.55 ℃ of incubations 3 hours, is cooled to room temperature, adds the slow mixing extracting of isopyknic phenol.In 4 ℃, the centrifugal 10min of 5500g gets and resets and add 0.6 times of volume Virahol, gently genomic dna is stirred with glass stick.Use 70% washing with alcohol.DNA is dissolved in the 10mlTE damping fluid.Add 4 ℃ of the slow mixing extractings of isopyknic phenol, the centrifugal 10min of 5500g, repeat to use phenol: chloroform: the slow extracting of primary isoamyl alcohol disappears up to egg white layer.Supernatant adds the equal-volume chloroform: primary isoamyl alcohol extracting, 4 ℃, the centrifugal 10min of 5500g.The 3mol/LNaAc (pH5.2) that adds 1/10 volume, 2 times of volume dehydrated alcohols, room temperature 20min.Gently genomic dna is stirred with glass stick.Use 70% washing with alcohol, desalination.The DNA that is wrapped on the glass stick is dissolved among the 0.5ml TE, and 4 ℃ of preservations are standby.Get 1 μ lDNA simultaneously and carry out 0.3% agarose gel electrophoresis, detect dna fragmentation size and integrity.
5. the structure of genomic library:
The structure of Radix Dauci Sativae genomic library carries out according to Promega EMBL3 specification sheets:
[1] the partially digested condition of DNA determines
Get the 150ul genomic dna and add 30ul10 * Sau3AI damping fluid (React3), 30ul 10 * BSA and 90ulddH
2O, mixing is sub-packed in 10 pipes gently.With Sau3AI enzyme Dilution Buffer gradient dilution by a certain percentage.Every pipe adds the enzyme liquid of 1ul gradient dilution respectively, and 37 ℃ of enzymes are cut 30min, adds 2 μ lEDTA termination reactions, and in 65 ℃ of deactivations 15 minutes, detects with 0.4% agarose gel electrophoresis whether enzyme is cut master tape between molecular weight 15kb-23kb.To determine the optimum proportion of genomic dna and Sau3AI.
[2] genomic dna is partially digested in a large number
By above-mentioned condition, add 1/2 of best enzyme amount, carry out a large amount of enzymes and cut.37 ℃ of enzymes are cut 30min, add 0.5MEDTA to final concentration 5mM termination reaction, and in 65 ℃ of deactivations 15 minutes, get in a small amount to detect with 0.4% agarose gel electrophoresis whether enzyme is cut master tape between molecular weight 15kb-23kb.Use phenol: chloroform: primary isoamyl alcohol, chloroform: each extracting of primary isoamyl alcohol once, supernatant adds the 3mol/L NaAc of 1/10 volume, 2 times of volume dehydrated alcohols ,-20 ℃ the precipitation 30 minutes.With 70% washing with alcohol precipitation, be dissolved among the aseptic TE of 100ul.
[3] reclaim the suitably dna fragmentation of size
With preparation type 0.3% agarose gel electrophoresis, cutting-out enzyme between molecular weight 15kb-23kb is cut master tape, the dialysis tubing electroelution method
9Reclaim.
[4] connect
The 15kb-23kbDNA fragment that reclaims is connected with the λ EMBL-3 arm of BamHI
The connection ratio is mol ratio 2: 1, and linked system is as follows:
1.DNA insert fragment 4ul
2.2ul?Lambda?EMBL-3 2ul(lug)
3.ligase(Promega) 1ul
4.ATP 1ul
5.ddH
2O 2ul
Total 10ul
Mix the back and connect 2 days in 16 ℃
[5] connect the product packing
From-70 ℃ of taking-up Packagen Extract, melting on ice.Add 8-10 μ l rapidly and connect product,, avoid bubble with rifle head mixing.The centrifugal 3-5 of 3000rpm second, be placed on 22 ℃ of water bath heat preservation 3h.Packing adds 500 μ lSM damping fluids and 20 μ l chloroforms after finishing, the soft mixing.Low-speed centrifugal is removed residue (albumen that does not have normal packing), shifts supernatant to new pipe.
[6] titre of mensuration genomic library
Frozen bacterial strain KW251 is rule 37 ℃ of overnight incubation on the LB flat board that contains tsiklomitsin 10 μ g/ml.Choose single bacterium colony and (contain 10mM MgSO to liquid LB
4With 1% maltose) in the substratum, 37 ℃ of shaking table overnight incubation.Transfer and (contain 10mM MgSO in fresh liquid LB
4With 1% maltose) in the substratum, 37 ℃ of shaking tables are cultivated 4-6hr. (OD
600=0.6).Get 1 μ l packing product and carry out 10 times of gradient dilutions with the SM damping fluid, it is undiluted to get 1 μ l, 1 μ l1: 10 dilution and 1 μ l1: 100 dilute packing end reaction thing respectively with 200 μ l KW251 competent cell (OD
600=0.6) mix, 30min is placed in 37 ℃ of water-baths.
In each EP pipe, add the Top agarose of 48 ℃ of preheatings of 3ml, pour into rapidly on the flat board that contains LB bottom substratum, leave standstill 10min, treat that Top agarose solidifies back 37 ℃ of cultivations fully.Visible plaque behind the common 6-8hr.
[7] preservation in library:
To pack product and add DMSO to final concentration 7%, mixing is sub-packed in the EP pipe gently, can preserve more than 2 years for-70 ℃.
Utilize control of the present invention to ooze the nucleus extraction genomic dna that legal system is equipped with carrot callus, the genomic dna molecular weight of extraction had both been avoided the pollution of chloroplast DNA more than 200kb, and having reduced again has inhibiting polysaccharose substance to ligase enzyme.Each packing in a small amount can reach 6 * 10
5Above storage capacity need not to build in the past a large amount of connections and packing in the storehouse, packs product in a small amount as long as merge twice, and the storage capacity of genomic library can reach 10
6, on average insert fragment 19kb, can cover Radix Dauci Sativae genome 4.8 * 10
5More than 30 times of kb have reached the standard that makes up genomic library fully.We have screened and have cloned the genome full length sequence of carrot somatic embryogenesis development related genes such as Radix Dauci Sativae LEA, XET, DNAJ, GP1 from this genomic library at present.
Reference
1 Wu is a tiger, 1998. " Principles of Gene Engineering " (second edition) science tech publishing house
Doubly put forth energy in 2 Shen, 2001. " molecular libraries " (863 biological high-technology book series) science tech publishing house
3 yellow U.S.s are beautiful etc., 1995. " biotechnology " Shanghai science tech publishing house
4 yellow U.S. beautiful 1986 aquatic protoplast regenerated plants " Science Bulletin " 31,1729 such as grade
5 Xu Zhihongs etc., 1997. " plant protoplast is cultivated and genetic manipulation " Shanghai science tech publishing house
1,982 one kinds of protoplast culture medium that suitability is wider such as 6 yellow U.S. beautiful grades " Institute of Genetics, Academia Sinica's work annual report "
7?Murray?M.G，et?al.1980?Rapid?isolation?of?high?molecular?weight?plantDNA?Nucl.Acids?Res.8，4321
8?Tower，P.(1991)Essential?Molecular?Biology，A?Practial?Approach，Volume?I，IRL?Press，Oxford.
9?Joseph?Sambrook，David?W.Russell?2001.Molecular?Cloning：ALaboratory?manual(3rd?Edition)，Cold?Spring?Harbor?Laboratory?Press，NewYork.
Claims (4)
1. method that makes up genomic library, this method may further comprise the steps:
(1) uses the plant explants evoked callus;
(2) use cellulase, polygalacturonase, the plant callus cell that the mixture enzyme liquid digestion process of macerozyme obtains, preparation protoplastis;
(3) utilizing concentration is greater than the non-ionic type osmotic pressure regulator solution swelling protoplastis of 0M less than 0.1M, discharges nucleus;
(4) concentration with the non-ionic type osmotic pressure regulator solution in the step (3) is adjusted to 0.5-0.8M:
(5) nucleus is washed and enrichment, and from nucleus, extract macromole nuclear DNA;
(6) with restriction enzyme high molecular nuclear DNA is carried out partially digestedly, reclaim suitable big or small fragment, make up genomic library.
2. in accordance with the method for claim 1, wherein, described non-ionic type osmotic pressure regulator is a N.F,USP MANNITOL, sorbyl alcohol, sucrose and/or PEG.
3. in accordance with the method for claim 1, wherein, the concentration of non-ionic type osmotic pressure regulator solution is 0.08-0.1M in the step (3).
4. in accordance with the method for claim 1, wherein, the restriction enzyme in the step (6) is Sau3AI, MobI, perhaps Bsp1431.
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