CN108913669A - A kind of drought resisting protein, the nucleic acid molecules of separation and application - Google Patents
A kind of drought resisting protein, the nucleic acid molecules of separation and application Download PDFInfo
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- CN108913669A CN108913669A CN201810921513.4A CN201810921513A CN108913669A CN 108913669 A CN108913669 A CN 108913669A CN 201810921513 A CN201810921513 A CN 201810921513A CN 108913669 A CN108913669 A CN 108913669A
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
- C12N9/93—Ligases (6)
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- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
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- C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
- C12N15/09—Recombinant DNA-technology
- C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
- C12N15/79—Vectors or expression systems specially adapted for eukaryotic hosts
- C12N15/82—Vectors or expression systems specially adapted for eukaryotic hosts for plant cells, e.g. plant artificial chromosomes (PACs)
- C12N15/8241—Phenotypically and genetically modified plants via recombinant DNA technology
- C12N15/8261—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield
- C12N15/8271—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance
- C12N15/8273—Phenotypically and genetically modified plants via recombinant DNA technology with agronomic (input) traits, e.g. crop yield for stress resistance, e.g. heavy metal resistance for drought, cold, salt resistance
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- C12Y—ENZYMES
- C12Y603/00—Ligases forming carbon-nitrogen bonds (6.3)
- C12Y603/02—Acid—amino-acid ligases (peptide synthases)(6.3.2)
- C12Y603/02019—Ubiquitin-protein ligase (6.3.2.19), i.e. ubiquitin-conjugating enzyme
Abstract
The invention discloses a kind of drought resisting protein, the nucleic acid molecules of separation and applications, are related to field of plant genetic project technology.Drought resisting protein disclosed by the invention, amino acid sequence is as shown in SEQ ID NO.2；The drought resisting protein has drought resistance, can be improved the drought-resistant ability of plant.Conversion encodes the plant of the gene of the drought resisting protein, compared to WT lines, has stronger drought-resistant ability.
Description
Technical field
The present invention relates to field of plant genetic project technology, in particular to the nucleic acid point of a kind of drought resisting protein, separation
Son and application.
Background technique
With the destruction of global ecological environment, the canceration of weather exacerbates the shortage of global water resources.The whole world has closely at present
1/3 area is in arid or semiarid zone, the desertification to be tilled the land as caused by water shortage are got worse.Arid is
More become the principal element for restricting survival and development of mankind, especially production estimation and grain security.Rice is as three big grains
One of food crop provides the staple food of the nearly half population in the whole world especially people of under-developed area.And rice is water requirement
Great crops, drought stress become the insoluble problem of current facing mankind naturally.China is due to precipitation space
Distributed pole is uneven, northwestwards successively decreases from the southeast, at the same year border Abrupt Precipitation change it is big, in addition industrial water and domestic water sharply increase
It is long, the current Rice Production phenomenon in poor harvest due to drought and water shortage Severe Reduction is caused, the grain security in China has been seriously affected.
The drought resistance that rice how is improved under drought condition, guarantees the production of rice to greatest extent, becomes rice and grind
The key subjects studied carefully.Drought resisting new varieties are cultivated, the function and drought resisting adjustment mechanism for studying gene related to drought tolerance are ground as the whole world
The hot spot studied carefully.With the research of genetic engineering, molecular genetics, transcription group, proteomics and gene expression regulation, anti-
The discovery and excavation of non-irrigated gene achieve preliminary progress in terms of the research of drought resisting mechanism.Rice faces the external source of drought stress
Signal, by the identification of signal and receptor, the transductory cascade of signal reacts, finally generated in effector cell Physiology and biochemistry and
The change of gene expression, to adjust the drought stress reaction of cell.It participates in entire drought stress reaction and is related to osmotic adjustment
Object synthesis and metabolism, plant arid hormone ABA synthesis and metabolism, the closing of stomata, the change of cell growth, cellular respiration
Activation, photosynthetic decrease etc..
In eucaryote, RING (really interesting new gene) finger factor is a kind of containing E3 company
Meet the big gene family member of enzymatic activity.Arabidopsis is about containing 350 or so RING finger genes.Literature research
Show that this kind of RING finger albumen influences the growth and development of plant in a manner of plant and environmental interaction.RING
Finger albumen takes part in the seed dormancy of plant, seed is sprouted, seedling growth, and especially take part in plant depends on ABA
Drought stress responsing reaction.Ko in 2006 et al. have found the XERICO factor take part in arabidopsis drought stress response it is anti-
It answers, wherein the drought-resistant ability of arabidopsis can be significantly improved by being overexpressed XERICO gene in arabidopsis, while also improving quasi-
The content of arid hormone ABA in southern mustard body.Zhang in 2007 et al. has found RING finger factor S DIR, takes part in dependence
In arid and the salt stress reaction of ABA.Bu in 2009 et al. has found that RING finger factor R HA2a is sprouted in arabidopsis seed
Hair and Seedling Stage participate in positive regulator ABA signal, and Li et al. people has found the autoploid RHA2b of RHA2a within 2011 later, discloses
RHA2b and RHA2a adjusts ABA signal in a manner of interacting, and improves the drought-resistant ability of arabidopsis.Ryu in 2010 etc.
People has found that arabidopsis RING finger factors A IRP takes part in drought stress responsing reaction.Subsequent Xia et al. was sent out in 2012
Now and a RING finger factor Z mRFP1 of corn is cloned, encoded an E3 ligase, has participated in the arid that ABA is adjusted
Stress responses.Clone and functional study before 2014 about this kind of RING finger factor, are concentrated mainly on model plant
On arabidopsis, the clone of the relevant RING finger gene such as other model plants such as corn, sorghum, wheat and functional study
It is fewer.
In conjunction with current reality, arid, semiarid water-deficient area accounts for China's national territorial area half or so, especially we
North China, northeast and the area of northwest three of important grain base, the exactly area of water shortage arid most serious are annual by dry farming
Kind area is close to 300,000,000 mu.Therefore, using plant gene engineering technology and modern molecular biology means, excellent drought resisting is cloned
Gene, by genetic transformation in rice and other crops heterogenous expression, to improve arid, semiarid zone vegetative coverage,
The production and reduction production loss of rice and other crops are all very important.
In consideration of it, the present invention is specifically proposed.
Summary of the invention
The purpose of the present invention is to provide a kind of drought resisting protein, which has drought resistance, can be improved plant
Drought-resistant ability.
Another object of the present invention is to provide a kind of isolated nucleic acid molecules, encode above-mentioned drought resisting protein, conversion
The plant of the nucleic acid molecules of the separation has higher drought-resistant ability.
Another object of the present invention is to provide a kind of carriers.
Another object of the present invention is to provide recombinant cells.
Another object of the present invention is to provide above-mentioned drought resisting protein, the nucleic acid molecules of separation, carrier and recombinant cells
Using.
Another object of the present invention is to provide a kind of methods for improving plant drought ability, using this method, Ke Yiti
The drought-resistant ability of high plant.
The invention is realized in this way：
On the one hand, the present invention provides a kind of drought resisting proteins, and amino acid sequence is as shown in SEQ ID NO.2.
On the one hand, the present invention provides a kind of isolated nucleic acid molecules, above-mentioned drought resisting protein is encoded.
Further, in some embodiments of the present invention, base sequence is as shown in SEQ ID NO.1.
The present invention is for the first time using the conserved sequence RING finger structural domain of arabidopsis XERICO as molecular probe, search two
EST the and nucleotide sequence of fringe false bromegrass (two fringe false bromegrass) carries out splicing and evolutionary analysis to obtained matching sequence,
It was found that a RING finger factor of two fringe false bromegrass, is named as BdRHP1.By the comparison of protein amino acid sequence and
Evolution homogeneous assays primarily determine that BdRHP1 may be ortholog of the arabidopsis XERICO in two fringe false bromegrass.
By constructing the over-express vector rice transformation of rice BdRHP1 gene (SEQ ID NO.1) and obtaining homozygous T2
For plant, by carrying out drought stress experiment with reproductive stage in seedling stage, demonstrate the RING finger of two fringe false bromegrass because
Sub- BdRHP1 takes part in ABA Signal Regulation approach and improves the reaction of rice drought stress response, and obtains drought-resistant ability than wild
The transgenic paddy rice strain for the BdRHP1 gene overexpression that raw type rice plant is significantly increased.The discovery of BdRHP1 anti-drought gene
And advantage, the drought resisting molecular breeding for rice in agricultural production, wheat, seeding corn and other crops provide new material and approach.
In addition, those skilled in the art are easy the nucleic acid shown in SEQ ID NO.1 point according to the degeneracy of codon
Subsequence basis carries out the replacement of one or more nucleotide, obtains corresponding derived sequence, so as to encode out present invention offer
SEQ ID NO.2 shown in drought resisting protein.Therefore, the sequence basis of the nucleic acid molecules shown in above-mentioned SEQ ID NO.1 into
The replacement of row one or more nucleotide, is encoded out the derived sequence of drought resisting protein shown in SEQ ID NO.2 accordingly
Also belong to protection scope of the present invention.
On the one hand, the present invention provides a kind of carriers, contain above-mentioned isolated nucleic acid molecules.
Further, in some embodiments of the present invention, above-mentioned carrier is pHB, and above-mentioned nucleic acid molecules pass through BamH
I and Sac I restriction enzyme site is connected on above-mentioned carrier.
It is readily appreciated that, those skilled in the art are as needed, can choose suitable carrier, divide as above-mentioned nucleic acid is delivered
Which kind of carrier no matter the tool of son, select, all belong to the scope of protection of the present invention.
On the one hand, the present invention provides a kind of recombinant cells, contain above-mentioned isolated nucleic acid molecules or above-mentioned load
Body.
Recombinant cell can be also possible to cell such as plant cell with bacterium such as Escherichia coli；Plant cell can be
Dicotyledonous plant cells are also possible to monocot plant cell.The specific category of recombinant cell or host cell, art technology
Personnel can select according to the actual situation.
On the one hand, the present invention provides above-mentioned drought resisting protein, above-mentioned isolated nucleic acid molecules, above-mentioned carrier or
The above-mentioned recombinant cell of person is cultivating the application in drought resistant plant variety.
Further, in some embodiments of the present invention, above-mentioned plant is rice, corn, wheat, soybean, cotton
Or sorghum.
The experimental verification of the embodiment of the present invention above-mentioned drought resisting protein, above-mentioned isolated nucleic acid molecules are transferred to rice
Afterwards, rice drought-resistant ability is improved.Therefore, above-mentioned drought resisting protein, above-mentioned isolated nucleic acid molecules, above-mentioned carrier with
And above-mentioned recombinant cell may be used to cultivate drought resistant plant variety.
It being readily appreciated that, those skilled in the art are as needed, and it can choose and need to cultivate drought-resistant plant new varieties, as long as
Apply drought resisting protein shown in SEQ ID NO.2 provided by the invention and/or its nucleic acid coding sequence belong to it is of the invention
Protection scope.
For example, the method for cultivating drought resistant plant variety can be, above-mentioned isolated nucleic acid molecules are situated between by Agrobacterium
Inducing defecation by enema and suppository, particle bombardment etc. import in plant the genetically modified plants new product clock for obtaining drought resisting, or for example by gene editing technology
Zinc finger nuclease technology (zinc-finger nucleases, ZFN), the activating transcription factor sample effect of artificial nuclease mediation
Object nucleic acid zymotechnic (transceription activator-like effector nucleases, TALEN) or RNA are mediated
CRISPR/Cas nucleic acid zymotechnic so that the endogenous gene group of target plant is changed to give expression to shown in SEQ ID NO.2
Drought resisting protein obtains non-transgenic drought-resistant plant new varieties.
On the one hand, the present invention provides a kind of methods for improving plant drought ability comprising：By above-mentioned isolated core
Acid molecule, above-mentioned carrier or above-mentioned recombinant cell import target plant.
Further, in some embodiments of the present invention, above-mentioned target plant be rice, corn, wheat, soybean,
Cotton or sorghum.
To sum up, the present invention has broken the mode of the conventional same species endogenous gene of conversion, to rice, wheat, corn etc.
The nearly source kind two fringe false bromegrass of crops has carried out deep related research, thus have found in two fringe false bromegrass with plant drought
Relevant gene order (SEQ ID NO.1) is acted on, to provide a kind of drought resisting protein coded sequence (SEQ ID NO.2).
The complete translation area (coding sequence) of the gene is transferred to generally in conjunction with the composition type expression promoter 35S of CaMV
Crops such as rice, it will thus provide the drought-resistant ability of the crops.
Therefore, by the RING finger gene nucleic acid sequence of the two fringe false bromegrass (coding SEQ ID NO.2 albumen, such as
SEQ ID NO.1) and protein sequence (SEQ ID NO.2), it is applied in the research of plant stress-resistance transgenic technology and molecular breeding,
Make the plant that there is apparent drought resistance function, can reduce injury of the drought stress to plant growth and development, and reduce farming
The loss of object production yields.
Detailed description of the invention
In order to illustrate the technical solution of the embodiments of the present invention more clearly, below will be to needed in the embodiment attached
Figure is briefly described, it should be understood that the following drawings illustrates only certain embodiments of the present invention, therefore is not construed as pair
The restriction of range for those of ordinary skill in the art without creative efforts, can also be according to this
A little attached drawings obtain other relevant attached drawings.
Fig. 1 is the structure of BdRHP1 albumen；
Fig. 2 is BdRHP1 gene overexpression vector construction flow chart；
Fig. 3 is the genomic DNA positive test symbol of transgenic plant；
Fig. 4 is the testing result of Total RNAs extraction
Fig. 5 is the RT-PCR testing result of transgenic plant；
Fig. 6 is the drought resisting phenotype of Seedling Stage drought stress wild type and transgenic line；
Fig. 7 is that Seedling Stage meets with drought stress, the survival rate of wild type and transgenic line after resuming water supply；
Fig. 8 is that Seedling Stage meets with drought stress wild type and the leaf water content of transgenic line compares；
Fig. 9 is the comparison that wild type and BdRHP1 are overexpressed plant excised leaf percentage of water loss；
Figure 10 is the table of wild type and transgenic line after meeting with drought stress 28 days in water requirement most urgent boot stage
Type；
Figure 11 is wild type and the last solid comparison of transgenic plant after meeting with drought stress 28 days in boot stage；
Figure 12 is the Yield comparison of wild type and transgenic plant under arid and normal condition.
Specific embodiment
It in order to make the object, technical scheme and advantages of the embodiment of the invention clearer, below will be in the embodiment of the present invention
Technical solution be clearly and completely described.The person that is not specified actual conditions in embodiment, according to normal conditions or manufacturer builds
The condition of view carries out.Reagents or instruments used without specified manufacturer is the conventional production that can be obtained by commercially available purchase
Product.
Feature and performance of the invention are described in further detail with reference to embodiments.
Vegetable material：
Two fringe false bromegrass wild type seeds and OryzasativaLcv.Nipponbare wild type seeds are all from Sichuan University, and plantation is in Anqing normal school
University greenhouse greenhouse breeds sowing, and 4 DEG C save backup.
Bacterial strain material：
Coli strain DH5 α, Agrobacterium strain EHA105
Plasmid material：
PMD18-T, pEASY-blunt are purchased from Beijing Quanshijin Biotechnology Co., Ltd.
Reagent：
DEPC is purchased from Sigma company.
RNA extracts reagent Trizol, is purchased from Invitrogen company.
CDNA synthetic agent box is purchased from Toyobo company.
RNase H enzyme, DNA marker are purchased from Takara company.
Restriction enzyme, Taq archaeal dna polymerase, T4DNA ligase, PrimeStar thermal starting high-fidelity DNA polymerase
Etc. being purchased from Takara company.
DNTP reagent is Time Technology Co., Ltd purchased from Beijing day.
Dimethyl sulfoxide (DMSO), Sigma company.
Plasmid extracts and DNA QIAquick Gel Extraction Kit is purchased from OMEGA company.Remaining reagent is all that domestic analysis is pure except especially indicating
Product.
Culture medium and solution
LB culture medium：Tryptone 10g/L, yeast powder 5g/L, NaCl 10g/L.With NaOH tune pH to 7.0, high pressure is gone out
Bacterium.
SOB culture medium：Tryptone 20g/L, yeast powder 5g/L, NaCl 0.58g/L, KCl 0.19g/L, 100 × Mg2+
10mL.With NaOH tune pH to 7.0, high pressure sterilization.
SOC culture medium：SOB+20mM glucose.
TB buffer (is configured) before use:1M KCl 4mL, 0.45M MnCl22.4mL, 0.50M CaCl20.6mL,
0.50M K-MES 0.5mL, ddH2O 12.5mL (total volume 20mL).
100×Mg2+Solution:20.33g MgCl2·6H20 and 24.65g MgSO47H20 constant volume is high in 100mL H2O
Pressure sterilizing.
20% glucose solution:20g glucose constant volume is in 100mL H2O, filtration sterilization.
1M KCl solution:7.45g KCl constant volume is in 100mL H2O, high pressure sterilization.
0.45M MnCl2Solution:8.9g MnCl2·4H20 constant volume is in 100mL H2O, high pressure sterilization.
0.50M CaCl2Solution：7.35g CaCl2·2H20 constant volume is in 100mL H2O, high pressure sterilization.
0.50M K-MES solution：9.76g MES constant volume is in 100mL H2O, with KOH tune pH to 6.3, filtration sterilization, packing
At the every pipe of 0.5mL, -20 DEG C of storages.
DMSO：Dispense the fresh DMSO of 300 μ l, -20 DEG C of storages.
Key instrument
Superclean bench (Suzhou, China), BioRed low temperature ultracentrifuge (BioRed Products), icematic
N35s ice machine, micropipettor (BioRed Products), PCR instrument (BioRed Products), CHB-100 thermostat metal
Bathe (Hangzhou, China), HZQ-F160 warm shaken cultivation case (Harbin, China), IF-RAD pressure stabilizing electrophoresis apparatus (BioRed public affairs entirely
Take charge of product), liquid nitrogen container (Chengdu, China), the multi-functional ultraviolet analyzer of gel (Shanghai, China), constant incubator (Shanghai,
China), BIO- glue imaging system, Spectramax M2 microplate reader.
Embodiment 1
The clone of BdRHP1 gene
1 extracts two fringe false bromegrass total serum IgE
1) liquid nitrogen grinds rapidly two fringe false bromegrass blade and stem tissue, is added by 50-100mg tissue/ml Trizol
Trizol acutely shakes, is placed at room temperature for 5min.
2) 12,000rpm is centrifuged 5min.
3) supernatant is taken, chloroform is added by 200ul chloroform/ml Trizol, acutely shakes 15sec, is placed at room temperature for 3min.
4) 4 DEG C of 12,000g are centrifuged 15min.
5) supernatant is taken, isopropanol is added by 0.5ml isopropanol/ml Trizol and mixes, is placed at room temperature for 10min.
6) 4 DEG C of 12,000g are centrifuged 10min, abandon supernatant, and RNA is sunken to tube bottom.
7) 75% ethyl alcohol is added by 75% ethyl alcohol of 1ml/ml Trizol, mildly vibrates centrifuge tube, suspend precipitating.
8) 4 DEG C of 8,000g are centrifuged 5min, as far as possible abandoning supernatant.
9) drying at room temperature 5-10min (RNA sample not dried excessively, otherwise be difficult to dissolve).
10) 50ul DEPC-H is used2O dissolution RNA sample, 55-60 DEG C, 5-10min.
2RT-PCR reaction obtains the complementary strand cDNA of RNA
It is placed under environment on ice, following components is added in the 200 μ lEP pipes of RNase-free：
5×RT Buffer 4μl；2 μ l of dNTP Mixture (each 10mM)；RNase Inhibitor(10U/μl)1μl；
Oligo(dT)20(10pmol/μl)1μl；Total RNA Xμl；RNase-Free H2O(11-X)μl；ReverTra Ace 1
μl。
Reverse transcription is carried out by following procedure：42℃40min；95℃5min；4℃5min.
PCR amplification BdRHP1 genetic fragment
Using the first chain cDNA of RNA reverse transcription as the template of amplifying target genes, pcr amplification reaction is carried out, amplification is drawn
Object sequence is：
Upstream primer：5'-CGGATCCGATGGGCATCTCCAGC-3’
Downstream primer：5'-CGAGCTCGCTAGTAGTGAATCAG-3’
The end of upstream primer 5 ' adds BamH I restriction enzyme site (underscore), and the end of downstream primer 5 ' adds Sac I restriction enzyme site
(underscore) so that amplified production is correctly connected on next carrier by digestion, and adds at 5 ' ends of two primers
Add a protection base C.
PCR reaction system：10×Buffer 2.5μL；dNTP 2μL(2.5mmol/L)；cDNA1μL(50ng)；Draw upstream
0.5 μ L of object (10 μm of ol/L)；0.5 μ L of downstream primer (10 μm of ol/L)；0.5 μ L (10000U/mL) of Taq polymerase；ddH2O supplement
To 25 μ L systems.
It mixes, tube bottom is arrived in centrifugation, carries out following PCR response procedures：
94 DEG C of initial denaturation 5min；94 DEG C of denaturation 30s；56 DEG C of annealing 30s；72 DEG C of extension 40s；25 circulations；72 DEG C of extensions
10min；4 DEG C of preservations.
Obtain the target fragment that fragment length is 520bp.Its base sequence is named as shown in SEQ ID NO.1
BdRHP1 gene；BdRHP1 gene coding BdRHP1 protein sequence as shown in SEQ ID NO.2, structure as shown in Figure 1,
Contain a RING-H2 Zinc finger domain and a transferring film structural domain.
PCR amplification purpose band is recycled, and is then sequenced by following system connection PMD18-T carrier, verifies it
Accuracy.
Linked system：The 3 μ L of target DNA of purifying；pMD18-T 1μL；10×Buffer1μL；1 μ L of T4 ligase；
ddH2O 4μL；
Condition of contact：16 DEG C of water-baths are stayed overnight.
Embodiment 2
The building of BdRHP1 gene plant over-express vector
BdRHP1 plant over-express vector pHB-BdRHP1 building process is referring to fig. 2.With restriction enzyme BamH I and
Sac I distinguishes the pcr amplification product of double digestion carrier pHB and target gene BdRHP1, and digestion products are carried out with T4DNA ligase
The connection of cohesive end carries out PCR detection with connection product conversion bacillus coli DH 5 alpha, and the plant for obtaining BdRHP1 gene crosses table
Up to carrier, it is named as pHB-BdRHP1.
Embodiment 3
BdRHP1 plant over-express vector agrobacterium mediation converted rice
1. the conversion of Agrobacterium and the identification of positive colony：
1) 150 μ, 1 Agrobacterium competent cell is taken, is thawed on ice；
2) carrier for adding l0 μ l to build, flicks mixing, ice bath 5min；
3) then 1m1YEB culture medium, 28 DEG C of shaken cultivations at a slow speed are added in quick-frozen l min in liquid nitrogen, 37 DEG C of water-bath 5min
2-4h；
4) culture is coated on the YEB plate containing 50 μ g/ml Kan and 50 μ g/ml Rif, 28 DEG C of culture about 36h
5) single colonie grown on picking plate is inoculated in and trains containing the YEB liquid of 50 μ g/ml Kan and 50 μ g/ml Rif
It supports in base, 28 DEG C of shaken cultivations are stayed overnight, and carry out PCR amplification identification by template of bacterium solution.
2. Agrobacterium-mediated Transformation infects and regrowth culture：
Picking Agrobacterium positive colony is in YEB (or YEP) fluid nutrient medium (Rif containing 50mg/L and 50mg/L Kan), and 28
DEG C, 180rpm cultivates to OD600=0.6-0.8.Bacterium solution is taken, in 1% ratio, is transferred to the Liquid Culture of fresh antibiotic-free
In base, add 100-500 μM of AS (can add can be not added), 28 DEG C, 180rpm culture about 4h to OD600=0.3-0.5 can be used to turn
Change.Bacterium solution is collected by centrifugation, bacterium solution is added in the 50ml AAM culture solution containing 100 μM/L acetosyringone, is shaken up, stands
Suspension 1h.
The Agrobacterium bacterium solution of above-mentioned processing is added in the culture bottle of sterilizing in the callus for preculture of learning from else's experience, after slightly shaking
30min is stood, co-cultivation base, 25 DEG C of dark culture 2-3d are inoculated in after drying callus on aseptic filter paper.After picking co-cultures
Callus is in wide-mouth culture bottle, with aseptic water washing 3-5 times, shakes every time for several times, until loseing filamentous cell in water.Last
The secondary sterile water with the Bian penicillin of carboxylic containing 250mg/L stands 1h, is subsequently placed on aseptic filter paper and dries.
Callus is transferred to Selective agar medium screening kanamycin-resistant callus tissue, transfers 1 time, is screened altogether three times every two weeks.Every two weeks will
Callus is transferred on new Selective agar medium, about needs three weeks i.e. visible warty foresythia kanamycin-resistant callus tissues long from the shrivelled callus of browning
Out.A part that kanamycin-resistant callus tissue is selected after callus is grown up is transferred on differential medium.Callus starts to turn green after 2 weeks, after 3 weeks
It can put out new shoots, subsequent root is also grown.Seedling is moved on root media, 1, every culture bottle clone.It takes root length to seedling
Cheng Hou removes culture bottle, after cleaning the culture medium on root, moves to greenhouse pot culture.
3. the positive identification of transgenic paddy rice：
The total DNA and RNA of transgenic paddy rice blade are extracted, PCR and RT-PCR detection is carried out.
PCR reaction system is：
10×Buffer 2μL；dNTP 2μL(2.5mmol/L)；1 μ L (50ng) of template；0.5 μ L (10 μ of upstream primer
mol/L)；0.5 μ L of downstream primer (10 μm of ol/L)；0.5 μ L (10000U/mL) of Taq polymerase；ddH2O adds to 20 μ L systems.
Reaction condition is：94 DEG C of initial denaturation 5min；94 DEG C of 30s, 56 DEG C of 30s, 72 DEG C of 40s, 26 circulations；Then 72 DEG C
10min.4 DEG C of preservations.
Using the DNA of extraction as template, PCR reaction result as shown in figure 3, B1, B2, B3, B5, B8, B9, B10 etc. all
There is the band of purpose genetic fragment, it was demonstrated that target gene has been transferred in rice genome.
In order to which testing goal gene has transcriptional expression in rice in mRNA level in-site, we extract transgenic rice plant
RNA carry out RT-PCR detection.BdRHP1 transgenic paddy rice total serum IgE agarose gel electrophoresis testing result is as shown in figure 4, mentioned
RNA structural integrity meets next RT-PCR testing requirements.BdRHP1 transgenic paddy rice RT-PCR testing result such as Fig. 5 institute
Show, according to RT-PCR as a result, transgenic rice plant (B2, B3, the B8, B9) rna level selected has the table of BdRHP1 gene
It reaches.RT-PCR detects the primer reacted：
Upstream primer：5'-GATGGGCATCTCCAGC-3'；
Downstream primer：5'-GCTAGTAGTGAATCAG-3'.
The drought resistance of 4 Transgenic Rice Seedlings phase of embodiment is tested
The stopping in two weeks after insemination and emergence of wild rice and transgenic paddy rice is watered, and it is real to carry out natural drought stress
It tests.Not stop watering within two weeks after emergence, continue the conduct control for keeping watering.Stop before supplying water, in each culture vessel
Soil is identical with water tariff collection, carries out the measurement of soil moisture content daily and observes the variation of plant.It is wild before Osmotic treatment
The growth of type and transgenosis and phenotype do not have difference, and after continuous drought 10 days, the blade of WT lines occurs wilting and clot
Phenotype, and the blade of transgenic plant is intact, until wilting symptom similar with WT lines occur in 14 talentes.The arid side of body
After compeling 19 days, the blade and plant drought severity of WT lines are significantly greater than transgenic plant (Fig. 6).Blade it is aqueous
Rate measurement display transgenic plant is able to maintain higher leaf water content after meeting with drought stress, under drought stress conditions,
The leaf water content of WT lines is 42.5%, and the average moisture content of transgenic plant is 58.7%, and transgenic plant contains
Water rate increases significantly (t is examined, P < 0.05) (Fig. 8) compared with wild type.After resuming water supply 5 days, observes WT lines and turn
The restoration ecosystem situation of gene plant, the survival rate of transgenic plant are apparently higher than WT lines, the survival of transgenic plant
Rate is 51.6%, and the survival rate of WT lines is 24.5% (t is examined, P < 0.05) (Fig. 7).It is wild in order to further compare
The drought-resistant ability of type and transgenic plant, we detect the percentage of water loss of wild type and transgenic plant excised leaf, can be with from Fig. 9
Find out, transgenic plant has stronger water holding capacity.
The drought resistance in 5 transgenosis boot stage of embodiment is tested
For the drought-resistant ability of integrated survey wild type and transgenic plant, we select water requirement of rice most urgent and quick
The period of sense carries out Osmotic treatment of cutting off the water supply.At preceding ten days of boot stage, start not pour water process, until maintaining restore again for 28 days
It supplies water, observes wild type and the phenotype and booting of transgenic plant, heading and plant development situation.Wild type is recorded simultaneously and is turned
Growth, development and mature condition of the gene plant under the conditions of normal water supply, and make correlation statistical analysis.It can from Figure 10
It arrives, after drought stress 28 days, heading rate is significantly increased transgenic plant than wild type, and wild type heading rate is 30%, turns
The heading rate of gene plant is 52.5%.The spike length of transgenic plant is 13.7cm, and the spike length of WT lines is 11.1cm；Turn
The grain number per spike of gene plant is 85, and the grain number per spike of WT lines is 62 (t is examined, P < 0.05)；Transgenic plant drought stress
Setting percentage afterwards is 52.2%, and the setting percentage of WT lines is 36.5%；Grain number per spike, spike length, setting percentage Isoquant refer to simultaneously
Mark is all remarkably higher than wild type, and grain number per spike, spike length, setting percentage Isoquant index are all remarkably higher than wild type (Figure 11) simultaneously.Dry
Under non-irrigated stress conditions, the single plant yield of transgenic plant is 5.85g, wild type 2.90g, is increased significantly (t compared with wild type
Examine, P < 0.05), production loss is few (Figure 12).
The above experiment shows to meet under drought stress conditions, is overexpressed the wound of the transgenic plant drought stress of BdRHP1
Evil degree is low compared with wild type, and restoration ecosystem is more preferable than wild type after rehydration, and the survival rate of corresponding plant is higher；In reproductive growth
Period especially needs water most urgent sensitive boot stage to meet with a degree of drought and water shortage processing, the booting of transgenic line
It blossoms and bears fruit and is performed better than than wild type.The spike length of transgenic line is longer than wild type, and the grain number of every fringe is more than wild type, solid
Rate is higher than wild type, while the yield after drought stress is higher than wild type.Illustrate the plant for being overexpressed two fringe false bromegrass BdRHP1
Strain, can significantly improve drought-resistant ability.
The foregoing is only a preferred embodiment of the present invention, is not intended to restrict the invention, for the skill of this field
For art personnel, the invention may be variously modified and varied.All within the spirits and principles of the present invention, made any to repair
Change, equivalent replacement, improvement etc., should all be included in the protection scope of the present invention.
SEQUENCE LISTING
<110>Anqing normal university
<120>A kind of drought resisting protein, the nucleic acid molecules of separation and application
<160> 2
<170> PatentIn version 3.5
<210> 1
<211> 477
<212> DNA
<213>Artificial sequence
<400> 1
atgggcatct ccagcatgcc ggcgcccaag gacagcctct tcatcttcct cctgtacaac 60
acggcggtgt ccatcgccgt gctctccaac ctgctgcgcg gcgccatggc cttcctcggc 120
atccccgtgc cgggggaaga cggcgacgag cacatcttcg cgatggtggg gtcgtcggct 180
tccacggcgg cggccgcggg gccgagcctg gcggacaggt tccggagcag cttcaggccg 240
gcgctgttcg ggcggcgggc gcacggcgcg gcggcggcgg actgccgcgt gtgcctggcc 300
agcttcgagc cggagtccgt ggtgaaccgc ctcccctgcg gccacctctt ccaccgggac 360
tgcctcgaga agtggctcgg ctacgacaac gccacctgcc cgctctgccg cctccgcctt 420
ctccccgccg ccgccgaccc ctcgccgccg gtcgcgcccg ccctgattca ctactag 477
<210> 2
<211> 158
<212> PRT
<213>Artificial sequence
<400> 2
Met Gly Ile Ser Ser Met Pro Ala Pro Lys Asp Ser Leu Phe Ile Phe
1 5 10 15
Leu Leu Tyr Asn Thr Ala Val Ser Ile Ala Val Leu Ser Asn Leu Leu
20 25 30
Arg Gly Ala Met Ala Phe Leu Gly Ile Pro Val Pro Gly Glu Asp Gly
35 40 45
Asp Glu His Ile Phe Ala Met Val Gly Ser Ser Ala Ser Thr Ala Ala
50 55 60
Ala Ala Gly Pro Ser Leu Ala Asp Arg Phe Arg Ser Ser Phe Arg Pro
65 70 75 80
Ala Leu Phe Gly Arg Arg Ala His Gly Ala Ala Ala Ala Asp Cys Arg
85 90 95
Val Cys Leu Ala Ser Phe Glu Pro Glu Ser Val Val Asn Arg Leu Pro
100 105 110
Cys Gly His Leu Phe His Arg Asp Cys Leu Glu Lys Trp Leu Gly Tyr
115 120 125
Asp Asn Ala Thr Cys Pro Leu Cys Arg Leu Arg Leu Leu Pro Ala Ala
130 135 140
Ala Asp Pro Ser Pro Pro Val Ala Pro Ala Leu Ile His Tyr
145 150 155
Claims (10)
1. a kind of drought resisting protein, which is characterized in that its amino acid sequence is as shown in SEQ ID NO.2.
2. a kind of isolated nucleic acid molecules, which is characterized in that it encodes drought resisting protein described in claim 1.
3. isolated nucleic acid molecules according to claim 2, which is characterized in that its base sequence such as SEQ ID NO.1 institute
Show.
4. a kind of carrier, which is characterized in that it contains isolated nucleic acid molecules described in claim 2 or 3.
5. carrier according to claim 4, which is characterized in that the carrier is pHB, and the nucleic acid molecules pass through BamH I
It is connected on the carrier with Sac I restriction enzyme site.
6. a kind of recombinant cell, which is characterized in that it contains isolated nucleic acid molecules described in claim 2 or 3 or it contains
Carrier described in having the right to require 4 or 5.
7. drought resisting protein described in claim 1, isolated nucleic acid molecules described in claim 2 or 3, claim 4 or 5 institutes
The carrier or recombinant cell as claimed in claim 6 stated are cultivating the application in drought resistant plant variety.
8. application according to claim 7, which is characterized in that the plant be rice, corn, wheat, soybean, cotton or
Sorghum.
9. a kind of method for improving plant drought ability, which is characterized in that it includes：By separation described in claim 2 or 3
Nucleic acid molecules, carrier described in claim 4 or 5 or recombinant cell as claimed in claim 6 import target plant.
10. according to the method described in claim 9, it is characterized in that, the target plant be rice, corn, wheat, soybean,
Cotton or sorghum.
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Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355300A (en) * | 2018-11-26 | 2019-02-19 | 成都中医药大学 | Coptis SDIR transcription factor is improving the application in plant drought |
Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2013056000A1 (en) * | 2011-10-13 | 2013-04-18 | Pioneer Hi-Bred International, Inc. | Drought tolerant genes and methods of use |
| EP2666867A1 (en) * | 2006-07-12 | 2013-11-27 | The Board Of Trustees Operating Michigan State University | DNA encoding ring zinc-finger protein and the use of the DNA in vectors and bacteria and in plants |
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2018
- 2018-08-14 CN CN201810921513.4A patent/CN108913669A/en active Pending
Patent Citations (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| EP2666867A1 (en) * | 2006-07-12 | 2013-11-27 | The Board Of Trustees Operating Michigan State University | DNA encoding ring zinc-finger protein and the use of the DNA in vectors and bacteria and in plants |
| WO2013056000A1 (en) * | 2011-10-13 | 2013-04-18 | Pioneer Hi-Bred International, Inc. | Drought tolerant genes and methods of use |
Non-Patent Citations (6)
| Title |
|---|
| D.-E. ZENG，ET AL: "Overexpression of BdRHP1 improves drought tolerance and reduces yield loss in rice", 《BIOLOGIA PLANTARUM》 * |
| DE-ER ZENG，ET AL: "Overexpression of Arabidopsis XERICO gene confers enhanced drought and salt stress tolerance in rice (Oryza Sativa L.)", 《J. PLANT BIOCHEM. BIOTECHNOL》 * |
| GENBANK: "XM_003572279.4", 《GENBANK》 * |
| GENBANK: "XP_003572327.1", 《GENBANK》 * |
| NORBERT BRUGIÈRE ,ET AL: "Overexpression of RING Domain E3 Ligase ZmXerico1 Confers Drought Tolerance through Regulation of ABA Homeostasis", 《PLANT PHYSIOL》 * |
| 侯 佩 等: "拟南芥XERICO基因诱导表达提高", 《应用与环境生物学报》 * |
Cited By (2)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN109355300A (en) * | 2018-11-26 | 2019-02-19 | 成都中医药大学 | Coptis SDIR transcription factor is improving the application in plant drought |
| CN109355300B (en) * | 2018-11-26 | 2022-01-28 | 成都中医药大学 | Application of coptis SDAR transcription factor in improving drought resistance of plants |
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