CN103421784A - Identification and utilization of drought and high-salt induced paddy rice promoter PDS1 - Google Patents

Abstract

The invention belongs to the technical field of paddy rice gene engineering, and particularly relates to application of a drought and high-salt induced paddy rice promoter PDS1 which is obtained through separation, clone and function verification to the low-temperature resistant genetic improvement of paddy rice. The nucleotide sequence of the PDS1 gene promoter is shown in SEQ ID NO: 1. Stress expression profile data analysis shows that the PDS1 gene is subject to strong high-salt inducible expression. The low-temperature induced paddy rice promoter PDS1 is obtained through cloning by adopting the PCR method, a DX2181GFP-a-PDS1 expression vector is built, enzyme activity reporting the gene protein GUS of paddy rice middle flower 11 under drought and high-salt stress is transformed, and that the promoter is subject to strong drought and high-salt inducible expression is testified.

Description

Evaluation and the utilization of rice drought and high salt inducible promoter PDS1

Technical field

The invention belongs to the plant gene engineering technology field.Relate to isolation identification and the application of the special inducible promoter of a kind of plant adverse circumstance.By separating and identify the promotor of a rice drought and high salt inducible genes, can be applied to, in the genetically engineered of plant, particularly improve the purpose of plant drought resistance and salt tolerance to reach genetic modification of plants.

Background technology

Along with global environment worsens, climatic anomaly, drought stress is one of major reason caused crop failure, is day by day endangering grain-production safety.For the country of the such shortage of fresh water of China, arid high salt pair agriculture production, even social life has been caused to extremely serious impact.Improve plant drought resistance and salt resistance all the time section be a long-term and difficult task.Along with molecular biological development, transgenosis has become the effective means of research functional genomics, and just progressively obtains in recent years feasibility study and generally accept by transgenosis approach improvement stress resistance of plant.There is the lot of documents report can improve to a certain extent resistance (Saijo etc., the Over-expression of a single Ca of plant by overexpression Stress response genes involved in plant ²⁺-dependent protein kinase confers both cold and salt/drought tolerance on rice plants.Plant J23:319-327,2000; Zhang etc., Two cysteine proteinase inhibitors from Arabidopsis thaliana, AtCYSa and AtCYSb, increasing the salt, drought, oxidation and cold tolerance.Plant Mol Biol68 (1-2): 131-143,2008; Hou etc., A homolog of human ski-interacting protein in rice positively regulates cell viability and stress tolerance.Proc Natl Acad Sci106 (15): 6410-6415,2009; Ma etc., Enhanced tolerance to chilling stress in OsMYB3R-2transgenic rice is mediated by alteration in cell cycle and ectopic expression of stress genes.Plant Physiol150 (1): 244-56,2009; Asano etc., Functional characterisation of OsCPK21, a calcium-dependent protein kinase that confers salt tolerance in rice.Plant Mol Biol75,179-191.2011).But the common use of these overexpressions in report is all constitutive promoter, although constitutive promoter overexpression effect is high, usually can be accompanied by inevitable negative effect.Although the promotor (Yamaguchi etc. that had bibliographical information to isolate from plant to be subject to Stress response, Characterization of the expression of adesiccation-responsive rd29gene of Arabidopsis thaliana and analysis of its promoter in transgenic plants.Mol Gen Genet23:331-340,1993; Kasuga etc., A combination oftheArabidopsis DREB1A gene and stress-induciblerd29A promoter improved drought-and low-temperature stress tolerance in tobacco by gene transfer.Plant Cell Physiol45:346-350,2004; Xiao etc., Over-expression ofa LEA gene in rice improves drought resistance under the field conditions.TheorAppl Genet115 (1): 35-46,2007), even but rare very strong adverse circumstance inducible promoter is applied to plant genetic engineering in important food crop paddy rice.

The present invention identifies a rice starter PDS1 who is subject to Drought and salt to coerce induced strong, the active level of adverse circumstance express spectra by native gene gene DS1 and the promotor PDS1 of this gene the analysis showed that, this promotor can be used for to the expression of resistance genes involved crops such as paddy rice, thereby effectively improve the degeneration-resistant border performance of plant.

Summary of the invention

The objective of the invention is the clone, identify a plant endogenous promotor that is subject to arid and high-salt stress abduction delivering, and utilize this promotor to build the anti-salt-related gene expression vector of drought resisting, reach and control the purpose that target gene is expressed by adverse circumstance specifically by genetic transforming method.

The present invention implements by the following technical programs:

At first be the promotor of separating the candidate gene that is subject to arid and the expression of high salt induced strong.Selected candidate gene is the paddy rice native gene, its concrete function also be not in the news (KOME annotation AK073435).The promotor of this gene separated derives from rice varieties " Japan is fine ", and we are by its called after PDS1.The PDS1 promotor is the sequence with base 1-2082 position in accompanying drawing 1, contain the ABA response element ABREATCONSENSUS (Choi etc. relevant to adverse circumstance in 447-453,1187-1193 base site, ABFs, a family ofABA-responsive element binding factors J Biol Chem.275:1723-1730,2000, Kang etc., Arabidopsis basic leucine zipper proteins that mediate stress-responsive abscisic acid signaling Plant Cell.14:343-357,2002, Oh etc., Arabidopsis CBF3/DREB1A and ABF3in transgenic rice increased tolerance to abiotic stress without stunting growth.Plant Physiology138:341-351,2005, Choi etc., Arabidopsis Calcium-Dependent Protein Kinase AtCPK32 Interacts with ABF4, a Transcriptional Regulator of Abscisic Acid-Responsive Gene Expression, and Modulates Its Activity.Plant Physiol.139:1750-1761, 2005), at 450-454, 1188-1192, 1446-1450, the ABA response element ABRELATERD1 (Simpson etc. relevant to adverse circumstance are contained in 1948-1952 base site, Two different novel cis-acting elements oferd1, a clpA homologous Arabidopsis gene function in induction by dehydration stress and dark-induced senescence.Plant J33:259-270, 2003, Nakashima etc., Transcriptional regulation of ABI3-and ABA-responsive genes including RD29B and RD29A in seeds, germinating embryos, and seedlings of rabidopsis.Plant Mol Biol.60:51-68,2006), contain the ABA response element ABREOSRAB21 (Marcotte etc. that adverse circumstance is relevant in 944-951 base site, Abscisic acid-responsive sequences from the Em gene of wheat.Plant Cell1:969-976,1989, Busk etc., Regulation of abscisic acid-induced transcription, Plant Mol Biol37:425-435,1998, Skriver etc., Gene expression in response to abscisic acid and osmotic stress, Plant Cell2:503-512, 1990), at 449-455, 1187-1193, 1947-1953, 1965-1971 base site is contained to adverse circumstance and calcium and is regulated and controled relevant response element ABRERATCAL (Kaplan etc., Rapid Transcriptome Changes Induced by Cytosolic Ca2+Transients Reveal ABRE-Related Sequences as Ca2+-Responsive cis Elements inArabidopsis.PlantCell.18:2733-2748, 2006), at 596-601, 602-607, 1541-1546, (the Xue etc. with dehydration response associated responses element CBFHV are contained in 1583-1588 base site, Characterisation ofthe DNA-binding profile ofbarley HvCBF1 using an enzymatic method for rapid, quantitative and high-throughput analysis of the DNA-binding activity.Nucleic Acids Res.30:e77, 2002, Svensson etc., Transcriptome analysis ofcold acclimation in barley albina and xantha mutants.PlantPhysiol.141:257-270.2006), at 596-601, (the Dubouzet etc. with dehydration response associated responses element DRECRTCOREAT are contained in 602-607 base site, OsDREB genes in rice, Oryza sativa L., encode transcription activators that function in drought-, high-salt-and cold-responsive gene expression.PlantJ33:751-763, 2003, Qin etc., Cloning and functional analysis ofa novel DREB1/CBF transcription factor involved in cold-responsive gene expression in Zea mays L. Plant Cell Physiol.45:1042-1052,2004, Diaz-Martin etc., Functional Interaction between Two Transcription Factors Involved in the Developmental Regulation of a Small Heat Stress Protein Gene Promoter Plant Physiology, 139:1483-1494,2005, Suzuki etc., Quantitative statistical analysis of cis-regulatory sequences in ABA/VP1-and CBF/DREB1-regulated genes ofArabidopsis.Plant Physiol.139:437-447,2005, Skinner etc., Structural, functional, and phylogenetic characterization of a large CBF gene family in barley.Plant Mol Biol.59:533-551,2005), at 186-191, response element MYB2CONSENSUSAT (the Abe etc. relevant to dehydration are contained in 1495-1500 base site, Arabidopsis AtMYC2 (bHLH) and AtMYB2 (MYB) function as transcriptional activators in abscisic acid signaling.Plant Cell15:63-78, 2003), at 23-28, 70-75, 186-191, 449-454, 807-812, 860-865, 920-925, 927-932, 1187-1192, 1368-1373, 1642-1647, 1836-1841, response element MYCCONSENSUSAT (the Oh etc. relevant to dehydration are contained in 1947-1952 base site, Arabidopsis CBF3/DREB1A and ABF3in transgenic rice increased tolerance to abiotic stress without stunting growth..Plant Physiology138:341-351,2005, Chinnusamy etc., Molecular genetic perspectives on cross-talk and specificity in abiotic stress signalling in plants.J Exp Bot.55:225-236,2004).

Promotor PDS1 provided by the present invention contains in zone a plurality of cis-acting elements relevant to adverse circumstance (as shown in Figure 1), can be specifically to adverse circumstance, dormin (ABA), the responsing reaction that dewatered (as shown in Figure 2).Spend 11 in GUS expression vector (as shown in Figure 3) the rice transformation acceptor kind that the applicant utilizes the PDS1 promotor to build, can when transfer-gen plant is subject to environment stress, induce consumingly the expression (as shown in accompanying drawing 4,5) of reporter gene GUS.Effect of the present invention refers to embodiment.

Detailed technical scheme is as described below:

A kind of isolated paddy DNA molecule, its nucleotide sequence as shown in SEQ ID NO:1 and accompanying drawing 1, the nucleotide sequence that wherein promotor PDS1 comprises the 1-2082 bit base in the sequence shown in accompanying drawing 1.

The DNA molecular of described a kind of separation, it is characterized in that zone is interior except basic promoter element (TATA-box), also comprise the combination of a plurality of Stress response cis-acting elements (ABREATCONSENSUS, ABRELATERD1, ABREOSRAB21, ABRERATCAL, CBFHV, DRECRTCOREAT, MYB2CONSENSUSAT, MYCCONSENSUSAT).

The scope of protection of the invention also comprises following aspect:

The paddy rice expression vector of all or part of sequence construct of described PDS1 promotor.

The all or part of sequence construct paddy rice of described PDS1 promotor expression vector, the method for cultivating the improvement plant by genetic transformation.

The all or part of sequence construct paddy rice of described PDS1 promotor expression vector, the adversity resistant plant material of cultivating by genetic transformation.The material of described adversity resistant plant refers to plant, seed or cell clone.

According to above technical scheme, obviously, the expression vector that other people beyond applicant or applicant can utilize PDS1 promotor provided by the present invention to build anti contravariance related gene transforms the resistance that improves the plants such as paddy rice.Recipient plant can be the cereal crop that comprises paddy rice, wheat, corn etc., can certainly be to comprise some other important cash crop, for example corn, cotton, rape or tomato.

The accompanying drawing explanation

Sequence table SEQ ID NO:1 is the nucleotide sequence of the rice starter PDS1 that clones of the present invention, and the sequence total length is 2082bp.

Fig. 1: be the PDS1 promoter sequence that the present invention clones.The underscore sequence is amplification PDS1 promotor primer sequence used.What shade showed is basic promoter element sequence.Stress response ABRE element core sequence is by italic and add wavy line and mean.

Fig. 2: demonstration be the PDS1 gene in Rice Seedlings kind " in spend 11 ", can be by abduction delivering consumingly when plant is subject to arid, high salt and ABA and coerces.

Fig. 3: demonstration be the expression vector DX2181GFP-a-PDS1 that the present invention builds.This carrier is containing hygromycin resistance screening-gene (HRG), and promotor PDS1 is fused to gus gene 5 ' end non-translational region.

Fig. 4: demonstration be that the PDS1 promotor is controlled the GUS activity of lower gus gene under the drought stress processing.DX2181 DX2181GFP-a empty carrier spends 11 in transforming, in contrast family.PDS1-13, PDS1-14, PDS1-15, PDS1-17, PDS1-21 and PDS1-24 spend independently transgenic positive family of 11 6 of obtaining during DX2181GFP-a-PDS1 transforms.3 time points are successively: d0 (before drought stress); D6 (blade half volume); D18 (blade is rolled up entirely).

Fig. 5: demonstration be the GUS activity of gus gene under 400mM NaCl Stress treatment under the PDS1 promotor is controlled.During transforming, DX2181 DX2181GFP-a empty carrier spends 11, family in contrast, and PDS1-15, PDS1-17, PDS1-21 and PDS1-24 spend independently transgenic positive family of 11 4 of obtaining during DX2181G-PDS1 transforms.3 time points are respectively salt stress 0h (S0), 6h (S6), 18h (S24).

Embodiment

Embodiment 1PDS1 isolation of promoter and evaluation

Arid and high salt inducible gene expression spectrum analysis by rice varieties " in spend 11 " (Chinese Academy of Agricultural Sciences's crop investigations provides), found that one is subject to arid and high salt induced strong (the expression amount raising of drought stress later stage is more than 20 times, NaCl coerces rear expression amount and improves more than 5 times) gene, its TIGR (http://rice.plantbiology.msu.edu/) ID is LOC_Os01g04590, we are by its called after PDS1, corresponding BAC clone number is NC_008396, full-length cDNA corresponding in KOME database (http://cdna01.dna.affrc.go.jp/cDNA/) is numbered AK073435.

Next step is exactly the promotor of separating this gene.Concrete steps are as follows: the scope that finds the genome sequence (AP004868) of the japonica rice that this gene pairs answers " Japan is fine " and choose the transcription initiation site upstream 2.5Kb of this gene at NCBI (http://www.ncbi.nlm.nih.gov/) is carried out pcr amplification as candidate's promoter region.Design primer PDS1-F (5 '-ACG

CAGCGGTCAGTGTCGTGT-3 ') and PDS1-R (5 '-ACG

ATAGGGAGGTTTAGTGGAGTTT-3 '), and at primer 5 ' end add restriction enzyme site HindIII (underline and mean by italic, three bases before restriction enzyme site are the protection base).At first utilize primer PDS1-F and PDS1-R with " Japan is fine " genomic dna (CTAB method extracting, Zhang etc., genetic diversity and differentiation of indica anjaponica rice detected by RFLP analysis, 1992, Theor Appl Genet, 83,495-499) for template, increased, reaction system is 20uL regular-PCR system, and reaction conditions is: 94 ℃ of denaturation 5min; 94 ℃ of 30sec; Annealing temperature is 61 ℃, and each circulation reduces by 0.2 ℃, 30sec; 72 ℃ of 2mim, 32 circulations; 72 ℃ are extended 7min.The PCR product is connected on pGEM-T Easy carrier (purchased from Promega company), screening positive clone order-checking (ABI3730 sequenator, Applied Biosystem, order-checking completes at national plant gene center [Wuhan]), result confirms: institute's extension increasing sequence is the PDS1 promoter sequence of expection, and it comprises a plurality of arids, ABA Stress response cis-acting elements (as Fig. 1).

Embodiment 2: the abduction delivering that detects paddy rice native gene Oshox24

The rice varieties " in spend 11 " of take is material, plants in the little square box of the root media in containing embodiment 3 to grow to 3,4 leaves and carry out arid, high salt and ABA during the phase and coerce.The drought stress method is that plant is placed in to the natural dehydration of air, gets 0h, 3h, 6h, 12h, 24h time point sample; High-salt stress is to add 100mL200mM NaCl in little square box, gets 0h, 3h, 6h, 12h time point sample; It is to add 70mL100uMABA in each little square box that ABA coerces, and gets 0h, 1h, 3h, 6h, 24h time point sample.Total RNA adopts TRIZOL reagent (purchased from Invitrogen company) to extract (extracting method is according to above-mentioned TRIZOL reagent specification sheets), utilizes ThermoScript II SSIII (purchased from Invitrogen company) by the synthetic cDNA (method is according to Invitrogen company ThermoScript II reagent specification sheets) of its reverse transcription.The synthetic cDNA of the above-mentioned reverse transcription of take is template, with primer (5 '-ACGAAAGCGAATCAACGAGAA-3 ' and 5 '-GGAGGCTCGGATTCCAGAA-3 '), the PDS1 gene is carried out to special pcr amplification (the long 67bp of amplified production).Use primer (5 '-TGGCATCTCTCAGCACATTCC-3 ' and 5 '-TGCACAAT GGATGGGTCAGA-3 ') to do specific amplified (the long 76bp of amplified production) to paddy rice Actin1 gene simultaneously, using and carry out quantitative analysis as internal reference.Reaction conditions is: 95 ℃ of 10sec, 95 ℃ of 5sec, 60 ℃ of 34sec, 40 circulations.Carry out the fluoroscopic examination real-time quantitative analysis in reaction process.Result shows that this gene is subject to strong rising abduction delivering in Drought at seedling stage, high salt and the ABA of rice varieties " in spend 11 " coerce, and the rising multiple is about respectively 80 times, 110 times and 100 times (as Fig. 2).

The rice transformation of embodiment 3:PDS1 promoters driven reporter gene

Embodiment of the present invention are exactly build the gus gene expression vector of PDS1 promotor and be transformed in rice varieties " in spend 11 ", detect quantitatively the arid of PDS1 promotor and the abduction delivering activity of high-salt stress.Concrete operations are as follows:

At first the PCR product of the PDS1 promotor of separation in embodiment 1 is connected into to pGEM-T Easy carrier, transforms bacillus coli DH 5 alpha (purchased from Promega company) and also obtain positive colony.Reclaim PC1 by the HindIII single endonuclease digestion from pGEM-T Easy positive colony and be connected to again GUS expression vector DX2181GFP-a (come by pBI101.1 and pCAMBIA1381GFP transformation, carrier contains gus reporter gene).Enzyme cut the checking positive colony and detect direction of insertion correct after, import to rice varieties " in spend in 11 " by agriculture bacillus mediated rice transformation system, through preculture, infect, cultivate altogether, callus that screening has hygromycin resistance, break up, take root, practice transplantation of seedlings, obtain transfer-gen plant.Agriculture bacillus mediated paddy rice (japonica rice subspecies) genetic conversion system is mainly applied the method for the people such as Hiei report (referring to Efficient transformation of rice, Oryza sativa L., mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA, 1994, Plant Journal6:271-282) also improve on its basis optimization.To not connect the DX2181GFP-a empty carrier rice transformation kind " in spend in 11 " of external source fragment in contrast simultaneously.Key step and reagent are as follows:

(1) reagent and solution abbreviation

In the present invention, the abbreviation of substratum plant hormone used is expressed as follows: 6-BA (6-BenzylaminoPurine, 6-benzyladenine); CN (Carbenicillin, Pyocianil); KT (Kinetin, kinetin); NAA (Napthalene acetic acid, naphthylacetic acid); IAA (Indole-3-acetic acid, indolylacetic acid); 2,4-D (2,4-Dichlorophenoxyacetic acid, 2,4 dichlorophenoxyacetic acid); AS (Acetosringone, Syringylethanone); CH (Casein Enzymatic Hydrolysate, caseinhydrolysate); HN (HygromycinB, Totomycin); DMSO (Dimethyl Sulfoxide, dimethyl sulfoxide (DMSO)); N6max (a large amount of composition solution of N6); N6mix (N6 trace ingredients solution); MSmax (a large amount of composition solution of MS); MSmix (MS trace ingredients solution).

(2) main solution formula

1) preparation of N6 substratum macroelement mother liquor [10 times of concentrated solutions (10X)]:

Dissolve one by one, then under room temperature, be settled to 1000ml.

2) preparation of N6 substratum trace element mother liquor [100 times of concentrated solutions (100X)]

Dissolve under room temperature and be settled to 1000ml.

3) molysite (Fe ₂EDTA) preparation of stock solution (100X)

Prepare the 800ml distilled water and be heated to 70 ℃, adding b diammonium disodium edta (Na2EDTA2H ₂O) 3.73 grams keep 2 hours after fully dissolving in 70 ℃ of water-baths, are settled to 1000ml, and 4 ℃ save backup.

4) VITAMIN stock solution (100X) preparation

Add water and be settled to 1000ml, 4 ℃ save backup.

5) preparation of MS substratum macroelement mother liquor (10X)

Dissolve under room temperature and be settled to 1000ml.

6) preparation of MS substratum trace element mother liquor (100X)

Dissolve under room temperature and be settled to 1000ml.

7) 2, the preparation of 4-D stock solution (1mg/ml):

Weigh 2,4-D100mg, dissolve 5 minutes with 1ml1N potassium hydroxide, be settled to 100ml after then adding 10ml distilled water dissolve complete, preserve under room temperature.

8) preparation of 6-BA stock solution (1mg/ml):

Weigh 6-BA100mg, dissolve 5 minutes with 1ml1N potassium hydroxide, be settled to 100ml, room temperature preservation after then adding 10ml distilled water dissolve complete.

9) preparation of naphthylacetic acid (NAA) stock solution (1mg/ml):

Weigh NAA100mg, dissolve 5 minutes with 1ml1N potassium hydroxide, be settled to 100ml after then adding 10ml distilled water dissolve complete, 4 ℃ save backup.

10) preparation of indolylacetic acid (IAA) stock solution (1mg/ml):

Weigh IAA100mg, dissolve 5 minutes with 1ml1N potassium hydroxide, be settled to 100ml, 4 ℃ of standby 300ml distilled water and ferric sulfate (FeSO of adding of preservations after then adding 10ml distilled water dissolve complete in a large triangular flask ₄7H ₂O) 2.78g.In another large triangular flask, add 300ml distilled water to use.

11) preparation of glucose stock solution (0.5g/ml):

Weigh glucose 125g, then with distilled water, dissolve and be settled to 250ml, after sterilizing, 4 ℃ save backup.

12) preparation of AS stock solution:

Weigh AS0.392g, DMSO10ml, divide and be filled in the 1.5ml centrifuge tube, and 4 ℃ save backup.

13) 1N potassium hydroxide stock solution

Weigh potassium hydroxide 5.6g, and dissolve and be settled to 100ml with distilled water, room temperature preservation is standby.

(3) for the culture medium prescription of rice transformation

1) inducing culture

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides and installs to 50ml triangular flask (25ml/ bottle), the sealing sterilizing.

2) subculture medium

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.9, boils and is settled to 1000ml, divides and installs to 50ml triangular flask (25ml/ bottle), the sealing sterilizing.

3) pre-culture medium

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, the sealing sterilizing.

Use front heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (25ml/ ware) in culture dish poured in packing into.

4) be total to substratum

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.6, the sealing sterilizing.

Use front heating for dissolving substratum and add 5ml glucose stock solution and 250 μ l AS stock solutions, (the every ware of 25ml/) in culture dish poured in packing into.

5) suspension medium

Adding distil water, to 100ml, is regulated pH value to 5.4, divides and installs in the triangular flask of two 100ml, the sealing sterilizing.

Add 1ml glucose stock solution and 100 μ l AS stock solutions before use.

6) select substratum

Adding distil water, to 250ml, is regulated pH value to 6.0, the sealing sterilizing.

Dissolve substratum before using, add 250 μ l HN and 400ppm CN, (25ml/ ware) in culture dish poured in packing into.

7) pre-division culture medium

Adding distil water is to 250ml, and 1N potassium hydroxide is regulated pH value to 5.9, the sealing sterilizing.

Dissolve substratum before using, add 250 μ l HN and 200ppm CN, (25ml/ ware) in culture dish poured in packing into.

8) division culture medium

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 6.0.

Boil and be settled to 1000ml, dividing and install to 50ml triangular flask (50ml/ bottle), the sealing sterilizing.

9) root media

Adding distil water is to 900ml, and 1N potassium hydroxide is regulated pH value to 5.8.

Boil and be settled to 1000ml, dividing and install to (25ml/ pipe) in the pipe of taking root, the sealing sterilizing.

(4) agriculture bacillus mediated genetic transformation step

3.1 callus of induce

(1) ripe rice paddy seed " in spend 11 " is shelled, then use successively 70% Ethanol Treatment 1 minute, 0.15% mercury chloride (HgCl2) seed-coat sterilization 15 minutes;

(2) wash seed 4-5 time with sterilizing;

(3) seed is placed on inducing culture;

(4) postvaccinal substratum is placed in to dark place and cultivates 4 weeks, 25 ± 1 ℃ of temperature.

3.2 callus subculture

Select the embryo callus subculture of glassy yellow, consolidation and relatively dry, be put in dark lower the cultivation 2 weeks on subculture medium, 25 ± 1 ℃ of temperature.

3.3 preculture

Select the embryo callus subculture of consolidation and relatively dry, be put in dark lower the cultivation 2 weeks on pre-culture medium, 25 ± 1 ℃ of temperature.

3.4 Agrobacterium is cultivated

(1) preculture Agrobacterium EHA105 (deriving from Australian CAMBIA laboratory, commercial bacterial strain) two days on the LA substratum of selecting with corresponding resistance, 28 ℃ of temperature;

(2) Agrobacterium is transferred in suspension medium, cultivates 2-3 hour on 28 ℃ of shaking tables.

3.5 Agrobacterium is infected

(1) pre-incubated callus is transferred in the bottle that sterilizing is good;

(2) regulate the suspension of Agrobacterium to OD ₆₀₀0.8-1.0;

(3) callus is soaked 30 minutes in agrobacterium suspension;

(4) shift callus blots to the good filter paper of sterilizing; Then be placed on common substratum and cultivate 2 days, temperature 19-20 ℃.

3.6 callus washing and selection are cultivated

(1) aqua sterilisa washing callus is to cannot see Agrobacterium;

(2) be immersed in containing in the aqua sterilisa of 400ppm Pyocianil (CN) 30 minutes;

(3) shift callus blots to the good filter paper of sterilizing;

(4) shift callus and select 2-3 time on substratum to selecting, each 2 weeks.(hygromycin selection concentration is 400ppm for the first time, is 250ppm for the second time later).

3.7 differentiation

Kanamycin-resistant callus tissue is transferred to dark place on pre-division culture medium and cultivates 5-7 week;

Shift the callus of pre-differentiation culture to division culture medium, cultivate under illumination, 26 ℃ of temperature.

3.8 take root

(1) cut the root that differentiation phase produces;

(2) then transfer them in root media, cultivate 2-3 week, 26 ℃ of temperature under illumination.

3.9 transplant

Wash the residual substratum on root off, the seedling that will have good root system proceeds to greenhouse, at initial several days, keeps moisture moistening simultaneously.

4, the evaluation of promotor PDS1 Drought and salt stress-inducing expression activity

The applicant adopts the DX2181GFP-a-PDS1 carrier rice transformation " in spend in 11 " of structure, obtains 25 strains of transgenic positive plant; The DX2181G empty carrier rice transformation " in spend in 11 " that does not connect the external source fragment obtains 8 strains of transgenic positive plant.Choosing 6 turns the positive family of promotor and 1 and turns empty carrier positive control family T2 is carried out to Totomycin (HN for seed, 50mg/mL, 1/1000 ratio adds in root media) resistance screening germinates, and the young plant kind of germination grown in the sandy soil of little red bucket to 4 leaf phases were done arid and high salt (4 transgenic positive familys) is coerced.The drought stress method is that plant is placed in to the natural dehydration of air, and high-salt stress is that plant is placed in to 1L400mMNaCl.Get the sample of coercing 0h, 6h, 18h.What every duplicate samples was got is the compound sample (being no less than 10 strains) of this family.

Sample liquid nitrogen grind away, by GUS extract (50mM Na ₂HPO4, pH7.0,10mM β-mercaptoethanol, 10mMNa ₂EDTA, 0.1%Sarkosyl, 0.1%Triton-100) the extracting total protein, from sample, a certain amount of albumen of taking-up and is carried out the analysis of GUS active level.The GUS that obtains raw sample by the analysis of fluorescence result is more alive than enzyme.Concrete steps are as follows: by the Bradford method (referring to A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.1976, Anal Biochem, 72:248-254) record the total protein concentration of sample, measure again the total protein of the equal in quality of certain volume with pipettor according to concentration, with the gross protein extract of same amount+0.4mlGUS Extraction buffer (seeing the end of writing)+10 μ l40mM substrate MUG (4-methylumbelifferyl-β-D-glucuronide) after 37 ℃ of water-bath certain hours (in 1h), add 1.6ml reaction terminating liquid (0.2MNa ₂CO ₃), each reaction arranges three technology and repeats.Measure each sample at exciting light 365nm with Tecan grating type microplate reader infinite M200, the fluorescent value at utilizing emitted light 455nm place is usingd the fluorescent value of 50nM MU as reference simultaneously.And then live by the ratio enzyme that above data calculate gus protein in each sample, i.e. the amount of 1ug total protein per minute reaction substrate MU (the pmol MU/min/ug protein of unit).

Measure and find by the gus protein active level, gus protein activity under the control of PDS1 promotor is subject to the induced strong of arid and high-salt stress, gus protein approximately 6 to 20 times (as Fig. 4) before the drought stress activity of 18 hours is to coerce in six independent transgenosis familys, gus protein approximately 3 to 6 times (as Fig. 5) before the high-salt stress activity of 18 hours is to coerce in four independent transgenosis familys.

Appendix: GUS Extraction buffer formula

GUS Extraction buffer (cumulative volume 1L uses the distilled water constant volume): pH=7.0

Claims (2)

1. one kind by arid and the promotor PDS1 of the special abduction delivering of high salt, and its nucleotide sequence is as shown in SEQ ID NO:1.

2. the application of promotor PDS1 claimed in claim 1 in controlling gene is expressed.

Images