CN107827963A - Application of the arabidopsis IDD14 genes in plant drouhgt stress patience is lifted - Google Patents
Application of the arabidopsis IDD14 genes in plant drouhgt stress patience is lifted Download PDFInfo
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Abstract
The invention discloses application of the arabidopsis IDD14 genes in plant drouhgt stress patience is lifted, the SEQ ID No in the IDD14 gene coded sequences table：The protein of amino acid sequence shown in 1.Overexpression arabidopsis IDD14 genes improve the drought tolerance of Arabidopsis plant to the present invention in arabidopsis first, and new thinking is provided to cultivate high drought resisting arabidopsis kind, and also improving drought resistance for other crop utilization homologous gene technologies provides theories integration.The IDD14 genes of application can provide support for the cereal crops such as corn, rice, wheat and other crop drought resistance Journal of Sex Research.
Description
Technical field
The present invention relates to field of plant genetic, and it is more particularly related to a kind of lifting plant arid side of body
Compel the arabidopsis gene of patience and the purposes of the gene.
Background technology
Arid has been global problem, and world's arid, semiarid zone have accounted for more than the 1/3 of land area, and arid is right
The influence of plant accounts for first place in many natural stress factors.Grain loss caused by drought will account for whole natural calamity grains
More than half of loss.Under field conditions (factors), drought stress has not only had a strong impact on crop growth and yield, and limits
The distribution of plant is made.Therefore, the molecular mechanism of Plant Tolerance drought stress is parsed, cultivates high yield, degeneration-resistant new crop varieties
The significant problem highly urgent into one.With the development of molecular biology technology, genetic engineering turns into current germplasm
Resource innovation and the strong weapon of improvement.
Gene currently used for drought stress tolerance engineering mainly includes following several classes.
First, participate in the gene of osmotic protection material (such as proline, mannitol, glycine betaine, trehalose) synthesis.This
A little genes can make plant to synthesize more osmotic adjustments under water stress, to improve the osmotic adjustment energy of plant
Power, so as to strengthen the drought resistance of plant.Key gene such as in rice in overexpression Proline synthesis approach
(P5CS, deltal-pyrroline-5-carboxylatesynthase) improves the drought resistance (Zhu of transfer-gen plant
Deng, Plant Sci, 1998,199：41-48).
Second, the related gene with Scavenger of ROS (ROS, Reactive oxygen species).This genoid
Expression enhancing plant to the Scavenging activity of active oxygen radical, make plant under water stress some enzymes of overexpression (such as SOD,
POD, CAT etc.), effectively to exclude harmful active oxygen radical, so as to improve the ability of cell dehydration tolerance.Such as arabidopsis
Responses of drought stress NAC transcription factor NTL4, can be by being directly incorporated in the promoter regions of ROS synthase genes, drought-induced
Leaf Senescence in promote active oxygen production.In contrast, ROS levels are in the ntl4 mutant for lacking NTL4 genes
Show to reduce, Delaying Leaf-Senescence, and enhancing drought resistance (Lee etc., Plant Journal, 2012,70：831-
844)。
3rd, encode the gene of drought induced-protein.This albuminoid can be divided mainly into two classes, and (i) plays indirect protection
Regulatory protein, mainly including G-protein, calmodulin, protein kinase, phosphatidase, transcription factor protein etc.；(ii) functional protein,
Including ionophorous protein, LEA protein (Late Embryogenesis Abundant protein), heat shock protein, aquaporin
Albumen, osmotic adjustment albumen, metabolic enzyme etc..During drought tolerance, these albumen directly can play important in the cell
Protective effect, improve plant to arid tolerance.Such as the overexpression of LEA genes result in transfer-gen plant to dry
The tolerance enhancing of drought, although its precise mechanism is unclear.LEA protein can also be used as molecular chaperones protection molecule confrontation thin
Cellular damage (Umezawa etc., Current Opinion in Biotechnology, 2006,17：113-122).
4th, controlling gene.This genoid includes the gene related to ABA approach, including ABA biological metabolism dependency basis
Because (such as NCED and ABAox) and the related gene of ABA signal transduction paths are (as encoded bZIP classes, Myb classes, zinc-finger
The gene of class transcription factor).For example, isolated ERF/AP2 class transcription factor families CBF/DREB1 and DREB2 can be with
Cis-acting elements DRE/CRT is combined, and is overexpressed tolerance of the CBF/DREB1 transfer-gen plants to freezing, arid and salt stress
Property increase (Liu etc., Plant Cell, 1998,10: 1391-1406).The trans-acting factor DREB2 of activation form can swash
It is living arabidopsis is improved by the expression of stress inducible gene drought resistance (Sakuma etc., PNAS, 2006,103：18822-
18827).The regulation and control of other endogenous ABAs gene such as RD22 (Abe etc., Plant Cell, 2003,15：63-78)、RD29B
(Fuiita etc., Plant Cell, 2005,17：3470-3488), ABF3 or AREB2/ABF4 (Kang etc., 2002, Plant
Cell, 14：343-357) etc. overexpression can also improve the Osmotic Stress Tolerance ability of genetically modified plants.Model plant intends south
The albumen of its coding of the HRD genes (HARDY) of mustard is AP2/ERF like transcription factors, is mutated in arabidopsis gain-of-function
The drought resistance and salt tolerance of enhancing are shown as in body hrd-D, the genetic transformation is also shown and resisted into crops rice
Phenotype that the consistent WUEL of drought improves (Karaba etc., PNAS, 2007,104： 15270-15275).
Because people are short in understanding to the molecular mechanism of plant drought, drought resisting molecular breeding also bears the character of much blindness.And
And plant drought effect is typically the result of numerous anti-drought gene co expressions, the drought resisting of plant is improved using single-gene strategy
Property in production application DeGrain, if change a gene expression can integrally regulate and control drought tolerance in plants reaction
Ability, that will be a preferably selection.
Early in 1998, Colasanti et al. was found that the gene I/D 1 that a control corn is bloomed
(INDETERMINATE1), its encoding proteins is the transcription factor containing four zinc fingerses.Follow-up study finds multiple transcriptions
The factor contains the functional domain of zinc fingers in similar ID1, is named as INDETERMINATE Domain, these transcriptions because
Son is also designated as IDD.IDDs is the peculiar a kind of transcription factor of plant institute, and corn, rice and arabidopsis contain 21,15 respectively
With 16 IDD genes.Rice Os ID1/Ehd2/RID1 protein sequence has the homology of height, and control with corn ID1
Rice is into colored factor of determination.OsIDD14/LPA1 (Loose PlantArchitecture1) then participate in rice stem to weight
The regulation and control of property and plant type.Different, the IDD bases of nearly half arabidopsis are resolved from the function of rare several IDD in corn and rice
Because being studied.IDD8/NUC (NUTCRACKER) regulates and controls to bloom by adjusting Sucrose Metabolism, IDD3/MAG (MAGPIE),
The development of IDD8/NUC and IDD10/JKD (JACKDAW) regulation and control roots, and IDD1/ENY (ENHYDROUS) participates in regulation and control seed
Ripe and sprouting.
The drought resisting crops that the excavation of excellent anti-drought gene can carry out field production application for cultivation are most important.
The content of the invention
It is an object of the invention to provide a kind of arabidopsis gene, overexpression vector for lifting plant drouhgt stress patience
And the encoding proteins of arabidopsis gene.
To reach above-mentioned purpose, the present invention adopts the following technical scheme that：
The gene of lifting plant drouhgt stress patience provided by the present invention, entitled IDD14, from arabidopsis, is compiled
The following protein of code：SEQ ID No in sequence table：The protein of amino acid sequence shown in 1.
The nucleotide sequence of the arabidopsis IDD14 genes of the lifting plant drouhgt stress patience of the present invention can be the gene
CDS sequences either with the sequence have more than 90% uniformity and encode identical functional protein DNA sequence dna.Sequence
SEQ ID No in table：Shown in 2 is the sequence of IDD14 genes.
The present invention is also provided comprising above-mentioned nucleotide sequence and the expression regulation sequence being operatively connected with the nucleotide sequence
Expression vector.Further, the expression regulation sequence includes the regulating and controlling sequence of the high expression of composing type, can be cauliflower
Mosaic virus 35 S promoter.
Present invention also offers a kind of method for lifting plant drouhgt stress patience.
Expression IDD14 genes can lift the drought stress patience of the plants such as arabidopsis, and this method is by the IDD14
Gene transfered plant cell, tissue or organ, by the plant cell after importing, tissue or organ culture into plant, make described
IDD14 genes are expressed in plant, obtain the plant of drought stress patience lifting.
Further, the IDD14 genes can be that the CDS sequences of the gene either have more than 90% with the sequence
Uniformity and the DNA sequence dna for encoding identical functional protein.There is more than 90% uniformity and coding identical work(with the sequence
The DNA sequence dna of energy albumen is to be separated, modified and/or designed what is obtained by CDS sequences method known to of the gene.
It will be understood by those skilled in the art that gene order change or the method shortened, and test these bases to change
Because the method for validity is well known to those skilled in the art.
The IDD14 genes of the present invention can import plant cell, tissue or organ by plant expression vector.The plant
Expression vector be pVIPMyc (Cui etc., PLOS Genetics, 2013,9：E1003759), pVIPMyc is conventional in the world
Genetic Transformation in Higher Plants carrier, transform and form on the basis of pVIP96.Use the IDD14 genes or its homologous sequence of the present invention
During row component plant expression vector, any composing type or inducible promoter can be added before its transcription initiation nucleotides.
Constitutive promoter can be cauliflower mosaic virus (CAMV) 35S promoter, rice Actin promoters or corn Ubiquitin
Promoter etc.；Inducible promoter can be by low temperature, arid, ABA, ethene, saline and alkaline or the induction such as chemical promoter.It is above-mentioned
Promoter can be used in combination individually or with other plant promoters.
The plant expression vector for carrying above-mentioned IDD14 genes or its homologous sequence can be by agriculture bacillus mediated, Ti matter
The combination of any one of conventional biology methods such as grain, plant viral vector, microinjection, particle gun or several method makes
With conversion plant cell, tissue or organ, and by the plant cell after importing, tissue or organ culture into plant；In the present invention
Seed of the plant tissue or organ being related to comprising plant, bud, Fruit pod, blade, scape etc..Plant can be corn, water
Rice, wheat or arabidopsis.
Present invention overexpression in arabidopsis by the nucleotide sequence of IDD14 genes, the results showed that in 2 independent turns
In gene Arabidopsis plant, the level of IDD14 protein is all apparently higher than wild type.
The present invention has obtained the mutant of a drought resistance enhancing by the screening in arabidopsis activated mutant body storehouse
Idd14-1D, the drought resisting phenotype of the mutant is due to caused by a gene I/D D14 for encoding IDD transcription factors is overexpressed.Cause
This, overexpresses IDD14 genes in arabidopsis, significant for improving arabidopsis drought stress patience, and this is cultivation
High drought resistance new varieties provide new thinking.
The beneficial effects of the present invention are：
(1) the invention provides a kind of application for the gene I/D D14 for improving arabidopsis drought stress patience.The present invention exists
In Arabidopsisecotype Colombia, after overexpressing IDD14 genes, it is found that the drought stress patience of mutant plants substantially carries
It is high.After dehydration and rehydration are handled under similarity condition, it is found that 100% mutant plants can restore normal growth, and it is corresponding
WT lines only 2% or so restore normal growth.
(2) the overexpression arabidopsis IDD14 genes in arabidopsis first of the invention.To cultivate high drought resisting arabidopsis product
Kind provides new thinking, and also improving drought resistance for other crop utilization homologous gene technologies provides theories integration.
(3) the IDD14 genes applied in the present invention can be cereal crops and other crops such as corn, rice, wheat
Study on drought resistance provides support.
Brief description of the drawings
Figure 1A and Figure 1B is idd14-1D plant of the invention and the comparison figure of the drought stress patience of WT lines；
Fig. 2A and Fig. 2 B are the comparison figure of the rate-of-loss of coolant of the blade of idd14-1D plant and WT lines；
Fig. 3 compares figure for the stomatal frequency of idd14-1D plant and WT lines；
Fig. 4 A and Fig. 4 B are the transfer-gen plant of overexpression IDD14 genes and the drought stress patience ratio of WT lines
Compared with figure；
Fig. 5 is to detect T3 for the horizontal result of IDDl4 protein expressions in transgenic positive plant with Western blot
Figure.
Embodiment
In order to make the purpose , technical scheme and advantage of the present invention be clearer, below in conjunction with the accompanying drawings and embodiment,
The present invention will be described in further detail.It should be appreciated that specific embodiment described herein is only to explain the present invention,
It is not intended to limit the present invention.
Following examples define the present invention, and describe the present invention in separation clone's IDD14 genes and authentication function
In used method.According to following description and these embodiments, those skilled in the art can determine that the present invention's is substantially special
Sign, and without departing from the spirit and scope of the invention, various changes and modifications can be made to the present invention, so that
It uses different purposes and condition.
Embodiment 1：Screening overexpression IDD14 gene plants
Mutant idd14-1D used in the present invention be from containing activation label arabidopsis T-DNA mutant libraries (Cui etc.,
PLOS Genetics, 2013,9：E1003759 the mutant for the leaf development exception that screening obtains in), has under obvious leaf
Roll up phenotype.Shown by genetic analysis early stage, idd14-1D is the dominant mutant of a single T-DNA insertion, mutant
Idd14-1D phenotype is as caused by IDD14 overexpression.
Embodiment 2：IDD transcription factor subfamily positive regulation arabidopsis drought stress tolerance reacts
Tested using Osmotic treatment, isolated chloroplasts measure, Stoma of Leaves density comparative analysis overexpression
Tolerance response of the idd14-1D mutant plants and control WT lines of IDD14 genes to drought stress.
(1) the idd14-1D mutant plants for overexpressing IDD14 genes have stronger drought stress patience
The seed of a certain amount of idd14-1D mutant plants and wild-type Arabidopsis plants is weighed, after sterilization, 4 DEG C low
Temperature 2~3 days, broadcasts continuous illumination culture 7 days on MS culture mediums by seed, transplants seedlings.Vermiculite: Nutrition Soil is claimed by 1: 1 mixing
Identical weight Nutrition Soil (about 70~85 grams) is taken in the smooth small basin in bottom, after bottom watering makes soil complete wetting, per small
Basin plants 9 seedlings, epiphragma, often 12 small basins of disk.Growth room's condition of culture is：21 DEG C of (light)/18 DEG C (dark), photoperiod：12 h
(light)/12h (dark), intensity of illumination are 80~120 μm of olm-2·s-1, relative humidity 50% or so.Film is taken off after 3 days, is continued
Growth 7 days, choose the consistent small basin of growth and carry out Osmotic treatment, the small basin of different strains is randomly placed, often converts small basin
Position, reduce position effect.Identical experiment is repeated 3 times the above, the results showed that, after stopping watering 20 days, wildtype Arabidopsis thaliana is planted
There is wilting phenomenon earlier than the idd14-1D mutant plants for overexpressing IDD14 genes in strain, is wilted to all plant
After phenomenon, add water recover 3 days after statistical result showed, absolutely overexpress IDD14 genes idd14-1D mutant plant
Strain can restore normal growth, and corresponding WT lines only 2% or so restore normal growth (as seen in figs. 1 a-1b, its
Middle Figure 1A is WT (wild-type Arabidopsis plants) and idd14-1D (the idd14-1D mutant plants of overexpression IDD14 genes)
Control, Osmotic treatment and rehydration phenotype.Figure 1B be WT and idd14-1D rehydrations after survival rate, * * represent each genetic stocks with
WT, which is compared, has pole significant difference (p ＜ 0.01)).
(2) overexpressing the rate-of-loss of coolant of the idd14-1D mutant plants blades of IDD14 genes reduces
Excised leaf dehydration experiment specific method is as follows：Idd14-1D mutant plants and wild-type Arabidopsis plants
Low temperature 3 days after seed is sterilized, broadcasts on 1/2MS culture mediums, after continuous illumination culture 7 days, moves into soil, at 21 DEG C
(light)/18 DEG C (dark), about surrounding is grown in 12h (light)/12h (dark) growth room.Before not blooming, overground part is cut off immediately
It is put into culture dish and weighs, is placed on progress leaves water loss experiment under the conditions of 22 ± 1 DEG C of temperature, is claimed at interval of certain time
Weight, each strain are repeated 4 times, and leaves water loss speed is calculated to lose the percentage of initial fresh weight.(Fig. 2A as seen in figs. 2a-2b
Represent WT and idd14-1D excised leaf dehydration phenotypes, Bar=1cm；Fig. 2 B represent WT and idd14-1D excised leaf dehydrations
Rate.* represent each genetic stocks and significant difference (p ＜ 0.05) compared with WT be present), the results showed that overexpression IDD14 genes
The rate-of-loss of coolant of idd14-1D mutant plants excised leafs is substantially less than WT lines (p ＜ 0.05).
(3) overexpressing the stomatal frequency of the idd14-1D mutant plants of IDD14 genes reduces
The lotus that the same area of 5~6 weeks idd14-1D mutant of clip and wild-type Arabidopsis plants is fully deployed respectively
Seat blade (being grown under 12h (light)/12h (dark)), is placed in buffer solution.Each processing takes 3 blades to do parallel reality
Test, epidermis bar of tearing respectively, brush away mesophyll cell, in 10 × 20 times of Leica optical microphotograph Microscopic observations.Each epidermis is random
Take 3 to 5 visuals field, measurement and record of stomatal number.Each processing repeats 4~6 times, counts its stoma number average value and parallel laboratory test
In error.(wherein idd14-1D represents the idd14-1D mutant plants of overexpression IDD14 genes as shown in Figure 3.* generations
Pole significant difference (p ＜ 0.01) compared with WT (wild-type Arabidopsis plants) be present in each genetic stocks of table), the results showed that it is super
The stomatal frequency for expressing the idd14-1D mutant plants blades of IDD14 genes is less than WT lines, and this species diversity pole
Significantly (p ＜ 0.01).
Guard cell's stomatic observation buffer solution：
500mM KCl(10×)：It is 3.7275g molten to 100mL H2In O.
5.0mM CaCl2(50×)：0.0555g is molten to 100mL H2In O.
100mM Mes/KOH(10×)：In the molten distilled waters to 100mL of 1.952g, pH to 6.1 is adjusted with 1.0M KOH.
1.0M KOH：In the molten distilled waters to 100mL of 5.61g.
Embodiment 3：Separation clone is used for the DNA fragmentation for building IDD14 gene plant expression vectors
Total serum IgE is extracted from the blade of arabidopsis using TRIzol reagents (buying from Invitrogen companies).Specific step
It is rapid as follows：Precooling centrifuge, take 50~100mg samples to add liquid nitrogen and be fully ground, add 1mL TRIzol solution, at room temperature
Stand 5min；200 μ L chloroforms are added, 15s is acutely shaken, stands 3min at room temperature；4 DEG C, 12,000g centrifugation 15min；Draw
Supernatant adds 1 times into new 1.5mL centrifuge tubes (RNase-free, deoxyribonuclease) (buying from AXYGEN companies)
The isopropanol of volume, after mixing, 10min is stood at room temperature；4 DEG C, 12,000g centrifugation 10min；Supernatant is outwelled, is added
1mL70% ethanol solutions, precipitation is upspring；4 DEG C, 7,500 g centrifugations 5min；Supernatant is outwelled, remaining second is sucked after centrifuging in short-term
Alcohol；After room temperature under shed places 10 min, add 50 μ L RNase-free water and fully dissolved, utilize ultraviolet spectrometry
Photometric determination RNA concentration.
Its reverse transcription is comprised the following steps that into cDNA using reverse transcriptase (buying from Invitrogen companies)：Successively
Add 2 μ g total serum IgEs, 1 μ l 10 × digestion buffer solution, 1 μ L DNase I (deoxyribonuclease I) (RNase-free) and
DEPC water (treated and through autoclave sterilization the MiliQ pure water with pyrocarbonic acid diethyl ester) is prepared into DNA to 10 μ l and digested instead
Liquid is answered, is placed in 37 DEG C of digestion half an hour, adds 1 μ L 25mM EDTA, 65 DEG C of inactivation processing 10min；Add into digestion product
Enter 1 μ L 50mM Oligo (dT) 18 and 1 μ L 10mM dNTP (deoxy-ribonucleoside triphosphate, deoxidation
Ribonucleotide triphosphate), 65 DEG C of denaturation 5min are placed in after mixing, reaction is immediately placed in ice bath after terminating and cools down at least 1min.
Then the chain buffer solution of 4 μ l, 5 times of density controls one, 1 μ l 0.1M DTT, 0.4 μ l RNase (ribalgilase) is sequentially added to suppress
Agent and 0.6 μ L reverse transcriptase (200U/ μ L), 1h is incubated after mixing at 50 DEG C.Then 70 DEG C of water-baths inactivate enzyme in 15 minutes, eventually
Only react, thus synthesized the first chain cDNA, using the first chain cDNA as template amplification target gene.
With with restriction enzyme site sense primer IDD14F (5 '-gggcccccATGCATAGAAGACGACATAAAG-3 ',
The additional ApaI sites of sequence specific primers and two protection bases, SEQ ID NO：3) and anti-sense primer IDD14R (5 '-
GagctccTGAAGATGCTCTATCACTCG-3 ', the additional XhoI sites of sequence specific primers and a protection base, SEQ ID
NO：4).Amplification purpose fragment, PCR reactions are carried out using high-fidelity Phusion archaeal dna polymerases (buying from Thermo companies)
Condition is 98 DEG C of pre-degenerations 30 seconds；98 DEG C 10 seconds, 51 DEG C 30 seconds, 72 DEG C 30 seconds, 30 circulation；72 DEG C 10 minutes.Agarose
Detected through gel electrophoresis purpose band, corresponding purpose is reclaimed using DNA gel QIAquick Gel Extraction Kit (buying from Vigorous companies)
Band；Connect sequencing vector(buying from TransGen companies).Screening positive clone is simultaneously sequenced, and obtains
Required DNA fragmentation, it is pEASY-IDD14cDNA by the clone designation.
Embodiment 4：The structure and genetic transformation of IDD14 gene 35S overexpression vectors
In order to preferably analyze the function of IDD14 genes, applicant overexpresses technology by 35S makes IDD14 genes exist
Expression rise in arabidopsis.The function of the gene is studied according to the phenotype of transfer-gen plant and physiological characteristic.35S surpasses
The construction method for expressing IDD14 gene plant expression vectors is as follows：Use cauliflower mosaic virus (CAMV) 35S promoter；It is first
The positive colony pEASY-IDD14cDNA ApaI and XhoI double digestions that will first be obtained in embodiment 3, reclaim Insert Fragment；
Equally, with same method digestion pVIPMyc plant expression vector, carrier segments are reclaimed.With the Insert Fragment and load of recovery
Body fragment does coupled reaction, converts bacillus coli DH 5 alpha.By digestion screening positive clone, plant expression vector, name are obtained
For pVIPMyc-IDD14.PVIPMyc be in the conventional Genetic Transformation in Higher Plants carrier in the world, be transformed on the basis of pVIP96 and
Into.PVIPMyc-IDD14 is converted to EHA105 Host Strains (EHA105 Host Strains are one kind of Agrobacterium).
It is conducted into by agriculture bacillus mediated arabidopsis genetic transformation in arabidopsis Colombia type, conversion obtains altogether
Obtain 30 plants of independent transgenic Arabidopsis plants.Specific steps：The flower opened on plant that bolting is bloomed and silique are cut,
Retain unopened bud；The Agrobacterium inoculation of plant will be converted to the LB (Luria-Bertani) containing corresponding antibiotic
In fluid nutrient medium, 28 DEG C of 220rpm are incubated overnight to OD600About 1.8；Room temperature 6,000rpm centrifugations 10min collects thalline；
Supernatant is outwelled, isometric dip dyeing liquid for shell (5.0% sucrose solution+0.025% (v/v) Silwet-L77) is added and thalline is resuspended；Weight
The bacterium solution hanged is put into culture dish, and the scape of arabidopsis is immersed in dip dyeing liquid for shell, fully immersion；Convert the plant finished
Water and cover moisturizing with polybag, remove polybag after about 24h and normally cultivate；Converting the material after 3 weeks will be slowly ripe,
Collect transgenic line seed, on the 1/2MS culture mediums of the resistance containing respective carrier screening obtain T1 for transgenic positive seedling,
T1 is further selfed for material, be may determine whether to insert for single copy for Resistant segregation ratio by T2, is then obtained in T3 generations
Homozygous transgenic line.
As shown in figs. 4 a-4b, it is above-mentioned IDD14 gene transgenics plant and the drought stress patience ratio of WT lines
Compared with figure.Wherein 35S-IDD14 represents the transfer-gen plant of overexpression IDD14 genes.Fig. 4 A are at WT and 35S-IDD14 arids
Reason and rehydration phenotype, 12-1 and 3-2 represent different positive transgenic strains, Bar=1cm.Fig. 4 B be WT and 35S-IDD14 not
With the survival rate after positive transgenic strain rehydration, * * represent each genetic stocks and significant difference (p ＜ in pole compared with WT be present
0.01).Choose and grow consistent transfer-gen plant and WT lines progress Osmotic treatment, the small basin of different strains is random
Place, often convert small basin position, reduce position effect.Identical experiment is repeated 3 times the above, the results showed that, stop watering 20 days
Afterwards, WT lines occur wilting phenomenon than transfer-gen plant earlier, after there is wilting phenomenon to all plant, add water extensive
Statistical result showed after multiple 3 days, Fig. 4 B show that the recovery rate of 35S-IDD14 plant reaches 100%, i.e., absolutely surpassed
The transfer-gen plant of expression IDD14 genes can restore normal growth, and corresponding WT lines only 2% or so recover just
It is frequently grown.
Gene I/D D14 is connected into the sequence of PVIPMyc carriers：
IDD14ApaI F 5′-gggcccccATGCATAGAAGACGACATAAAG-3′
IDD14XhoI R 5′-gagctccTGAAGATGCTCTATCACTCG-3′
52.1 DEG C/50.6 DEG C PCR primer length of Tm：999bp
Embodiment 5：Detect the IDD14 protein levels of transfer-gen plant and wildtype Arabidopsis thaliana
With arabidopsis Colombia wild type and the 2 independent T3 obtained by embodiment 4 for transgenic Arabidopsis plants
For material, the soluble protein of blade is extracted, IDD14 protein water in Arabidopsis leaf is detected using Western blot
It is flat.Specific method is as follows：2g blades are collected from above-mentioned material, powder is fully ground into liquid nitrogen, appropriate protein is added and carries
Buffer solution (0.5M Tris-MES, pH=8.0,0.5mM EDTA and protease inhibitor cocktail) is taken, mixes, is put on ice
Put 30 minutes.Then 4 DEG C of 12000g is centrifuged 20 minutes, after supernatant filter-cloth filtering subzero 80 DEG C freeze it is standby.Utilize 10%
SDS-PAGE carries out protein electrophorese.After electrophoresis terminates, by blotting be transferred to pvdf membrane (Millipore,
Billerica, MA).Then it is horizontal by detecting the MYC label proteins matter merged with IDD14 protein, to reflect
IDD14 protein expressions.(wherein 35S-IDD14 represents to turn the plant of IDD14 genes as shown in Figure 5；12-1 and 3-2 generations
Table difference positive transgenic strain), in 2 independent transgenic Arabidopsis plants (12-1 and 3-2), IDD14 protein
Level is all apparently higher than arabidopsis Colombia WT lines.
Mutant idd14-1D used in the present invention is screened from the arabidopsis T-DNA mutant libraries containing activation label
The abnormal mutant of the leaf development of acquisition, has obvious leaf last volume phenotype.Shown by genetic analysis early stage, idd14-1D
It is the dominant mutant of a single T-DNA insertion, its phenotype is as caused by IDD14 overexpression.In the T3 generations of mutant
Found in plant, the drought stress patience of Arabidopsis plant is apparently higher than WT lines：Dehydration and rehydration under similarity condition
After processing, finding 100% overexpression IDD14 gene plants can restore normal growth, and corresponding WT lines are only
Only 2% or so restore normal growth.Therefore, IDD14 genes are overexpressed in arabidopsis, for improving arabidopsis drought stress
Patience is significant, and the cereal crops such as this is corn, rice, wheat and other crop drought resistance Journal of Sex Research provide support.
All reagents, operating procedure and method used in the present invention be the common agents of this area, operating procedure with
Method.
Presently preferred embodiments of the present invention is the foregoing is only, is not used for limiting the practical range of the present invention；If do not take off
From the spirit and scope of the present invention, the present invention is modified or equivalent substitution, all should covered in the claims in the present invention
Among protection domain.
Sequence table
<110>Shandong college of education
<120>Application of the arabidopsis IDD14 genes in plant drouhgt stress patience is lifted
<160> 4
<170> PatentIn version 3.3
<210> 1
<211> 333
<212> PRT
<213>Arabidopsis
<400> 1
Met His Arg Arg Arg His Lys Val Pro Trp Lys Leu Leu Lys Arg Glu
1 5 10 15
Thr Asn Glu Glu Val Arg Lys Arg Val Tyr Val Cys Pro Glu Pro Thr
20 25 30
Cys Leu His His Asn Pro Cys His Ala Leu Gly Asp Leu Val Gly Ile
35 40 45
Lys Lys His Phe Arg Arg Lys His Ser Asn His Lys Gln Trp Ile Cys
50 55 60
Glu Arg Cys Ser Lys Gly Tyr Ala Val Gln Ser Asp Tyr Lys Ala His
65 70 75 80
Leu Lys Thr Cys Gly Thr Arg Gly His Ser Cys Asp Cys Gly Arg Val
85 90 95
Phe Ser Arg Val Glu Ser Phe Ile Glu His Gln Asp Thr Cys Thr Val
100 105 110
Arg Arg Ser Gln Pro Ser Asn His Arg Leu His Glu Gln Gln Gln His
115 120 125
Thr Thr Asn Ala Thr Gln Thr Ala Ser Thr Ala Glu Asn Asn Glu Asn
130 135 140
Gly Asp Leu Ser Ile Gly Pro Ile Leu Pro Gly His Pro Leu Gln Arg
145 150 155 160
Arg Gln Ser Pro Pro Ser Glu Gln Gln Pro Ser Thr Leu Leu Tyr Pro
165 170 175
Phe Val Thr Asn Gly Ser Ile Glu Leu Gln Leu Leu Pro Ser Arg Asn
180 185 190
Cys Ala Asp Glu Thr Ser Leu Ser Leu Ser Ile Gly Thr Met Asp Gln
195 200 205
Lys Thr Met Ser Glu Val Glu Lys Lys Ser Tyr Glu Lys Gly Glu Thr
210 215 220
Ser Leu Glu Arg Glu Glu Ala Arg Arg Glu Thr Lys Arg Gln Ile Glu
225 230 235 240
Ile Ala Glu Leu Glu Phe Ala Glu Ala Lys Arg Ile Arg Gln His Ala
245 250 255
Arg Ala Glu Leu His Lys Ala His Leu Phe Arg Glu Glu Ala Ser Arg
260 265 270
Arg Ile Ser Ala Thr Met Met Gln Ile Thr Cys His Asn Cys Lys Gln
275 280 285
His Phe Gln Ala Pro Ala Ala Leu Val Pro Pro Pro Pro Gln Thr His
290 295 300
Cys Thr Asp Glu Ser Thr Ser Leu Ala Val Ser Tyr Met Ser Ser Ala
305 310 315 320
Thr Thr Glu Gly Glu Lys Ala Ser Asp Arg Ala Ser Ser
325 330
<210> 2
<211> 1002
<212> DNA
<213>Arabidopsis
<400> 2
atgcatagaa gacgacataa agttccatgg aagcttctta aaagagagac caacgaggaa 60
gtgaggaaaa gagtctacgt ctgtccggag ccgacatgtc tccaccacaa cccttgccac 120
gcgctcggag atctcgtggg aatcaagaaa cacttccgac ggaaacatag taaccacaag 180
caatggatct gcgagcgatg ctcaaaaggc tacgcagttc aatctgatta caaagctcat 240
ctcaaaacct gtggcactcg cggccactcc tgtgactgcg gccgagtttt ttccagagtg 300
gaaagtttca tagagcacca agacacttgc accgtacgcc gatcccaacc ctccaaccac 360
cgtttacatg agcagcaaca acataccaca aacgctacac aaaccgcttc aaccgcggaa 420
aacaacgaga acggggacct ctccattggt cctatattgc ctggacatcc tttacaaaga 480
agacaatccc caccgtcgga acaacaacca tccactttgc tctatccctt cgttactaat 540
ggtagtatcg agcttcagct acttccatcg aggaattgtg ctgatgagac cagccttagt 600
ctgtctatag ggacaatgga tcaaaagaca atgtcggaag ttgagaagaa gagctacgag 660
aagggagaaa cgagcctaga aagagaggag gcgagaagag aaacaaagag gcagatcgaa 720
atcgcggaat tggagtttgc tgaagccaag agaataaggc aacatgcgag agctgagctt 780
cacaaagctc atctttttag agaagaagca agtaggagga ttagtgcaac gatgatgcaa 840
ataacttgcc acaattgcaa gcaacatttt caagctccgg ctgctttggt tcctcctcct 900
cctcagacgc attgtaccga tgagagcacg tctctggccg tgagctacat gtcttcggcg 960
actaccgaag gagaaaaggc gagtgataga gcatcttcat ag 1002
<210> 3
<211> 30
<212> DNA
<213>Artificial sequence
<400> 3
gggcccccat gcatagaaga cgacataaag 30
<210> 4
<211> 27
<212> DNA
<213>Artificial sequence
<400> 4
gagctcctga agatgctcta tcactcg 27
Claims (9)
1. application of the arabidopsis IDD14 genes in plant drouhgt stress patience is lifted, in the IDD14 gene coded sequences table
SEQ ID No：The protein of amino acid sequence shown in 1.
2. application as claimed in claim 1, it is characterised in that the sequence of the IDD14 genes is the CDS sequences of the gene.
3. application as claimed in claim 2, it is characterised in that SEQ in the nucleotide sequence of the IDD14 genes such as sequence table
ID No：Shown in 2.
4. the application as described in claim any one of 1-3, it is characterised in that by the IDD14 gene transfered plant cells, group
Knit or organ, by the plant cell after importing, tissue or organ culture into plant, the IDD14 genes is expressed in plant,
Obtain the plant of drought stress patience lifting.
5. application as claimed in claim 4, it is characterised in that the IDD14 genes import plant by plant expression vector
Cell, tissue or organ.
6. application as claimed in claim 5, it is characterised in that the plant is corn, rice, wheat or arabidopsis.
7. application as claimed in claim 5, it is characterised in that the plant expression vector is pVIPMyc.
8. application as claimed in claim 5, it is characterised in that the plant expression vector passes through a kind of composing type or induction type
Promoter drives the expression of the IDD14 genes.
9. application as claimed in claim 8, it is characterised in that cauliflower mosaic virus is used in the plant expression vector
35S promoter drives the expression of the IDD14 genes.
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|---|---|---|---|---|
| CN110387382A (en) * | 2018-04-13 | 2019-10-29 | 中国农业大学 | Gene A RR5 is improving the application in plant drought ability |
Citations (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN101508728A (en) * | 2009-04-02 | 2009-08-19 | 中国农业大学 | Drought tolerant associated protein for plant, encoding gene and uses thereof |
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2017
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|---|---|---|---|---|
| CN101508728A (en) * | 2009-04-02 | 2009-08-19 | 中国农业大学 | Drought tolerant associated protein for plant, encoding gene and uses thereof |
Non-Patent Citations (3)
| Title |
|---|
| DAYONG CUI等: "The Arabidopsis IDD14, IDD15, and IDD16 Cooperatively Regulate Lateral Organ Morphogenesis and Gravitropism by Promoting Auxin Biosynthesis and Transport", 《PLOS GENETICS》 * |
| GENBANK: "indeterminate(ID)-domain 14 protein [Arabidopsis thaliana]", 《GENBANK》 * |
| PIL JOON SEO等: "Two splice variants of the IDD14 transcription factor competitively form nonfunctional heterodimers which may regulate starch metabolism", 《NATURE COMMUNICATIONS》 * |
Cited By (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN110387382A (en) * | 2018-04-13 | 2019-10-29 | 中国农业大学 | Gene A RR5 is improving the application in plant drought ability |
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