CN102424821B

CN102424821B - Extraction method for total RNA of feed type tetraploid robinia pseudoacacia leaves

Info

Publication number: CN102424821B
Application number: CN201110434648.6A
Authority: CN
Inventors: 刘磊; 孟凡娟; 黄凤兰
Original assignee: Northeast Forestry University
Current assignee: Northeast Forestry University
Priority date: 2011-12-22
Filing date: 2011-12-22
Publication date: 2014-06-11
Anticipated expiration: 2031-12-22
Also published as: CN102424821A

Abstract

The present invention relates to the field of plant molecular biology, specifically to an extraction method for total RNA of feed type tetraploid robinia pseudoacacia leaves. The extraction method is characterized by: filling tetraploid robinia pseudoacacia leaves in a centrifuge tube to carry out grinding; adding an ice bath buffer and beta-mercaptoethanol; centrifugating and removing supernatant; adding a CTAB extracting solution and beta-mercaptoethanol; centrifugating and taking supernatant; adding phenol and chloroform; uniformly mixing, centrifugating and taking supernatant; adding ethanol and a potassium acetate solution, uniformly mixing; carrying out ice bath, standing, and removing supernatant; cleaning by ethanol, and removing supernatant; carrying out air dry; adopting ddH2O to dissolve the RNA precipitation. The extraction method of the present invention has the following advantages that: the leaves are directly grinded in the centrifuge tube and are only subjected to extraction one time by the chloroform so as to reduce the operation time and the use amount of the material; bentonite is added to the extracting solution so as to well remove the protein interference; large amount of the potassium acetate is added to remove the polysaccharide so as to obtain the complete and stable RNA with high purity and high yield; the method has characteristics of simple operation, low cost, only requirements of ordinary equipment and domestic chemical reagents, and the like; the condition is provided for further molecular biology researches of the feed type robinia pseudoacacia and similar plants.

Description

The extracting method of the total RNA of feed form tetraploid locust blade

Technical field

The present invention relates to molecular biology of plants technical field, i.e. the extracting method of the total RNA of a kind of feed form tetraploid locust blade.

Background technology

Robinia pulse family (Leguminosae) Papillionoideae (Papilionaceae) plant, its blade is rich in crude protein, sugar, minerals and vitamins, it is the new feed that a kind of DEVELOPMENT PROSPECT is considerable, especially its albumen of the blade of tetraploid locust and sugared more horn of plenty of content, the biological characteristics of further investigation feed form tetraploid locust, for exploitation high-quality feed resource, promote animal husbandry level, have great importance.; owing to being rich in a large amount of protein and polysaccharide in feed form tetraploid locust blade; make the extraction of its total RNA very difficult, limited the research of feed form tetraploid locust molecular biology aspect, the seed selection of tetraploid locust and exploitation are limited to.

The extractive technique of RNA (plant total RNA) is one of basic experiment technology of a molecular biology of plants, is also the prerequisite of carrying out the research of molecular biology of plants aspect.Only have purity high quality high, that integrity is good total RNA of acquisition, could be used for mRNA purifying, Northern hybridization, construction cDNA library, by RT-PCR isolated genes and carry out protein In Vitro Translation equimolecular biological experiment and operate.

Conventional plant tissue method for extracting total RNA has guanidine isothiocyanate method, SDS method, phynol method, CTAB method, hot borate method, Trizol test kit method etc.These methods core reagent used is respectively guanidinium isothiocyanate, SDS, CTAB and boric acid.Guanidinium isothiocyanate is smudge cells rapidly, as strong denaturant, nucleic acid one protein structure is changed, and the complex body of efficient solution freestone albumen and nucleic acid, finally discharges nucleic acid.SDS (sodium lauryl sulphate) is a kind of anion surfactant or ion stain remover, can be from LISS precipitate nucleic acids and acidic polysaccharide.CTAB (palmityl trimethyl ammonium chloride) is as cats product, and energy dissolved cell film and nuclear membrane albumen, make nucleoprotein depolymerization, thereby makes RNA free out.Borate buffer system can be coupled at protease K digesting albumen with steps such as LiCl selective precipitation RNA together with.

A large amount of existence of the protein in plant tissue and polysaccharide, the purity on RNA and yield have important impact, and tetraploid locust is especially true.Experiment shows, adopts conventional equipment and general chemistry reagent, is difficult to effectively to remove or suppresses the interference of the materials such as protein in locust tree blade smudge cells, cannot extract high-quality RNA.Adopt high-end devices and imported materials and items, although can obtain RNA, but still exist process complexity, cost is high, and product is unstable, easily the problem of degraded.Therefore, extracting high quality RNA from be rich in the locust tree blade of crude protein and polysaccharide is a difficult problem of generally acknowledging in the industry always.

Summary of the invention

The object of the present invention is to provide a kind of can extraction and the total RNA of purifying from be rich in the feed form tetraploid locust blade of crude protein and polysaccharide, and the method that process is simple, easy to operate, with low cost, result is stable.

Above-mentioned purpose is realized by following technical scheme: the extracting method of the total RNA of a kind of feed form tetraploid locust blade, be characterized in: pack tetraploid locust blade into centrifuge tube grind into powder, add ice bath damping fluid and beta-mercaptoethanol, centrifugal, supernatant discarded, in centrifuge tube, add CTAB extracting solution and beta-mercaptoethanol to mix, centrifugal, get supernatant liquor, add water-saturated phenol and chloroform, mix, centrifugal, draw supernatant liquor, add dehydrated alcohol and liquor kalii acetici to mix, ice bath is centrifugal after leaving standstill, abandoning supernatant, after cleaning with 70% ethanol, carry out centrifugal, discard supernatant liquor, gas is dry, the ddH processing with appropriate DEPC ₂o thoroughly dissolves RNA precipitation.

The extracting method of the total RNA of said Feeding Robinia pseudoacacia blade, comprises following concrete steps:

(1) take 0.3g plant leaf and directly put into the centrifuge tube of 1.5mL, and put into rapidly after liquid nitrogen, with less centrifuge tube by blade grind into powder, add 700 μ l ice bath damping fluids, 60 μ l beta-mercaptoethanols, mix with vortex mixer vibration 2-3min, place on ice after 10min in 4 DEG C, 12000g, centrifugal 10min.

(2) supernatant discarded adds 700 μ l CTAB extracting solutions in centrifuge tube, 50 μ l beta-mercaptoethanols, and thermal agitation 2-3min mixes.

(3) centrifuge tube being placed in to temperature is the water-bath 7-9min of 65 DEG C, and shake therebetween several times.

(4) be placed in rapidly on ice after centrifuge tube is taken out from water, adding volume ratio is water-saturated phenol and the chloroform totally 700 μ l of 1: 1, after thermal agitation 2-3min mixes in 4 DEG C, 12000g, centrifugal 10min.

(5) get supernatant in another new centrifuge tube, add the dehydrated alcohol of 1/2 volume and the 5mol/L Potassium ethanoate of 1 volume (pH 5.8), slowly shake mixes, and places after 10min in 4 DEG C 12000g, centrifugal 10min on ice.

(6) supernatant discarded, by 75% ethanol washing and precipitating, slowly mixes, and 4 DEG C, 12000g, centrifugal 5min.

(7) supernatant discarded, moment is centrifugal thoroughly abandon dry, the dry 5min of gas in stink cupboard, the ddH processing with DEPC ₂o thoroughly dissolves RNA precipitation.

Said ice bath damping fluid is the NaCl composition by EDTA (pH, 8.0), the 2.0mol/L of the Tris-HCl of 100mmol/L (pH, 8.0), 25mmol/L.

In said CTAB extracting solution, add bentonite.

Said CTAB extracting solution is by 2% CTAB, 0.8% bentonite, the Tris-HCl (pH of 100mmol/L, 8.0), the NaCl of the EDTA of 25mmol/L (pH, 8.0), 1.4mol/L, 1% polyvinylpolypyrrolidone and 0.1% DEPC composition.

Said 75% ethanol is by 7.5 parts of dehydrated alcohols and 2.5 parts of ddH that DEPC processed ₂o preparation, the ddH that DEPC processed ₂o adds 99.9 parts of ddH by 0.1 part of DEPC ₂in O, whirlpool mixes the pure water through autoclave sterilization again.

The invention has the beneficial effects as follows: blade directly grinds in centrifuge tube, to reduce operating time and material usage, utilize and add the extracting solution of bentonite can better remove the interference that a large amount of albumen extracts RNA; Utilize the extracting of chloroform/primary isoamyl alcohol once, save operating time and expense; Add the Potassium ethanoate of high density and large volume with Polysaccharide removing, thereby obtain high purity, high yield and complete stable RNA, reverse transcription, gene clone be can meet, group analysis and the biological analysis of quantitative fluorescent PCR equimolecular transcribed, and have easy and simple to handle, success ratio is high, applied widely, without features such as degraded, common equipment and domestic chemical reagent can meet the demands, for the molecular biology research that deeply carries out Feeding Robinia pseudoacacia and similar plant provides condition.

Brief description of the drawings

Fig. 1 does not add total RNA of the tetraploid locust blade of the CTAB method extraction of bentonite;

Fig. 2 utilizes total RNA of the tetraploid locust blade of Trizol extraction;

Total RNA of the tetraploid locust blade that Fig. 3 SDS method is extracted;

Total RNA of the tetraploid locust blade that Fig. 4 SDS-CTAB method is extracted;

Total RNA of the tetraploid locust blade that Fig. 5 different concns bentonite extracts.

Embodiment

The total design of the present invention is: taking common equipment and domestic chemical reagent as main, taking the interference of eliminating albumen and polysaccharide as target, adopt add bentonite, precipitation in extracting solution before and added a large amount of Potassium ethanoates etc. to remove the gordian technique of too much protein and polysaccharide, and then obtained complete stable RNA product.Specific practice is: by directly grind into powder in centrifuge tube of locust tree blade, add ice bath damping fluid and beta-mercaptoethanol, and centrifugal; Abandoning supernatant, adds Extraction buffer and beta-mercaptoethanol, and vibration mixes, processed by hot bath; Add water-saturated phenol and chloroform, vibration mixes, centrifugal; Draw that supernatant liquor adds that dehydrated alcohol and liquor kalii acetici mix, ice bath is centrifugal again after leaving standstill, abandoning supernatant, carries out centrifugally with 70% ethanol after cleaning, discard supernatant liquor, and gas is done, the ddH processing with appropriate DEPC ₂o thoroughly dissolves RNA precipitation.

To be a lot of around the embodiment of above-mentioned technological line, in order describing the problem, only to introduce an embodiment below:

Preparation ddH ₂o: add 99.9 parts of ddH by 0.1 part of DEPC ₂in O, whirlpool mixes through autoclave sterilization again and get final product.

The ethanol of preparation 75%: 7.5 parts of dehydrated alcohols and 2.5 parts of ddH that DEPC processed ₂o is made into.

Prepare the Tris-HCl (pH, 8.0) of ice bath damping fluid: 100mmol/L, the EDTA (pH, 8.0) of 25mmol/L, the NaCl of 2.0mol/L.

Preparation CTAB extracting solution: 2% CTAB, 0.8% bentonite, the Tris-HCl (pH, 8.0) of 100mmol/L, the EDTA (pH, 8.0) of 25mmol/L, the NaCl of 1.4mol/L, 1% polyvinylpolypyrrolidone and 0.1% DEPC.

Get the centrifuge tube of 1.5mL, add in advance 600-800 μ l ice bath damping fluid, 60 μ l beta-mercaptoethanols, place for subsequent use on ice;

Take 0.3g plant leaf, directly put into 1.5ml centrifuge tube for subsequent use, do grinding rod by blade grind into powder with little centrifuge tube, can save time about approximately 0.5 hour compared with sending into the method for centrifuge tube after grinding.Mix with vortex mixer vibration 2-3min, place on ice after 10min in 4 DEG C, 12000g, centrifugal 10min,

Supernatant discarded adds 700 μ l CTA β extracting solutions in centrifuge tube, 50 μ l beta-mercaptoethanols, and thermal agitation 2-3min mixes;

It is the water-bath 7-9min of 65 DEG C that centrifuge tube is placed in to temperature, and shake therebetween several times;

After centrifuge tube is taken out from water, be placed in rapidly on ice, adding volume ratio is water-saturated phenol and the chloroform totally 700 μ l of 1: 1, after thermal agitation 2-3min mixes in 4 DEG C, 12000g, centrifugal 10min.Get supernatant in another new centrifuge tube, add the dehydrated alcohol of 1/2 volume and the 5mol/L Potassium ethanoate of 1 times of volume, slowly shake mixes, and places after 10min in 4 DEG C 12000g, centrifugal 10min on ice.The consumption of Potassium ethanoate is generally not more than 1/2 volume and 2mol/L's, and the consumption of the Potassium ethanoate in this step exceeds conventional amount used, experiment showed, that conventional amount used is difficult to reach the effect of Polysaccharide removing.

Supernatant discarded, by 75% ethanol washing and precipitating, slowly mixes, and 4 DEG C, 12000g, centrifugal 5min.

Supernatant discarded, moment is centrifugal thoroughly abandons dry object to reach.The dry 5min of gas in stink cupboard.The ddH processing with DEPC ₂o thoroughly dissolves RNA precipitation.

Present method experiment used is more, only exemplifies three experiments below:

1. bentonite concentration affects experiment:

Bentonite concentration, respectively with 0%, 0.2%, 0.4%, 0.6%, 0.8%, 1.0%, obtains the effect shown in Fig. 5.As can be seen from Figure 5: while not adding bentonite, although can propose RNA, but concentration is lower, and the bentonite concentration adding is while being 0.2% and 0.4%, the RNA banding pattern hangover of extracting, and in the time adding bentonite concentration to be 0.6% and 0.8%, the RNA banding pattern of extraction is comparatively clear, in the time that the concentration of bentonite is 1.0%, banding pattern thickens again unclear.Therefore the bentonite concentration adopting is in the present invention 0.6%.

2. the electrophoresis contrast experiment of Different Extraction Method:

Fig. 1 has shown total RNA of the tetraploid locust blade of the CTAB method extraction that does not add bentonite, the RNA band Fuzzy that adopts this method to extract, and more shallow, yield is lower, is 347.61% (seeing attached list); Fig. 2 has shown total RNA of the tetraploid locust blade that utilizes the extraction of Trizol method, and its banding pattern is not obvious, and has a large amount of albumen in point sample hole, adopts the RNA yield of the method also lower simultaneously, is 325.68% (seeing attached list); Fig. 3 has shown the total RNA of tetraploid locust blade that utilizes SDS method to extract, and the RNA banding pattern that utilizes this method to extract is not obvious, and yield lower be 347.96%; That Fig. 4 shows is the total RNA of tetraploid locust blade that utilizes SDS-CTAB method to extract, the RAN band Fuzzy that utilizes the method to extract, and yield lower be 659.54%, and in point sample hole, there is a large amount of albumen; Fig. 5 is total RNA of the tetraploid locust blade that extracts after utilization interpolation different concns bentonite.

3. the yield contrast experiment of Different Extraction Method:

Subordinate list explanation: the RNA purity that different methods extracts and yield (data in form are that three secondary pollutants are learned the mean value repeating)

As seen from the above table, adopt the yield of present method far above other extracting method.

Claims

1. the extracting method of the total RNA of feed form tetraploid locust blade, it is characterized in that: pack tetraploid locust blade into centrifuge tube grind into powder, add the Tris-HCl by the 100mmol/L of pH=8, the EDTA of the 25mmol/L of pH=8, ice bath damping fluid and the beta-mercaptoethanol of the NaCl composition of 2.0mol/L, centrifugal, supernatant discarded, to adding in centrifuge tube by 2% CTAB, 0.8% bentonite, the Tris-HCl of the 100mmol/L of pH=8.0, the EDTA of the 25mmol/L of pH=8.0, the NaCl of 1.4mol/L, CTAB extracting solution and the beta-mercaptoethanol of 1% polyvinylpolypyrrolidone and 0.1% DEPC composition mix, centrifugal, get supernatant liquor, adding volume ratio is water-saturated phenol and the chloroform of 1:1, mix, centrifugal, draw supernatant liquor, add the 5mol/L liquor kalii acetici of the dehydrated alcohol of 1/2 volume and the pH=5.8 of l volume to mix, ice bath is centrifugal after leaving standstill, abandoning supernatant, use by 7.5 parts of dehydrated alcohols and 2.5 parts of ddH that DEPC processed ₂after 75% ethanol of O preparation cleans, carry out centrifugally, discard supernatant liquor, gas is dry, the ddH processing with DEPC ₂o thoroughly dissolves RNA precipitation, the ddH that wherein said DEPC processed ₂o refers to and adds 99.9 parts of ddH with 0.1 part of DEPC ₂in O, whirlpool mixes the pure water making through autoclave sterilization again.