CN102443631A - Detection method of plant telomerase activity - Google Patents
Detection method of plant telomerase activity Download PDFInfo
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- CN102443631A CN102443631A CN2011103579934A CN201110357993A CN102443631A CN 102443631 A CN102443631 A CN 102443631A CN 2011103579934 A CN2011103579934 A CN 2011103579934A CN 201110357993 A CN201110357993 A CN 201110357993A CN 102443631 A CN102443631 A CN 102443631A
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Abstract
The invention discloses a method for detecting plant telomerase activity. The method comprises steps that: (1) a plant telomerase primary extract is prepared; (2) the plant telomerase is enriched, wherein 0.9-11g of DEPC-treated PEG6000 is added to every 10ml of the primary extract; (3) a protein content is determined, wherein the protein content of an enriched supernatant obtained in the step (2) is determined; (4) telomerase enzymatic reactions are carried out, and reaction products are recovered, wherein a deactivated enriched supernatant is adopted as a control group, the enriched supernatant is adopted as an experimental group, and a control group recovered product and an experimental group recovered product are obtained after processing; (5) DNA content determination is carried out, wherein the control group recovered product and the experimental group recovered product obtained in the steps (4) are respectively sufficiently dissolved in bacteria-free water, and the amounts of ssDNA are respectively determined. The ssDNA content of the control group recovered product is subtracted from the ssDNA content of the experimental group recovered product, such that the plant telomerase activity value is obtained.
Description
Technical field
The present invention relates to the authenticate technology of a kind of plant Telomerase, particularly the method for kind of plant Telomerase separation and detection.
Background technology
Telomere is a kind of special construction of eukaryote end of chromosome, and in recent years, big quantity research shows that fringes of chromosome and cytogenetics stablizes, and is closely related with cell fission power, cell viability, vitality.Its length is reduced with the division number of times of cell, but it also can synthesize under telomerase catalytic and extend, and keeps certain length.Discover that the human cancerous tumor cell more than 90% has telomerase activation; (normal human subject cell Telomerase Activity is very low to highlight vital role in the Telomerase pair cell canceration process; In case the phenomenon that telomerase activation improves will appear in cell carcinogenesis, be used for one of important physiological index of cell carcinogenesis at present).Especially the somatic sustainability division of plant biological, Telomerase is indispensable, and most important (lacked the effect of Telomerase, vegetable cell is difficult to maintain continuous breeding, process of growth).Because the activity of Telomerase is far below animal tissues in plant tissue, even in higher plant meristematic tissue of activity or callus, all be difficult to usually detect.Therefore, also there is not a kind of technological method can separate and detect the method for plant telomerase activation effectively and quickly even to this day.
The present existing detection that derives from the animal Telomerase is directly extracting solution to be carried out enzymatic reaction for " TRAP method ", then the amount of detecting reactant.Because in the animal tissues, telomerase activation is higher, directly extracting solution detects easily.But for the lower plant tissue of telomerase activation, just there is very big problem in so direct detection, mainly is detection sensitivity not enough (enzyme content is very low), even just can't detect telomerase activation to some SA tissues.
Summary of the invention
The technical problem that the present invention will solve provides that a kind of technology is succinct, the conclusion detection method of plant telomerase activation accurately.
In order to solve the problems of the technologies described above, the present invention provides the detection method of a kind of plant telomerase activation, may further comprise the steps:
1), plant Telomerase extracting solution preparation just:
After utilizing liquid nitrogen that plant tissue to be measured is fully ground, grind to form homogenate in 3～5 ℃ of adding extracting solution continued; Then, 3～5 ℃, the centrifugal 12～17min of 15000～17000g, the supernatant of gained is as first extract;
Said extracting solution is: 50mM pH 7.5Tris-HCl, 5.0mM MgCl2,100mM KCl, 20mM EGTA, 1.0mM DT T, 0.1mM PMS F, 1.5%PVP, 10% glycerine;
That is, in the said extracting solution of every 100ml, contain 50mM pH 7.5Tris-HCl, 5.0mM MgCl
2, 100mM KCl, 20mM EGTA, 1.0mM DTT, 0.1mM PMSF, 1.5g PVP and 10ml glycerine, all the other are water;
The amount ratio of plant tissue and extracting solution is: 2.5g plant tissue to be measured/8～12ml extracting solution;
2), the enrichment of plant Telomerase:
Every 10ml just adds 0.9～11 gram through the PEG6000 that DEPC handles in the extract, under 3～5 ℃, turns upside down to PEG6000 continued concussion (slight concussion) 30min that fully suspends;
In 3～5 ℃, the centrifugal 12～17min of 15000～17000g, behind the removal supernatant, add 3～4ml extracting solution then, 3～5 ℃ of stirrings that suspend again in 3～5 ℃, the centrifugal 12～17min of 15000～17000g, get the enrichment supernatant;
Contain the plant Telomerase in this enrichment supernatant, so far step has been accomplished the separation of plant Telomerase;
3), the mensuration of protein content:
Get step 2) gained enrichment supernatant mensuration protein content;
4), the recovery of the enzymatic reaction of Telomerase and product:
Get step 2) part of the enrichment supernatant of gained, through 65 ℃ of 15min deactivation pre-treatment, after the deactivation pre-treatment enrichment supernatant as control group; Step 2) another part of the enrichment supernatant of gained is as experimental group;
Control group and experimental group are carried out following processing respectively: get and contain enrichment supernatant after the proteic deactivation pre-treatment of 100mg/contain the proteic enrichment supernatant of 100mg to put into centrifuge tube, add 30 μ l, 10 * enzymatic reaction liquid, use ddH
2O is settled to 300 μ l, 18～20 ℃ of insulation 12～14min; 92～96 ℃ of enzyme-deactivatings are handled 0.8～1.2min then; The chloroform that adds 280～320 μ l again, abundant mixing is in 3～5 ℃; Centrifugal 12～the 17min of 20000～22000g; Get supernatant, add the 3M sodium acetate soln of 1/10 chloroform volume and add Virahol, mixing; Place-18～-22 ℃ of freezing 50～70min again, the volume of said Virahol is chloroform and 3M sodium acetate soln volume sum 0.8～1.2 times; 3～5 ℃ again, the centrifugal 18～22min of 20000～22000g, behind the removal supernatant, the use volumetric concentration is 70～80% washing with alcohol deposition; After waiting to precipitate seasoning, get control group respectively and reclaim product/experimental group recovery product;
The remarks explanation: above-mentioned deactivation pre-treatment can not make proteic content change;
5), dna content is measured:
Control group recovery product/experimental group recovery product of step 4) gained is added sterilized water respectively and fully dissolves; Measure the amount of ssDNA then respectively; The ssDNA content that experimental group is reclaimed product deducts the ssDNA content that control group reclaims product, gets the plant telomerase activation.
Annotate: the telomerase activation of plant tissue is defined as: contained Telomerase in every 100mg albumen, at 19 ℃, 13min can synthesize the amount of ssDNA.
Improvement as the detection method of plant telomerase activation of the present invention: step 3) is through protein content in the common spectrophotometric determination enrichment supernatant.
Further improvement as the detection method of plant telomerase activation of the present invention:
10 * enzymatic reaction liquid in the step 4) is: 0.5M pH 8.3Tris-HCl, 50mM MgCl
2, 0.5M KCl, 100mM EGTA, 0.1%Triton x-100,10mM DTT, 0.5mM dNTP-C, 0.1%BSA, 2.0mM leading primer;
Leading primer is 5 '-ATGATGTGCAACTCGACAACTT-3 '.
That is, contain in 10 * enzymatic reaction liquid of every 100ml: 0.5M pH 8.3Tris-HCl, 50mM MgCl
2, 0.5M KCl, 100mM EGTA, 0.1ml Triton x-100,10mM DTT, 0.5mM dNTP-C, the BSA of 0.1 gram and the leading primer of 2.0mM, all the other are water.
The remarks explanation: dNTP-C promptly removes dCTP and gets in dNTP.
Further improvement as the detection method of plant telomerase activation of the present invention: in the step 4): 4 bases of 3 ' of leading primer tasteless nucleotide end immutable (ACTT that is specific 3 ' end is immutable) in the enzymatic reaction, all the other bases admit of certain variation.
In step 4) of the present invention, the volume that is used for the ethanol (volumetric concentration is 70～80%) of washing precipitation is generally 0.8～1.5 times of chloroform volume.
The present invention specifically realizes through following technical scheme:
1, all apparatus, vessel are carried out the conventional processing of removing RNase.Because the activity of plant Telomerase, except its protein subunit, key is the integrity that keeps its RNA template sequence.Suggestion is extracted the condition of total RNA requirement and is carried out in haveing suffered sepn process by routine.
2, the plant Telomerase preparation of extracting solution just.After utilizing liquid nitrogen that plant tissue to be measured is fully ground, add extracting solution, continue to grind to form homogenate.Then, after the spinning, keep supernatant.
3, adopt polyoxyethylene glycol (polyethylene glycol, PEG) enrichment Telomerase albumen from extract sample liquid.Alternative adsorbed molecules amount of the PEG of different molecular weight and the different albumen of three-dimensional structure.Therefore, proteic enrichment is vital to the plant Telomerase to select the PEG of optimum molecular weight.Therefore, selected PEG6000 in the present invention for use.
4, the proteic wash-out of plant Telomerase.From the proteic PEG sample of enriching plant Telomerase, utilize the ratio difference of PEG/ extracting solution can be further selectively with plant Telomerase albumen wash-out from PEG.Therefore, this also is to the proteic enrichment committed step of plant Telomerase.
5, the quantitatively determined of plant Telomerase sample total protein.Through protein contnt in the common spectrophotometric determination sample, to confirm to add in the enzymatic reaction volume of sample extracting solution.
6, plant Telomerase enzymatic reaction.According to Kim, the Telomerase reaction principle of finding in 1996, Telomerase is to the leading primer effect of the DNA oligonucleotide of specified conditions, and can effectively extend its leading primer.Because any enzymatic reaction all relates to reaction conditionss such as temperature of reaction, reaction times.Therefore, it is important obtaining The optimum reaction conditions.
7, the recovery of reaction product and quantitatively determined.Reaction product still is a single stranded DNA, and fragment is shorter, and precipitate recovery process is adopted in suggestion.Because dna content is less in the recovery sample, adopt the DNA microanalyser to carry out quantitatively determined, record and assay determination result.
The detection method of plant telomerase activation of the present invention relates to separation of plant Telomerase and detection, and it has following advantage:
1) the most important advantage of the present invention's technology is that it can effectively detect being in SA Telomerase in the plant tissue;
2) can record the content of the reaction product of Telomerase accurately, fast through the minim DNA quantitative analysis.
In sum, the present invention has carried out the enrichment (being similar to concentration process) of Telomerase through beneficiation technologies with direct extracting solution, has reached in the SA tissue also can detect telomerase activation.Simultaneously, owing to effectively improved Telomerase concentration, the precision and the safety that detect have therefore been improved.
Embodiment
In each step of following examples, being under 3～5 ℃ low temperature of not specifying carried out.
Embodiment 1, Caulis et Folium Brassicae capitatae bud Telomerase separate and active the detection
1, the Telomerase preparation and the enrichment of extracting solution just:
Get 2.5g Caulis et Folium Brassicae capitatae bud and put into mortar, after fully grinding with liquid nitrogen,, continue to be ground to homogenate, be transferred in the 50ml centrifuge tube in 3～5 ℃ of adding 10ml extracting solutions, 4 ℃, the centrifugal 15min of 16000g; Purpose is in order to remove phytoclasts; Obtain the supernatant of 11ml altogether.
In the said extracted liquid of every 100ml, contain 50mM pH 7.5Tris-HCl, 5.0mM MgCl
2, 100mM KCl, 20mM EGTA, 1.0mM DTT, 0.1mM PMSF, 1.5g PVP and 10ml glycerine, all the other are water.
Get the 10ml supernatant and put into centrifuge tube, add the PEG6000 (going RNase to handle) of 1.0g through DEPC, turn upside down under 4 ℃ fully suspend to PEG after, continue slight concussion 30min (purpose is to carry out the proteic selective adsorption of plant Telomerase).4 ℃ then, the centrifugal 15min of 16000g removes supernatant, adds 3.50ml extracting solution (thereby purpose is eluted to the purpose that reaches enrichment the solution with the Telomerase albumen that adsorbs from PEG6000), 4 ℃ of stirring 30min that suspend, 4 ℃ again, the centrifugal 15min of 16000g; Get supernatant.This supernatant (being the enrichment supernatant) can directly be used for following step 2 and step 3; Also can be in-80 ℃ of preservations, take out when needing, thawing naturally to 4 ℃ is used further to following step 2 and step 3.
2, the mensuration of total protein content (mg/ml) in the sample.
Through protein contnt in the NANODROP 2000spectrophotometer working sample (being the supernatant of step 1) gained) of Thermo SCIENTIFIC company, triplicate.For the first time: 19.5; For the second time: 19.6; For the third time: 19.6; Average: 19.57.
3, enzymatic reaction and product reclaim
Enzymatic reaction need be handled, normal extraction enzyme liquid as contrast through the high-temperature inactivation to Telomerase; Specific as follows:
The about 20 μ l of sample thief (being the supernatant of step 1 gained) handle 15min deactivation pre-treatment through 65 ℃, enrichment supernatant after the deactivation pre-treatment, as control group.
With the enzyme liquid sample (being control group) of normal enzyme liquid (being the supernatant of step 1 gained, as experimental group) and inactivation treatment, protein content is respectively got 5.11 μ l (containing 100mg albumen) and is handled as follows respectively per sample:
Put into the 1.5ml centrifuge tube, add 10 * enzymatic reaction liquid, 30 μ l, add 264.89 μ l ddH
2Thereby O is settled to 300 μ l, mixing, 19 ℃ of insulation 13min; 94 ℃ then, 1min carries out enzyme-deactivating to be handled.The chloroform that adds equal-volume (i.e. 300 μ l), abundant mixing, 4 ℃, the centrifugal 15min of 21100g; Get supernatant, in the gained supernatant, add 30ul 3M sodium acetate soln and 330ul Virahol, mixing is placed on-20 ℃ of freezing 60min; 4 ℃ again, 21100g, centrifugal 20min removes supernatant, with the washing with alcohol deposition of 300ul 75% (volumetric concentration); The deposition seasoning gets control group and reclaims product/experimental group recovery product;
Contain in 10 * enzymatic reaction liquid of every 100ml: 0.5M pH 8.3Tris-HCl, 50mM MgCl
2, 0.5M KCl, 100mM EGTA, 0.1ml Triton x-100,10mM DTT, 0.5mM dNTP-C, the BSA of 0.1 gram and the leading primer of 2.0mM, all the other are water;
DNTP-C promptly removes dCTP and gets in dNTP.
Leading primer is 5 '-ATgATgTgCAACTCgACAACTT-3 '.
4, sample DNA assay
Control group recovery product/experimental group of above-mentioned steps gained is reclaimed product and operates as follows respectively:
Add the dissolving of 20.0ul sterilized water; Measure the content of DNA through the NANODROP 2000spectrophotometer of Thermo SCIENTIFIC company.Because Telomerase synthetic DNA is a single stranded DNA, and determining instrument is set to the ssDNA measurement function, triplicate is averaged.The difference of handling sample and control sample dna content is confirmed as contained Telomerase in certain protein mass, and the DNA to leading primer extension measures under certain condition.
Specific as follows:
Get 1.0ul product recovery sample (being the solution of above-mentioned 20.0ul sterilized water dissolving back gained), measure the content of ssDNA through the NANODROP 2000spectrophotometer of Thermo SCIENTIFIC company, the result is described in table 1 below (ng/ μ l):
Table 1
Caulis et Folium Brassicae capitatae bud telomerase activation is defined as: at 19 ℃, and 13min, per 100 μ g albumen synthesize the oligonucleotide of 781.4ng (39.07ng/ul * 20.0 μ l).
Telomerase separates and active the detection in embodiment 2, the rice callus tissue
1, the Telomerase preparation and the enrichment of extracting solution just
Get 2.5g rice callus tissue and put into mortar, fully grind the back in 4 ℃ of adding 10ml extracting solutions, continue to be ground to homogenate, be transferred in the 50ml centrifuge tube with liquid nitrogen, 4 ℃, the centrifugal 15min of 16000g, the supernatant of gained is as first extract; Extracting solution is with above-mentioned embodiment 1.
Get 10ml supernatant (promptly just extract) and put into centrifuge tube, add the PEG6000 (going RNase to handle) of 1.0g through DEPC, turn upside down under 4 ℃ fully suspend to PEG6000 after, continue slight concussion 30min.
Then, 4 ℃, the centrifugal 15min of 16000g removes supernatant, adds the 3.50ml extracting solution, and suspend and stir 30min, 4 ℃ again, the centrifugal 15min of 16000g, this supernatant (being the enrichment supernatant) can directly be used for following step 2 and step 3; Also can be in-80 ℃ of preservations, take out when needing, thawing naturally to 4 ℃ is used further to following step 2 and step 3.
2, the mensuration of total protein content (mg/ml) in the sample
Through protein contnt in the NANODROP 2000spectrophotometer working sample (being the supernatant of step 1 gained) of Thermo SCIENTIFIC company, triplicate.For the first time: 12.2; For the second time: 12.0; For the third time: 12.2; MV: 12.13.
3, enzymatic reaction and product reclaim
Sample thief (being the supernatant of step 1 gained) 20 μ l handle 15min deactivation pre-treatment through 65 ℃, enrichment supernatant after the deactivation pre-treatment, as control group.
With the enzyme liquid sample (being control group) of normal enzyme liquid (being the supernatant of step 1 gained, as experimental group) and inactivation treatment, protein content is respectively got 8.23 μ l (100mg albumen) and is handled as follows respectively per sample:
Put into the 1.5ml centrifuge tube, add 10 * enzymatic reaction liquid, 30 μ l, 261.77 μ l ddH
2O.19 ℃ of insulation 13min; 94 ℃ then, 1min carries out enzyme-deactivating to be handled.The chloroform that adds equal-volume (i.e. 300 μ l), abundant mixing, 4 ℃, the centrifugal 15min of 21100g; Get supernatant, add the Virahol of 330 μ l and the 3M sodium acetate soln of 30 μ l, mixing is placed on-20 ℃ of freezing 60min; 4 ℃ again, 21100g, centrifugal 20min, behind the removal supernatant, the washing with alcohol deposition with 75%; The deposition seasoning gets control group and reclaims product/experimental group recovery product;
10 * enzymatic reaction liquid is with above-mentioned embodiment 1.
4, sample DNA assay
Control group recovery product/experimental group of above-mentioned steps gained is reclaimed product and operates as follows respectively:
Add the dissolving of 20.0ul sterilized water; Amount through ssDNA after the NANODROP 2000spectrophotometer mensuration enzymatic reaction of Thermo SCIENTIFIC company.Measure result such as following table 2 (ng/ μ l):
Table 2
Rice callus organizes telomerase activation to be defined as: at 19 ℃, 13min, every 100ug albumen synthesize the oligonucleotide of 1118ng (55.9ng/ul * 20.0 μ l).
Embodiment 3, rice young panicle Telomerase separate and active the detection
1, the Telomerase preparation and the enrichment of extracting solution just
(1～2cm) puts into mortar, fully grinds the back in 4 ℃ of adding 10ml extracting solutions (extracting solution is with above-mentioned embodiment 1) with liquid nitrogen, continues to be ground to homogenate to get the 2.5g rice young panicle; Be transferred in the 50ml centrifuge tube; 4 ℃, the centrifugal 15min of 16000g, the supernatant of gained is as first extract;
Get 10ml supernatant (promptly just extract) and put into centrifuge tube, add the PEG6000 (going RNase to handle) of 1.0g through DEPC, turn upside down under 4 ℃ fully suspend to PEG6000 after, continue slight concussion 30min.Then, 4 ℃, the centrifugal 15min of 16000g removes supernatant, adds the 3.50ml extracting solution, suspends and stirs 30min.4 ℃ again, the centrifugal 15min of 16000g gets supernatant.This supernatant (being the enrichment supernatant) can directly be used for following step 2 and step 3; Also can be in-80 ℃ of preservations, take out when needing, thawing naturally to 4 ℃ is used further to following step 2 and step 3.
2, the mensuration of total protein content (mg/ml) in the sample.
Through protein contnt in the NANODROP 2000spectrophotometer working sample (being the supernatant of step 1 gained) of Thermo SCIENTIFIC company, triplicate.For the first time: 9.3; For the second time: 9.9; For the third time: 9.4; MV: 9.53.
3, enzymatic reaction and product reclaim
Sample thief (being the supernatant of step 1 gained) 20 μ l handle 15min deactivation pre-treatment through 65 ℃, enrichment supernatant after the deactivation pre-treatment, as control group.
With the enzyme liquid sample (being control group) of normal enzyme liquid (being the supernatant of step 1 gained, as experimental group) and inactivation treatment, protein content is respectively got 10.49 μ l (100mg albumen) and is handled as follows respectively per sample:
Put into the 1.5ml centrifuge tube, add 10 * enzymatic reaction liquid (with embodiment 1), 30 μ l, 259.51 μ l ddH
2O.19 ℃ of insulation 13min; 94 ℃ then, 1min carries out enzyme-deactivating to be handled.The chloroform that adds 300 μ l, abundant mixing, 4 ℃, 21100g, centrifugal 15min; Get supernatant, add 330 μ l Virahols and 30 μ l 3M sodium acetate solns again, mixing is placed on-20 ℃ of freezing 60min; 4 ℃ again, 21100g, centrifugal 20min, behind the removal supernatant, the washing with alcohol deposition with 75%; The deposition seasoning gets control group and reclaims product/experimental group recovery product;
4, sample DNA assay
Control group recovery product/experimental group of above-mentioned steps gained is reclaimed product and operates as follows respectively:
Add the dissolving of 20.0ul sterilized water; Amount through ssDNA after the NANODROP 2000spectrophotometer mensuration enzymatic reaction of Thermo SCIENTIFIC company.Measure result such as following table 3 (ng/ μ l):
Table 3
Rice callus organizes telomerase activation to be defined as: at 19 ℃, 13min, every 100ug albumen synthesize the oligonucleotide of 862ng (43.1ng/ul * 20.0 μ l).
Contrast experiment 1, contriver to fully with the Caulis et Folium Brassicae capitatae bud of embodiment 1, with the rice callus tissue of embodiment 2, detect according to the TRAP method with the rice young panicle of embodiment 3, the result distinguishes as follows: 20.5ng, 29.1ng and 31.3ng.
Contrast experiment 2, will be the oligonucleotide (the nude mice solid tumor tissue of breast cancer cell Bcap-37 transfection) of 435ng, operate that the gained result is 451ng, with the detected result basically identical of TRAP method according to TRAP method detected result according to method of the present invention.
Contrast experiment 3, PEG6000 used among the embodiment 1 is used PEG1000 respectively, PEG2000, PEG4000, PEG8000 and PEG10000 substitute, and all the other are fully with embodiment 1; The gained conclusion is respectively: 2.1ng, 4.5ng, 14.8ng, 18.3ng and 13.8ng.
Contrast experiment 4, make the consumption of the PEG6000 among the embodiment 1 into 0.7g by 1.0g, all the other are fully with embodiment 1; The gained conclusion is: 26.5ng.
Contrast experiment 5, make the consumption of the PEG6000 among the embodiment 1 into 1.3g by 1.0g, all the other are fully with embodiment 1; The gained conclusion is: 22.1ng.
At last, it is also to be noted that what more than enumerate only is several specific embodiments of the present invention.Obviously, the invention is not restricted to above embodiment, many distortion can also be arranged.All distortion that those of ordinary skill in the art can directly derive or associate from content disclosed by the invention all should be thought protection scope of the present invention.
Claims (4)
1. the detection method of plant telomerase activation is characterized in that may further comprise the steps successively:
1), plant Telomerase extracting solution preparation just:
After utilizing liquid nitrogen that plant tissue to be measured is fully ground, grind to form homogenate in 3～5 ℃ of adding extracting solution continued; Then, 3～5 ℃, the centrifugal 12～17min of 15000～17000g, the supernatant of gained is as first extract;
In the said extracting solution of every 100ml, contain 50mM pH 7.5Tris-HCl, 5.0mM MgCl
2, 100mM KCl, 20mM EGTA, 1.0mM DTT, 0.1mM PMSF, 1.5g PVP and 10ml glycerine, all the other are water;
The amount ratio of plant tissue and extracting solution is: 2.5g plant tissue to be measured/8～12ml extracting solution;
2), the enrichment of plant Telomerase:
Every 10ml just adds 0.9～11 gram through the PEG6000 that DEPC handles in the extract, under 3～5 ℃, turns upside down to the PEG6000 continued concussion 30min that fully suspends;
In 3～5 ℃, the centrifugal 12～17min of 15000～17000g, behind the removal supernatant, add 3～4ml extracting solution then, 3～5 ℃ of stirrings that suspend again in 3～5 ℃, the centrifugal 12～17min of 15000～17000g, get the enrichment supernatant;
3), the mensuration of protein content:
Get step 2) gained enrichment supernatant mensuration protein content;
4), the recovery of the enzymatic reaction of Telomerase and product:
Get step 2) part of the enrichment supernatant of gained, through 65 ℃ of 15min deactivation pre-treatment, after the deactivation pre-treatment enrichment supernatant as control group; Step 2) another part of the enrichment supernatant of gained is as experimental group;
Control group and experimental group are carried out following processing respectively: get and contain enrichment supernatant after the proteic deactivation pre-treatment of 100mg/contain the proteic enrichment supernatant of 100mg to put into centrifuge tube, add 30 μ l, 10 * enzymatic reaction liquid, use ddH
20 is settled to 300 μ l, 18～20 ℃ of insulation 12～14min; 92～96 ℃ of enzyme-deactivatings are handled 0.8～1.2min then; The chloroform that adds 280～320 μ l again, abundant mixing is in 3～5 ℃; Centrifugal 12～the 17min of 20000～22000g; Get supernatant, add the 3M sodium acetate soln of 1/10 chloroform volume and add Virahol, mixing; Place-18～-22 ℃ of freezing 50～70min again, the volume of said Virahol is chloroform and 3M sodium acetate soln volume sum 0.8～1.2 times; 3～5 ℃ again, the centrifugal 18～22min of 20000～22000g, behind the removal supernatant, the use volumetric concentration is 70～80% washing with alcohol deposition; After waiting to precipitate seasoning, get control group respectively and reclaim product/experimental group recovery product;
5), dna content is measured:
Control group recovery product/experimental group recovery product of step 4) gained is added sterilized water respectively and fully dissolves; Measure the amount of ssDNA then respectively; The ssDNA content that experimental group is reclaimed product deducts the ssDNA content that control group reclaims product, gets plant telomerase activation value.
2. the detection method of plant telomerase activation according to claim 1 is characterized in that: said step 3) is through protein content in the common spectrophotometric determination enrichment supernatant.
3. the detection method of plant telomerase activation according to claim 1 and 2 is characterized in that: the 10 * enzymatic reaction liquid in the said step 4) is:
Contain in 10 * enzymatic reaction liquid of every 100ml: 0.5M pH 8.3Tris-HCl, 50mM MgCl
2, 0.5M KCl, 100mM EGTA, 0.1ml Triton x-100,10mM DTT, 0.5mM dNTP-C, the BSA of 0.1 gram and the leading primer of 2.0mM, all the other are water;
Said leading primer is 5 '-ATGATGTGCAACTCGACAACTT-3 '.
4. the detection method of plant telomerase activation according to claim 3 is characterized in that: in the said step 4): 4 bases of 3 ' of leading primer tasteless nucleotide end are immutable in the enzymatic reaction, and all the other bases admit of certain variation.
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| CN103333958A (en) * | 2013-06-07 | 2013-10-02 | 浙江今复康生物科技有限公司 | Method for detecting telomerase through washing-free anchored-extension and telomeric-binding amplification and kit |
| CN104897756A (en) * | 2015-06-19 | 2015-09-09 | 青岛大学 | Electrochemical sensor for detecting telomerase activity and method for manufacturing electrochemical sensor |
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| FITZGERALD MS ET AL: "characterization and developmental patterns of telomerase expression in plants", 《PRO NATL ACD SCI》 * |
| 刘小川等: "水稻端粒酶活性定量分析研究", 《中国生物工程杂志》 * |
| 陈波等: "水稻愈伤组织端粒酶催化特征检测", 《浙江理工大学学报》 * |
Cited By (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103333958A (en) * | 2013-06-07 | 2013-10-02 | 浙江今复康生物科技有限公司 | Method for detecting telomerase through washing-free anchored-extension and telomeric-binding amplification and kit |
| CN103333958B (en) * | 2013-06-07 | 2014-08-13 | 浙江今复康生物科技有限公司 | Method for detecting telomerase through washing-free anchored-extension and telomeric-binding amplification and kit |
| CN104897756A (en) * | 2015-06-19 | 2015-09-09 | 青岛大学 | Electrochemical sensor for detecting telomerase activity and method for manufacturing electrochemical sensor |
| CN104897756B (en) * | 2015-06-19 | 2015-12-02 | 青岛大学 | A kind of electrochemical sensor for test side telomerase activity and preparation method thereof |
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