CN102732527A - Screening method of Ammopiptanthus mongolicus drought resistance gene - Google Patents
Screening method of Ammopiptanthus mongolicus drought resistance gene Download PDFInfo
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Abstract
The invention provides a screening method of an Ammopiptanthus mongolicus drought resistance gene, comprising the following steps of: 1) taking Ammopiptanthus mongolicus which has undergone drought treatment; 2) extracting RNA by the adoption of 2*CTAB method; 3) carrying out RNA purification; 4) synthesizing cDNA by reverse transcription reaction; 5) conducting high-throughput sequencing; 6) acquiring differential expression sequence; and 7) retrieving and conducting software analysis to obtain the drought resistance gene. According to the invention, Ammopiptanthus mongolicus is adopted for transcriptome analysis so as to screen the Ammopiptanthus mongolicus drought resistance gene. Therefore, the screening method is of great guiding significance for breeding of drought resistant plants.
Description
Technical field
The present invention relates to the screening method of gene, relate in particular to a kind of screening method of Stem and leaf of Mongolian Ammopiptanthus anti-drought gene.
Background technology
Abiotic stress is the serious harm of modern agriculture, and particularly arid becomes one of most important factor of influence agricultural.In recent years, because China's industrial development is rapid, environment is polluted, and Greenhouse effect are on the rise.Along with the continuous rising of temperature and the influence of Desertification expansion, China's agricultural has received seriously influencing of arid.At present China has and surpasses 50% cultivated farmland and receive drought impact, and the serious drought of 20% cultivated farmland is arranged.Even in quantity of precipitation more Central China and South China thereof; Because quantity of precipitation skewness; These geographic farm crop all receive drought impact in the critical period of flowering such as paddy rice is almost annual, thereby directly influence output, serious drought in addition No kernels or seeds are gathered, as in a year of scarcity; And the northern China most of areas, original quantity of precipitation is just on the low side, adds the damage caused by a drought that is on the rise, and the Agricultural Development situation allows of no optimist especially.China is as large agricultural country, supporting 1/4th population near world population, and therefore agricultural all is the most important thing for the China and even the whole world.
For drought resisting, the various phytology research workers in the world always with it as emphasis research topic, done a large amount of work for this reason, also obtained significant progress.At present, for plant drought, generally tend to screen anti-drought gene, the mode that adopts anti-drought gene to carry out the breeding of drought resisting plant then obtains the good plant of drought resistance, thereby through screening, obtains the key that good anti-drought gene just becomes the drought resisting breeding.
Summary of the invention
The objective of the invention is to overcome above-mentioned deficiency, a kind of screening method of Stem and leaf of Mongolian Ammopiptanthus anti-drought gene is provided, it can filter out the anti-drought gene of Stem and leaf of Mongolian Ammopiptanthus quickly and effectively.
In order to reach above-mentioned technical purpose, the present invention adopts following technical scheme:
A kind of screening method of Stem and leaf of Mongolian Ammopiptanthus anti-drought gene wherein, may further comprise the steps
1) gets the Stem and leaf of Mongolian Ammopiptanthus that arid is handled
2) adopt the method for 2 * CTAB to extract RNA
3) RNA purifying
4) the synthetic cDNA of reverse transcription
5) high-flux sequence
6) obtain the differential expression sequence
7) retrieval and software analysis obtain anti-drought gene.
Stem and leaf of Mongolian Ammopiptanthus has the very low flow of water and rising intensity, and bound water content is very high, and the Stem and leaf of Mongolian Ammopiptanthus not only flow of water is low, and water-retaining capacity is very strong, and from the leaf to the stem, all has the xeromorphy that suppresses rising dehydration.It is thus clear that the Stem and leaf of Mongolian Ammopiptanthus drought resistance is outstanding, select for use Stem and leaf of Mongolian Ammopiptanthus to screen anti-drought gene, can obtain good anti-drought gene.
The screening method of above-mentioned Stem and leaf of Mongolian Ammopiptanthus anti-drought gene, wherein, the seed of said Stem and leaf of Mongolian Ammopiptanthus uses the alcohol immersion of 70% volume ratio, after the Losantin sterilization with 10% mass ratio, uses aseptic water washing then.
The screening method of above-mentioned Stem and leaf of Mongolian Ammopiptanthus anti-drought gene, wherein, step 2) extract RNA through following flow process
(1) Stem and leaf of Mongolian Ammopiptanthus true leaf or stem Duan Yu-20 ℃ are pulverized, add the damping fluid of 65 ℃ of preheatings, concussion is in 65 ℃ of water-baths
(2) add phenol/chloroform/primary isoamyl alcohol, in 4 ℃ centrifugal, get supernatant
(3) add LiCl ,-20 ℃ of placements
(4) abandon supernatant, 70% volume ratio ethanol rinsing deposition
(5) remove ethanol, add absolute ethyl alcohol-20 ℃ placement
(6) in 4 ℃ of centrifugal, as to adopt DEPC to handle water dissolution depositions
(7) agarose gel electrophoresis, voltage detecting ,-70 ℃ of storages.
The screening method of above-mentioned Stem and leaf of Mongolian Ammopiptanthus anti-drought gene, wherein, repeat twice said step (2), (3).
The screening method of above-mentioned Stem and leaf of Mongolian Ammopiptanthus anti-drought gene wherein, carries out contamination analysis after the said step 5) order-checking, and some of picked at random are compared in NCBI NR DB after reading long assembling.
The screening method of above-mentioned Stem and leaf of Mongolian Ammopiptanthus anti-drought gene, wherein, said step 5) order-checking back uses DNASTARNGEN software that sequencing result and wild-type sequencing result are made up assembling.
The screening method of above-mentioned Stem and leaf of Mongolian Ammopiptanthus anti-drought gene wherein, will obtain difference expression gene and carry out preliminary screening in the said step 6), choose the gene of differential expression more than 4 times.
The screening method of above-mentioned Stem and leaf of Mongolian Ammopiptanthus anti-drought gene, wherein, said step 6), 7) Application of DNA STAR:ArrayStar software has carried out the differential expression analysis to transcribing group in.
The present invention compares prior art and has the following advantages:
(1) screening method of Stem and leaf of Mongolian Ammopiptanthus anti-drought gene of the present invention selects for use Stem and leaf of Mongolian Ammopiptanthus to screen anti-drought gene, can obtain good anti-drought gene.
(2) screening method of Stem and leaf of Mongolian Ammopiptanthus anti-drought gene of the present invention is analyzed with the screening anti-drought gene the group of transcribing of Stem and leaf of Mongolian Ammopiptanthus, can filter out good anti-drought gene comprehensively, exactly.
Description of drawings
Fig. 1 is that wild-type Stem and leaf of Mongolian Ammopiptanthus sequencing result pollutes evaluation figure
Fig. 2 handles the Stem and leaf of Mongolian Ammopiptanthus sequencing result for the present invention's arid and pollutes evaluation figure.
Embodiment
Below in conjunction with accompanying drawing and embodiment the present invention is further explained:
The preferable following steps that contain successively of the screening method of Stem and leaf of Mongolian Ammopiptanthus anti-drought gene of the present invention:
1, experiment material is handled
Select for use and be taken from Inner Mongol Alxa desert area Stem and leaf of Mongolian Ammopiptanthus (Ammopiptanthus mongolicus) seed as experiment material.The Stem and leaf of Mongolian Ammopiptanthus seed is preserved in 4 ℃ of sealings under obsolete situation.For the experiment material of using among this paper, the Stem and leaf of Mongolian Ammopiptanthus seed that at first will deposit in 4 ℃ of refrigerators takes out, in Bechtop; The ethanol of use 70% soaks 30s with it, with behind the 10% Losantin sterilization 20min, uses aseptic water washing 3-5 time then; Afterwards it is seeded on the MS solid medium, cultivated one month, seedling is taken out transplanting from substratum; Grow after one month, drought and water shortage one month is collected their blade and stem section then; Be placed in the liquid nitrogen, put into-80 ℃ of refrigerators then and preserve subsequent use.
2, RNA extracts
The method of 2 * CTAB is adopted in the extraction of RNA.RNA extracts reagent and sees table 1, table 2.It is following that RNA extracts flow process:
(1) get arid Stem and leaf of Mongolian Ammopiptanthus true leaf and the stem section of handling of 0.2g and be placed in the mortar of-20 ℃ of precoolings, pulverize in the liquid nitrogen, the RNA that adds 65 ℃ of preheatings of prior warp extracts damping fluid 800 μ l.Concussion, 65 ℃ of water-bath 5min shake 3 times.
(2) centrifuge tube is taken out from water-bath, be cooled to room temperature, add isopyknic phenol/chloroform/primary isoamyl alcohol, concussion 10min.4 ℃, the centrifugal 10min of 12000rpm.
(3) get supernatant, chloroform/primary isoamyl alcohol repeats extracting once.
(4) get supernatant, add the LiCl of 1/5 volume 10mmol/L, place 1h for-20 ℃.
(5) 4 ℃, the centrifugal 20min of 12000rpm.
(6) abandon supernatant, blot the residual liquid in clean bottom.
(7) 400 μ l DEPC water dissolution precipitate, and add the LiCl of 1/5 volume 10mmol/L again, place 1h for-20 ℃.
(8) 4 ℃, the centrifugal 15min of 12000rpm abandons supernatant, 70% ethanol rinsing deposition 2-3 time.
(9) 4 ℃, the centrifugal 10min of 12000rpm removes ethanol, natural air drying in the room temperature stink cupboard, about 15min.
(10) solution is gone in the centrifuge tube of 1.5mL, add the absolute ethyl alcohol of 2 times of volume precoolings, place 3h for-20 ℃.
(11) in 4 ℃, the centrifugal 20min of 12000rpm.
The water dissolution deposition that (12) 20 μ L DEPC handle.
(13) 1% agarose gel electrophoresis, the 120V voltage detecting.-70 ℃ of storages are subsequent use.
Table 12 * CTAB RNA extracts damping fluid
Table 2 SSTE damping fluid
3, RNA purifying
The RNA purification kit is bought from Takara company, and RNA purifying flow process is following:
(1) with OligotexTM-dT30 < Super>in 37 ℃ of insulations.When 2 * Binding Buffer has deposition to separate out, please in 37 ℃ of dissolving back uses fully.
(2) in 1.5ml Microtube, prepare reaction solution shown in the table 3 by following method.
Table 3 RNA purification reaction liquid
*By requirement in the table, use DEPC HO to prepare the suitable RNA aqueous solution.
(3) with behind the abundant mixing of the reaction solution of above-mentioned preparation, 70 ℃ of heating 3min (RNA sex change).
(4) room temperature is placed 10min (OligotexTM-dT30 and mRNA combine).
(5) the centrifugal 5min of 15000rpm.
(6) remove supernatant (trying not to suck OligotexTM-dT30), add the Wash Buffer of 35 μ l, after OligotexTM-dT30 is fully suspended, join among the Column Cup of Spin Column Set.
(7) the centrifugal 30s of 15000rpm.
(8) in Column Cup, add the Wash Buffer of 350 μ l, OligotexTM-dT30 is fully suspended after, Spin Column is moved to new Spin Column with on the centrifugal Tube.
(9) the centrifugal 30s of 15000rpm.
(10) Spin Column is moved to new Spin Column with on the centrifugal Tube.
(11) in Column Cup, add the DEPC H of 70 ℃ of preheatings of 20～50 μ l
2O, OligotexTM-dT30 fully suspends.
(12) the centrifugal 30s of 15000rpm reclaims mRNA.
(13) 2～3 (DEPC H that at every turn all should add 70 ℃ of preheatings of repeatable operation above-mentioned steps
2O).
4, cDNA is synthetic
CDNA synthetic agent box is bought from Agilent company, and the synthesis flow of cDNA is following:
1) cDNA first chain is synthetic
(1) bath of fetching boiling water, 42 ℃ of preheatings.
(2) from Ultralow Temperature Freezer, [α-32P] dNTP is got, place on ice.
(3) in the centrifuge tube that removes RNase of a 2ml, be sequentially added into reagent shown in the table 4:
The table 4 cDNA first chain synthesis reaction system
With table 4 gained mixed solution mixing, add X μ l polyA)+RNA (0.5-5 μ g).
(4) room temperature is placed 10min.Add 0.5 μ l of the [α-32P] dNTP (800Ci/mmol) in the put procedure.
(5) the AccuScript RT of adding 3 μ l in the first chain synthesis reaction system, the final volume of the first chain synthesis reaction system is 50 μ l.
(6) centrifugal 30min behind the mixing gently.
(7) therefrom take out 5 μ l and add in the new centrifuge tube that removes RNase, add [α-32P] dNTP (800Ci/mmol) of 0.5 μ l again.This radio active material will be analyzed with the product of second chain together.
(8) with first chain synthetic reaction system water-bath 1h in 42 ℃ of water-baths.
(9) open 16 ℃ of water-baths, prepare for cDNA second chain is synthetic.
(10) after the 1h, the cDNA first chain synthesis reaction system is taken out from 42 ℃ of water-baths, the reaction solution that will not add radioelement is put on ice, contains the radioelement reaction solution and puts into-20 ℃ (being used for electrophoretic analysis).
2) cDNA second chain is synthetic
(1) will be used for after the non-enzyme agent dissolves of second chain synthesis reaction centrifugally, place on ice.
(2) in 45 μ l, the first chain synthesis reaction systems ("dead" isotropic substance), add reagent in the table 5 in order.
(3) in above-mentioned reaction system, add 2 μ l RNase H (1.5U/ μ l) and 11 μ l DNA polymeraseI (9.0U/ μ l).
(4) centrifugal 30s behind the mixing gently, 16 ℃ of water-bath 2.5h.Constantly observe water temperature during this time, guarantee that water temperature is no more than 16 ℃.
(5) behind the 2.5h, reaction solution is taken out from 16 ℃ of water-baths, place on ice rapidly.
The table 5 cDNA second chain synthesis reaction system
3) the terminal passivation of cDNA
(1) in the second chain synthesis reaction system, adds 23 μ l blunting dNTP mix and 2 μ l cloned Pfu DNA polymerase (2.5U/ μ l).
(2) shake mixing rapidly, centrifugal 30s.72 ℃ of water-bath 30min (not surpassing 30min).
(3) dissolving 3M sodium-acetate.
(4) from 72 ℃ of water-baths, take out reaction solution, centrifugal rapidly, add the phenol-chloroform [1: 1 (v/v)] of 200 μ l, shook several minutes.
(5) the centrifugal 2min of room temperature gets supernatant.
(6) add isopyknic chloroform, the concussion mixing.
(7) with the at room temperature centrifugal 2min of the maximum speed of revolution of whizzer.
(8) add 20 μ l 3M sodium acetate and 400 μ l 100% (v/v) ethanol, deposition cDNA.Mixing.
(9)-20 a ℃ deposition is spent the night.
(10) with the maximum speed of revolution of whizzer at 4 ℃, centrifugal 2min abandons supernatant.
(11) add 500 μ l 70% (v/v) ethanol, washing and precipitating.
(12) under the maximum speed of revolution room temperature condition with whizzer, centrifugal 2min abandons supernatant.
(13) under the maximum speed of revolution room temperature condition with whizzer, empty centrifugal 2min abandons supernatant.Deposition is dried.
(14) in the air dried deposition, add 9 μ l TE buffer, place 30min for 4 ℃.
5, high-flux sequence
Adopt 454 sequenators to carry out 454 high-flux sequences in U.S. guest Western method Leah state university.
6, sequencing result contamination analysis
After obtaining sequencing result, need to confirm whether order-checking receives the pollution of bacterium and fungi.Therefore we from the sequence of order-checking gained random choose 1000 read long; Use DNASTAR NGEN software assembling back and NCBI NR DB to compare; And adopt Megan software to analyze; Result such as Fig. 1, shown in Figure 2, result find in the material that contrast and arid are handled, and the shared ratio of bacterium and fungi is all little.Bacterium and fungi one have 174 in contrast, and the sequence of the known Stem and leaf of Mongolian Ammopiptanthus of NCBI has 71; Material bacteria and fungi that arid is handled have 228, and the sequence of the known Stem and leaf of Mongolian Ammopiptanthus of NCBI has 19.Hence one can see that, and our sequencing result does not pollute, and is true and reliable.
7, long assembling is read in order-checking
We assemble respectively wild-type Stem and leaf of Mongolian Ammopiptanthus and the sequencing result that arid is handled to use DNASTAR NGEN software, and the sequencing result of the Stem and leaf of Mongolian Ammopiptanthus that wild-type and arid are handled combines and assembles, and we are referred to as both.Obtained 8480 sequences after the assembling of wild-type Stem and leaf of Mongolian Ammopiptanthus, obtained 7474 sequences after the Stem and leaf of Mongolian Ammopiptanthus assembling that arid is handled, with having obtained 15112 sequences after two colony's assemblings.The result is as shown in table 6 in assembling.
Table 6 wild-type, arid are handled and Both assembling information analysis
8, differential expression analysis
Application of DNA STAR:ArrayStar has carried out the differential expression analysis to whole 15112 sequences of transcribing group, obtains the differential expression sequence, filters out the sequence of differential expression more than 4 times, and the gene of part differential expression more than 4 times is as shown in table 7.
The portion gene of table 7 differential expression more than 4 times
9, anti-drought gene screening
Choose the gene of above-mentioned differential expression more than 4 times,, transcribe group analysis, found 9 in the albumen relevant with aging with the DB comparison; The protein 16 relevant with arid is individual, with 1 in cold relevant albumen, 5 in MYB, MYC-like albumen, 5 of HSPs; With 1 in the albumen of salt response, 1 of ultraviolet corresponding protein, 6 of pathogenesis-related proteins, 3 of adverse circumstance response proteins; 6 of growth hormone GAP-associated protein GAPs, the GA associated protein 1 is individual, and the cell walls correlated protein 12 is individual, 6 of channel proteins; 1 of aluminium inducible protein, and the albumen relevant with photosynthesis has 24, the result is as shown in table 8:
The screening of table 8 Stem and leaf of Mongolian Ammopiptanthus adversity gene
Aforesaid method adopts Stem and leaf of Mongolian Ammopiptanthus to screen anti-drought gene, can obtain good anti-drought gene, employing transcribe the anti-drought gene that group analyzing method can filter out Stem and leaf of Mongolian Ammopiptanthus more comprehensively and accurately.The anti-drought gene that filters out can be used to cultivate new drought resisting plant.For example, can the anti-drought gene that filter out be recombinated in the plant expression vector with just form, adopt transgenic technology that recombination is imported in the vegetable cell, obtain transfer-gen plant; Through detecting transgene expression and plant being carried out drought resistance identify, therefrom filter out transfer-gen plant and offspring thereof that drought resistance obviously improves again, create the new variety that have good drought-resistant ability in plant breeding.
Claims (8)
1. the screening method of a Stem and leaf of Mongolian Ammopiptanthus anti-drought gene is characterized in that: may further comprise the steps
1) gets the Stem and leaf of Mongolian Ammopiptanthus that arid is handled
2) adopt the method for 2 * CTAB to extract RNA
3) RNA purifying
4) the synthetic cDNA of reverse transcription
5) high-flux sequence
6) obtain the differential expression sequence
7) retrieval and software analysis obtain anti-drought gene.
2. according to the screening method of the said Stem and leaf of Mongolian Ammopiptanthus anti-drought gene of claim 1, it is characterized in that: the seed of said Stem and leaf of Mongolian Ammopiptanthus uses the alcohol immersion of 70% volume ratio, after the Losantin sterilization with 10% mass ratio, uses aseptic water washing then.
3. according to the screening method of the said Stem and leaf of Mongolian Ammopiptanthus anti-drought gene of claim 1, it is characterized in that: said step 2) extract RNA through following flow process
(1) Stem and leaf of Mongolian Ammopiptanthus true leaf or stem Duan Yu-20 ℃ are pulverized, add the damping fluid of 65 ℃ of preheatings, concussion is in 65 ℃ of water-baths
(2) add phenol/chloroform/primary isoamyl alcohol, in 4 ℃ centrifugal, get supernatant
(3) add LiCl ,-20 ℃ of placements
(4) abandon supernatant, 70% volume ratio ethanol rinsing deposition
(5) remove ethanol, add absolute ethyl alcohol-20 ℃ placement
(6) in 4 ℃ of centrifugal, as to adopt DEPC to handle water dissolution depositions
(7) agarose gel electrophoresis, voltage detecting ,-70 ℃ of storages.
4. according to the screening method of the said Stem and leaf of Mongolian Ammopiptanthus anti-drought gene of claim 3, it is characterized in that: repeat twice said step (2), (3).
5. according to the screening method of any said Stem and leaf of Mongolian Ammopiptanthus anti-drought gene of claim 1-4, it is characterized in that: carry out contamination analysis after the said step 5) order-checking, some of picked at random are compared in NCBI NR DB after reading long assembling.
6. according to the screening method of any said Stem and leaf of Mongolian Ammopiptanthus anti-drought gene of claim 1-4, it is characterized in that: said step 5) order-checking back uses DNASTAR NGEN software that sequencing result and wild-type sequencing result are made up assembling.
7. according to the screening method of any said Stem and leaf of Mongolian Ammopiptanthus anti-drought gene of claim 1-4, it is characterized in that: will obtain difference expression gene in the said step 6) and carry out preliminary screening, and choose the gene of differential expression more than 4 times.
8. according to the screening method of any said Stem and leaf of Mongolian Ammopiptanthus anti-drought gene of claim 1-4, it is characterized in that: Application of DNA STAR:ArrayStar software has carried out the differential expression analysis to transcribing group said step 6), 7).
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Cited By (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710355A (en) * | 2013-12-16 | 2014-04-09 | 中国农业科学院生物技术研究所 | Ammopiptanthus mongolicus drought induced related gene, and expression vector and application thereof |
| CN106282158A (en) * | 2015-12-02 | 2017-01-04 | 香港中文大学深圳研究院 | A kind of method extracting high-quality genomic DNA from Caulis et Folium Ammopiptanthi Mongolici |
| CN107287188A (en) * | 2017-04-25 | 2017-10-24 | 安徽安龙基因医学检验所有限公司 | A kind of improvement CTAB extracting rhodiola roots RNA method |
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2011
- 2011-04-14 CN CN2011100935138A patent/CN102732527A/en active Pending
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| 王丹丹等: "沙冬青cDNA AFLP体系的建立及引物筛选", 《安徽农业科学》 * |
| 王海光: "水分胁迫诱导表达基因在水、旱稻幼苗中的表达分析", 《中国农业大学博士学位论文》 * |
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN103710355A (en) * | 2013-12-16 | 2014-04-09 | 中国农业科学院生物技术研究所 | Ammopiptanthus mongolicus drought induced related gene, and expression vector and application thereof |
| CN103710355B (en) * | 2013-12-16 | 2015-05-06 | 中国农业科学院生物技术研究所 | Ammopiptanthus mongolicus drought induced related gene, and expression vector and application thereof |
| CN106282158A (en) * | 2015-12-02 | 2017-01-04 | 香港中文大学深圳研究院 | A kind of method extracting high-quality genomic DNA from Caulis et Folium Ammopiptanthi Mongolici |
| CN106282158B (en) * | 2015-12-02 | 2019-05-03 | 香港中文大学深圳研究院 | A method of genomic DNA is extracted from Ammopiptanthus mongolicus |
| CN107287188A (en) * | 2017-04-25 | 2017-10-24 | 安徽安龙基因医学检验所有限公司 | A kind of improvement CTAB extracting rhodiola roots RNA method |
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Application publication date: 20121017 |