CN102453083A

CN102453083A - Plant stress tolerance related protein ZmPMP3 and coding gene thereof and application thereof

Info

Publication number: CN102453083A
Application number: CN2010105255831A
Authority: CN
Inventors: 黎裕; 付静; 张登峰; 李会勇; 刘颖慧; 石云素; 宋燕春; 王天宇
Original assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Current assignee: Institute of Crop Sciences of Chinese Academy of Agricultural Sciences
Priority date: 2010-10-29
Filing date: 2010-10-29
Publication date: 2012-05-16
Anticipated expiration: 2030-10-29
Also published as: CN102453083B

Abstract

The invention discloses a plant stress tolerance related protein ZmPMP3 and a coding gene thereof and an application thereof. The protein provided by the invention is called ZmPMP3, and is from corns, and is protein shown in the following 1) or 2): 1), a protein composed of amino acid sequences shown in the sequence 2 of the sequence table; 2), a protein obtained by substituting and/or deleting and/or adding one or several amino acid residues for the amino acid sequence of the sequence 2, wherein the protein is relevant to the plant stress tolerance and/or water retention rate and is derived from the protein in the 1). The protein disclosed by the invention is proved by the experiment that the ZmPMP3 obtained by cloning can be introduced into arabidopsis to obtain genetically modified arabidopsis. In comparison with wild arabidopsis, the stress tolerance and/or the water retention rate of the genetically modified arabidopsis are higher than that of the wild arabidopsis. The ZmPMP3 gene and the genetically modified arabidopsis obtained by the experiment have important significance on plant genetic breeding aspect.

Description

With plant stress tolerance correlative protein ZmPMP3 and encoding sox and application

Technical field

The present invention relates to a kind of and plant stress tolerance correlative protein ZmPMP3 and encoding sox and application.

Background technology

The growth of crop that arid, salt marsh, low temperature, oxygen such as have coerced at the serious threat of many abiotic stress, and then cause the continuous deterioration of environment.Worldwide, abiotic stress all is the major cause that causes crop failure, annual 50% of the crop failure that on average occupies.Abiotic stress is coerced through plant being produced a series of morphology, the influence of Physiology and biochemistry state and molecular level, thereby growth and the output of the plant of influence.Plant when response abiotic stress, often produces the variation of a series of biochemical moleculars and gene level as the biosystem of a complicacy.The inside and outside cytolemma of contact cell in the plant materials; Directly perception comes from the physical chemistry signal of outside, in this simultaneously, and the medium that links to each other with the external world as cell; When cell receives the external world when coercing, often also cell membrane has produced irreversible destruction.Research in recent years discloses, and the protein ingredient of cytolemma and membrane structure under extraneous stress conditions variation have taken place, to defend external injury of coercing pair cell.Therefore think that some coerce relevant membranin in the perception outer signals, protection and repair cell film aspect play a role.

In recent years; From Arabidopis thaliana (Arabidopsis thaliana), wheat (Triticum aestivum), paddy rice (Oryza sativa L.); Barley (hordeum vulgure); Long fringe oat (fophopyrum elongatum), goatweed (Aneurolepidium chinense) is cloned respectively in the flower of Stinkgrass plants such as (Puccinellia tenuiflora) and has obtained coding and the higher membrane protein gene of yeast (Saccharomyces cerevisiae) Pmp3p consistence.These genes receive arid, and are cold, and Salt Stress-induced, the one type of low-molecular-weight membranin of encoding.Yeast (Saccharomyces cerevisiae) Pmp3p is a kind of 55 amino acid whose acid hydrophobins that contain.The yeast mutants that has lacked PMP3 is compared with the wild-type contrast, has strengthened the susceptibility to sodium ion and HYG, has also suppressed the dependency of Trk1p and Trk2p mutants which had growth to potassium ion simultaneously.Navarre and Goffeau think that Pmp3p plays as a kind of plasmalemma protein and keep the cytolemma electromotive force, guarantee the effect of the ion running balance that cell is inside and outside.Because MPM3 albumen is a kind of ubiquitous lower molecular weight transmembrane protein; And P77240 with Escherichia coli; P34655, Q20516, Q22702, Q22701, Q22700 in the nematode (Caenorhabditis elegans), and with plant in the albumen of some stress-inducings genes encoding of expressing have high homology.

Also from corn, do not find similar albumen at present.

Summary of the invention

An object of the present invention is to provide a kind of and plant stress tolerance correlative protein ZmPMP3 and encoding sox thereof.

Protein provided by the present invention, the name be called ZmPMP3, synthetic is following 1) or 2) or 3) protein:

1) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 2;

2) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 4;

3) with the aminoacid sequence of sequence 2 or sequence 4 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant with plant stress tolerance and/or moisture holding capacity by 1) deutero-protein.

Sequence 2 or sequence 4 are formed by 58 amino-acid residues in the above-mentioned sequence table.The replacement of said one or several amino-acid residue and/or disappearance and/or be added to replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.

The above-mentioned proteic encoding sox relevant with plant stress tolerance also belongs to protection scope of the present invention.

Proteinic encoding sox provided by the present invention is that said encoding sox is following 1)-8) in the gene shown in arbitrary:

1) dna molecular shown in the sequence 1 in the sequence table;

2) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 87th-283 Nucleotide;

3) in the sequence table sequence 1 from the dna molecular shown in 5 ' terminal the 88th-264 Nucleotide;

4) dna molecular shown in the sequence 3 in the sequence table;

5) in the sequence table sequence 3 from the dna molecular shown in 5 ' terminal the 88th-264 Nucleotide;

6) in the sequence table sequence 3 from the dna molecular shown in 5 ' terminal the 88th-283 Nucleotide;

7) under stringent condition with 1) or 2) or 3) or 4) or 5) or 6) dna molecule hybridize that limits and the dna molecular said and plant stress tolerance and/or moisture holding capacity related protein of encoding;

8) with 1) or 2) or 3) or 4) or 5) or 6) dna sequence dna that limits has 70% at least, have 75% at least, have 80% at least, have 85% at least, have 90% at least, have 95% at least, have 96% at least, have 97% at least, have 98% or have the dna molecular of 99% homology and coding and said plant stress tolerance and/or moisture holding capacity related protein at least at least.

Wherein, sequence 1 is made up of 389 deoxyribonucleotides in the sequence table, and its open reading frame (ORF) is from 5 ' terminal the 88th-264 bit base.

Sequence 4 is made up of 389 deoxyribonucleotides in the sequence table, and its open reading frame (ORF) is from 5 ' terminal the 88th-264 bit base.

Said stringent condition also can be at 6 * SSC, in the solution of 0.5% SDS, 65 ℃ of hybridization down, uses 2 * SSC then, 0.1%SDS and 1 * SSC, and 0.1% SDS respectively washes film once.

The recombinant expression vector, reorganization bacterium, transgenic cell line or the expression cassette that contain above-mentioned protein coding gene also belong to protection scope of the present invention.

Insert the pCAMBIA3301-ZmPMP3 that said encoding sox obtains between Nco I that said recombinant expression vector specifically can be at pCAMBIA3301 and BstE II site.

Increase above-mentioned arbitrary said encoding sox total length or its any segmental primer to also belonging to protection scope of the present invention.

Another object of the present invention provides the method for the transgenic plant of cultivating resistance of reverse and/or moisture holding capacity raising.

Method provided by the present invention is that described encoding sox is imported the transgenic plant that the purpose plant obtains, and resistance of reverse of said transgenic plant and/or moisture holding capacity are higher than said purpose plant.

The excised leaf water retention that it is said transgenic plant that the moisture holding capacity of said transgenic plant is higher than said purpose plant is higher than said purpose plant;

Said resistance of reverse is drought tolerance and/or salt tolerance.

Said encoding sox is to import in the said purpose plant through said recombinant expression vector.

Said plant is dicotyledons or monocotyledons, and said dicotyledons is preferably Arabidopis thaliana.

Experiment of the present invention proves; Method with the clone obtains the ZmPMP3 gene; It is imported the transgenic arabidopsis that obtains in the Arabidopis thaliana, compare with the wild-type Arabidopis thaliana, the resistance of reverse of transgenic arabidopsis and/or water retention are higher than the wild-type Arabidopis thaliana; Resistance of reverse shows drought tolerance and/or salt tolerance, and water retention mainly is that the water retention of blade is higher than the wild-type Arabidopis thaliana.ZmPMP3 gene that this experiment obtains and transgenic arabidopsis have important meaning aspect plant genetics and breeding.

Description of drawings

Fig. 1 is that ZmPMP3 gene crossing in yeast mutants expressed

Fig. 2 is the ZmPMP3 Subcellular Localization.

Fig. 3 is the pcr amplification figure of ZmPMP3

Fig. 4 cuts evaluation figure for the enzyme of pCAMBIA3301-ZmPMP3.

Fig. 5 is that the PCR of Agrobacterium GV3101/pCAMBIA3301-ZmPMP3 identifies figure.

Fig. 6 carries the transgenic arabidopsis genome for a short time for the SDS method.

Fig. 7 is that transgenic arabidopsis genome PCR detects bar gene result.

Fig. 8 is that transgenic arabidopsis genome PCR detects ZmPMP3 gene result.

Fig. 9 counts statistics for the sprouting of transgenic arabidopsis strain system

Figure 10 heavily adds up for the biology that is of transgenic arabidopsis strain system

Figure 11 is heavy for the biology of the strain system of transgenic arabidopsis strain system

Figure 12 counts statistical graph for accomplishing Arabidopis thaliana strain in the time of infertility

Figure 13 handles preceding Arabidopis thaliana for 300mM NaCl

Figure 14 handles Arabidopis thaliana two days later for 300mM NaCl

Figure 15 is for after 300mM NaCl handled for two weeks, and ck and strain under control group and 300mM NaCl handle are 6

Figure 16 is an excised leaf water retention statistical graph

Embodiment

Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.

Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.

The acquisition of embodiment 1, ZmPMP3 and the research of function thereof

One, the acquisition of ZmPMP3

Various consumptive materials, enzyme and reagent:

Restriction enzyme (NEB), T4 ligase enzyme (Promega) are available from the white Bioisystech Co., Ltd in Yuanping City, Beijing; Intestinal bacteria competence TOP10, kantlex, taq enzyme, 2 * Pfu PCR Master Mix purchase in TIANGEN Biotech (Beijing) Co., Ltd.; DNA marker is all available from the Beijing Quanshijin Biotechnology Co., Ltd; PEG3000 (polyethylene glycol, polyoxyethylene glycol), carrier DNA, YPD substratum, LiAc damping fluid, TE damping fluid are purchased in general Jino (Beijing) Bioisystech Co., Ltd; Aureobasidin A (TaKaRa), pAUR123 carrier (TaKaRa), pMD18-T plasmid (TaKaRa) are purchased in logical (Beijing) bio tech ltd of the six directions; VITAMIN B4, glucose, dNTP mix purchase the biological Ltd in ancient cooking vessel state.

Carrier and yeast strain:

YR93-31 bacterial strain (Mayumi Inada; Akihiro Ueda; Weiming Shi; Tetsuko Takabe (2005) .A stress-inducible plasma membrane protein 3 (AcPMP3) in a monocotyledonous halophyte; Aneurolepidium chinense, regulates cellular Na+and K+accumulation under salt stress.Planta 220:395-402, the public can obtain from crop science institute of the Chinese Academy of Agricultural Sciences.) be the two mutants that does not contain yeast PMP3 gene.

YR93-1 bacterial strain (Mayumi Inada; Akihiro Ueda; Weiming Shi, Tetsuko Takabe. (2005) A stress-inducible plasma membrane protein 3 (AcPMP3) in a monocotyledonous halophyte, Aneurolepidium chinense; Regulates cellular Na+and K+accumulation under salt stress.Planta 220:395-402, the public can obtain from crop science institute of the Chinese Academy of Agricultural Sciences.) be the bacterial strain that contains yeast PMP3 gene, with this bacterial strain as contrast.

Subcellular Localization is with transient expression carrier pBI221-GFP (Ying Fu; Ying Gu, Zhiliang Zheng, Geoffrey Wasteneys; Zhenbiao Yang. (2005) Arabidopsis Interdigitating Cell Growth Requires Two Antagonistic Pathways with Opposing Action on Cell Morphogenesis.Cell; Vol.120,687-700, the public can obtain from crop science institute of the Chinese Academy of Agricultural Sciences.)。

Contain the restriction enzyme site primer sequence:

Make up pBI221-GFP Subcellular Localization carrier primer:

GFP-F：5`-TATCTAGAATGTCGGAGGGGACTGCCAACTGCG-3`

GFP-R：5`-ATGCCGCGGTAGTTGTTCTTGGTGATGGCGTAG-3`

Make up pAUR123 expression vector primer:

M1-PAUR-F1：5′-TGGGGTACCAAGAAGCATCGGCAGCAT-3′

M1-PAUR-R1：5′-TATGAGCTCACGCAGATACAGAGACAT-3′

YPDA liquid nutrient medium (100ml):

YPD substratum 0.8g

0.2% VITAMIN B4 1.5ml

Add ddH ₂O to 95ml transfers pH to 6.5, autoclaving

40% glucose that adds the 5ml sterilization.

YPDA solid medium (100ml):, just need to add 2g agar, autoclaving again with the YPDA liquid nutrient medium.

AbA: be the abbreviation of microbiotic Aureobasidin A

Key instrument

Particle gun system (Bio-rad), (concentrator 5301 for vacuum Rotary drying appearance; Eppendorf), constant incubator, laser confocal microscope (LEICA TCS SP2 confocal laser scanning system), microscope (OLYMPUS, BH-2).

1, the acquisition of ZmPMP3

Extract the cDNA of the key self-mating system CN165 of corn (Zea mays L.) (initiative of king sky research institute of Institute of Crop Science, Chinese Academy of Agricultural Science); Carry out pcr amplification with the M1-full-F/M1-full-R primer; Obtain the fragment of 389bp; This fragment is connected on the pMD18-T (TaKaRa), obtains pMD18-T/ZmPMP3-1.

Also but artificial synthesized sequence 1, carries out pcr amplification with the M1-full-F/M1-full-R primer, and this fragment is connected on the pMD18-T (TaKaRa), obtains pMD18-T/ZmPMP3-1 equally.

M1-full-F：5`-AGCGAAAGGAGAGAAGGAATC-3`

M1-full-R：5`-CATGGGGTGGGTACGGTAG-3`

With pMD18-T/ZmPMP3-1 is template, carries out pcr amplification with M1-full-F and M1-full-R as primer, obtains the PCR product of about 389bp.This PCR product is sent to order-checking; The result has in the sequence table sequence 1 from 5 ' terminal 1-389 position Nucleotide for this PCR product; The unnamed gene of this PCR product is ZmPMP3; The coding region of this gene be in the sequence table sequence 1 ' terminal 88-264 position Nucleotide, the albumen called after ZmPMP3 of this genes encoding, this proteic aminoacid sequence are the sequence 2 in the sequence table from 5.Sequence 1 is made up of 389 Nucleotide, and sequence 2 is made up of 58 amino acid.

2, yeast genetic transformation:

A: the acquisition of Yeast expression carrier

Above-mentioned 1 pMD18-T/ZmPMP3-1 that obtains is carried out double digestion with Kpn I and Sac I; The fragment that obtains is cut the pAUR123 carrier segments that obtains with the same enzyme of process and is connected, and obtains connecting product and changes among the intestinal bacteria competence Top10, obtains transformant; Transformant is extracted plasmid; Send to order-checking, the result for this plasmid for ' terminal 1-389 position Nucleotide is inserted into the Kpn I of pAUR123 carrier and the carrier that Sac I restriction enzyme site obtains, called after pAUR123-ZmPMP3 from 5 with sequence in the sequence table 1; The coding region dna fragmentation of ZmPMP3 is positioned at promotor PADH1 downstream among the pAUR123-ZmPMP3, and this recombinant vectors is that yeast is crossed expression vector.

Specific as follows:

1) the coding region DNA of amplification ZmPMP3 introduces restriction enzyme site

Pcr amplification system (50 μ l):

pMD18-T/ZmPMP3-1 0.85μl

M1-PAUR-F1 2.5μl(10μM)

M1-PAUR-R1 2.5μl(10μM)

2×Pfu?PCR?Master?Mix 25μl

ddH ₂O 19.15μl

Pcr amplification program: 94 ℃ of 10min

94℃?30sec

56℃?30sec

72℃?1min

72℃?10min

4 ℃ of insulations, 35 circulations.

Purifying and recovering behind the electrophoresis detection purpose band.

2) pAUR123-ZmPMP3-1 recombinant vectors

50 μ l systems: the substep enzyme is cut:

PCR product behind the step 1) purifying (50ng/ μ l) 25 μ l

Buffer1 5μl

ddH ₂O 17.5μl

Kpn?I 1μl

Sac?I 1μl

BSA 0.5μl

30 ℃ of water-bath enzymes are cut 2h; Run 1% sepharose, cut glue and reclaim, obtain enzyme and cut dna fragmentation.

PAUR123 carrier enzyme is cut:

50 μ l systems (enzyme is cut three pipes simultaneously): the substep enzyme is cut:

Plasmid (100ng/ μ l) 30 μ l

Buffer1 5μl

Kpn?I 1μl

Sac?I 1μl

BSA 0.5μl

ddH ₂O 12.5μl

37 ℃ of water-bath enzymes are cut 2h, and 1% agarose gel electrophoresis detects, and cut glue and reclaim the big fragment of carrier.

Enzyme is cut back dna fragmentation and enzyme cuts afterwards that the big fragment of carrier is connected:

10 μ l systems:

Carrier (20ng/ μ l) 1 μ l

Fragment (50ng/ μ l) 7 μ l

T4?Buffer 1μl

T4 ligase enzyme 1 μ l

16 ℃ of airbath 12h; Transformed into escherichia coli competence Top10, bacterium liquid PCR screening positive clone obtains pAUR123-ZmPMP3.

Plasmid extracts: the little extraction reagent kit of plasmid (day root company):

(1) the single bacterium colony hickie of inoculation contains in the LB liquid nutrient medium of Amp resistance in 5ml, and 37 ℃ of 200rpm shaking culture are spent the night.

(2) column equilibration step: adsorption column is put into collection tube, in adsorption column, add 500 μ l balance liquids, the centrifugal 1min of 12000rpm outwells the waste liquid in the collection tube, adsorption column is relay reclaim in the collector.(annotating: use the adsorption column of handling the same day)

(3) bacterium liquid is changed in the Eppendorf pipe, the centrifugal 30sec of 12000rpm, the Ex-all supernatant is collected thalline as far as possible.Add 250 μ l solution P1, resuspended thalline.(annotate: this step must thoroughly be hanged and opened, otherwise can influence the cracking of thalline, cause plasmid extracted amount and purity on the low side)

(4) add 250 μ l solution P2, upset mixes 4～6 times gently up and down, makes the abundant cracking of thalline, and this moment, bacterium liquid should become limpid, as did not become limpid, explained that then thalline is too much, and cracking is inabundant.(annotate: inviolent during mixing, in order to avoid interrupt bacillus coli gene group DNA, cause in the plasmid of extraction and be mixed with bacillus coli gene group DNA)

(5) add 350 μ l solution P3, overturn gently up and down immediately and mix 6～8 times, white flocks should appear in this moment.The centrifugal 10min of 12000rpm, deposition is formed on the centrifuge tube bottom.

(6) get supernatant in the adsorption column that column equilibration was handled, the centrifugal 30sec of 12000rpm outwells the waste liquid in the collection tube, and this step can repeatedly collect.

(7) in adsorption column, add 700 μ l rinsing liquids, the centrifugal 30sec of 12000rpm outwells the waste liquid in the collection tube.

(8) in adsorption column, add 500 μ l rinsing liquids, the centrifugal 30sec of 12000rpm outwells the waste liquid in the collection tube.

(9) adsorption column is put back to collection tube, the centrifugal 2min of 12000rpm is to remove rinsing liquid as far as possible.

(10) room temperature was placed several minutes, removed residual rinsing liquid as far as possible.

(11) take out adsorption column and put into a clean centrifuge tube, be no less than the elution buffer of 65～70 ℃ of water-bath preheatings of 50 μ l in the adsorption film middle part, room temperature is placed 2min, the centrifugal 1min of 12000rpm.

(12) in order to obtain more purified product, the centrifugal solution that obtains in (11) is added adsorption column more again, the centrifugal 1min of 12000rpm.

(13) get 1 μ l plasmid and carry out electrophoresis detection, according to its quality of DNA marker preliminary judgement and concentration

B, yeast genetic transformation:

(1) picking YR93-31 yeast bacterial plaque is in the 10mlYPDA liquid nutrient medium, 30 ℃ of 220rpm incubated overnight.

(2) get in right amount in the 50ml liquid nutrient medium, it is 0.4 that 30 ℃ of 220rpm are cultured to OD600.

Under (3) 25 ℃, the centrifugal 5min of 1000rpm collects bacterium.

(4) abandon supernatant, with the resuspended thalline of 10ml sterilized water.

Under (5) 25 ℃, the centrifugal 5min of 1000rpm abandons supernatant, with the resuspended thalline of 1ml 1 * TE.

(6) 30 ℃ of 200rpm shake bacterium 30min.

(7) the pAUR123-ZmPMP3 recombinant plasmid that 2 μ g obtain is prepared in each reaction, and the carrier DNA 10 μ l of processing are in the 1.5ml centrifuge tube.

Annotate: carrier DNA uses pre-treatment: boiling water boils 10min, on ice 1min; Boiling water boils 10min again, on ice 1min.

(8) from (6), get 200 μ l in (7).

(9) each reaction adds 600 μ l PEG solution.

After (10) 30 ℃ of 200rpm shake bacterium 30min, add 70 μ l DMSO and put upside down mixing, not vortex gently.

(11) 42 ℃ of thermal shock 15min, 1min on ice.

(12) the centrifugal 30sec of 12000rpm collects thalline in the pipe end.

(13) with the resuspended thalline of 0.5ml 1 * TE.

(14) get 200 μ l and coat YPDA (AbA resistance) solid medium.

(15) yeast is inverted cultivation 3 days for dull and stereotyped 30 ℃.

Obtain transformant and be positive colony, called after YR93-31/pAUR123-ZmPMP3.

3, ZmPMP3-1 gene crossing in yeast mutants expressed:

(1) picking YR93-31/pAUR123-ZmPMP3 mono-clonal is in AbA resistance YPDA liquid nutrient medium, and 28 ℃ are shaken bacterium 48hr to saturated.

(2) by 1: 100 the switching above bacterium liquid once after, shake bacterium 36hr, this moment OD ₆₀₀Be about 1.0, dilution bacterium liquid is to OD ₆₀₀=0.3.(3) will dilute 10 times, 500 times, 1000 times of good bacterium liquid difference redilution, and get the good bacterium liquid 6 μ l of this dilution,

Be inoculated in the YPDA solid medium (YPDA+25mM NaCl+0.5 μ g/L AbA) that contains NaCl and AbA respectively and in the YPDA solid medium (YPDA+0.5 μ g/L AbA) that contains AbA, be inverted cultivation 48 hours for 30 ℃, observe the upgrowth situation of bacterial strain.

Adopting uses the same method changes paddy rice homologous gene OsLti6a over to and obtains recombinating bacterium YR93-31/pAUR123-OsLti6a among the bacterial strain YR93-31; There are some researches prove the function that OsLti6a can complementary bacterial strain YR93-31; To recombinate bacterium YR93-31/pAUR123-OsLti6a as positive control, be used for relatively weighing the additional degree of the function of ZmPMP3-1 gene pairs yeast mutants.

With YR93-1 (bacterial strain of pmp3 genetically deficient sudden change does not take place) is control strain 1; Be seeded in the YPDA substratum (YR93-1 can not be grown in the YPDA substratum that contains the AbA resistance, has only the YR93-31 bacterial strain that has transformed recombinant plasmid can be grown in the YPDA substratum that contains the AbA resistance) that does not contain the AbA resistance and contain in the YPDA solid medium (YPDA+25mM NaCl) of NaCl.

As control strain 2, be inoculated in the YPDA solid medium (YPDA+25mM NaCl+0.5 μ g/L AbA) that contains NaCl and AbA respectively and in the YPDA solid medium (YPDA+0.5 μ g/L AbA) that contains AbA with YR93-31.

The result is as shown in Figure 1, is YR93-1, YR93-31, YR93-31/pAUR123-OsLti6a (pAUR123-OsLti6b), the upgrowth situation of YR93-31/pAUR123-ZmPMP3 (pAUR123-ZmPMP3-1) bacterial strain in YPDA and 25mM NaCl+YPDA substratum among the figure; Be respectively the bacterium liquid of 1000,500,10 and 1 times of dilutions from left to right.

From figure, find out, in control medium (YPDA), four kinds of bacterial strain growing way basically identicals, as if the growing way of YR93-31 also be better than other bacterial strains; And under 25mM NaCl handled, the growing way of four kinds of bacterial strains all weakened, but the growing way of YR93-31 bacterial strain all is lower than other 3 bacterial strains, therefore, inferred that the ZmPMP3-1 gene maybe be relevant with intracellular Na+ metabolism.

3, Subcellular Localization in the onion epidermis

1), the acquisition of Subcellular Localization expression vector pBI221-ZmPMP3-GFP

PBI221-GFP carrier enzyme is cut: adopt Xba I and Sac II double digestion pBI221-GFP carrier, obtain enzyme and cut back BI221-GFP carrier.

30 μ l system enzymes are cut the purpose fragment:

pMD18-T/ZmPMP3-1(300ng/μl) 2μl

Buffer3 3μl

BSA 0.3μl

ddH ₂O 22.7μl

Xba?I 1μl

Sac?II 1μl

37 ℃ of 2hr; Run 1% sepharose, the time of race will be grown as far as possible, cuts glue and reclaims, and obtains enzyme and cuts the back fragment.

PBI221-GFP after enzyme is cut the back fragment and enzyme is cut is connected:

10 μ l systems:

Enzyme is cut back pBI221-GFP (20ng/ μ l) 0.8 μ l

Enzyme is cut back fragment (100ng/ μ l) 7.2 μ l

Buffer 1μl

T4 ligase enzyme 1 μ l

16 ℃ of 12hr; Transformed into escherichia coli competence Top10; Obtain transformant; Extract the plasmid of transformant and send to order-checking; The result for this plasmid for ' Xba I and Sac II that terminal 1-177 position Nucleotide is inserted into the pBI221-GFP carrier cut the carrier that the site obtains, called after pBI221-ZmPMP3-GFP from 5 with sequence in the sequence table 1.The coding region of ZmPMP3 is positioned at promotor CaMV35S downstream among the pBI221-ZmPMP3-GFP.

The preparation of onion material:

In Bechtop, onion is peelled off after the exterior skin, cut the bulb written treaty 4cm of third and fourth layer ²Square, peel epidermis gently, entocuticle and MS solid medium are adjacent to, less as far as possible bubble, petridish seals subsequent use with Parafilm.

Portable particle gun bullet is made:

(1) prepares 1ml, 200 μ l, 20 μ l liquid-transfering guns, one bottle of absolute ethyl alcohol, timer, marking pen, paper handkerchief.

(2) claim that 20mg (0.02g) PVP puts into the 1.5ml centrifuge tube of a dried and clean, add the 1ml absolute ethyl alcohol, make its dissolving, concentration is the mother liquor of 20mg/ml.

(3) sucking-off 12.5 μ l mother liquors are diluted to 5ml, and making its final concentration is 0.05mg/ml.

(4) claim 2.mg 1 μ M bronze, the 1.5ml centrifuge tube of putting into a dried and clean with the 1ml absolute ethyl alcohol carry out 3 cleanings (vortex, of short duration centrifugal, sucking-off.Attention: have a small amount of bronze to be bonded on the tube wall this moment).

(5) add 100 μ l 0.05M spermidines, vortex mixing, ultrasonication 5～10sec (removal electric charge).

(6) add the above-mentioned pBI221-ZmPMP3-GFP that obtain of 25 μ l (1 μ g/ μ l) mixing.Vortex limit, limit adds 100 μ l 1M CaCl ₂, room temperature leaves standstill 10min.

(7) clean 5 times with the 1ml absolute ethyl alcohol, method is the same.

(8) with 3ml 0.05mg/ml PVP bronze is diluted in the big centrifuge tube of a 10ml.

(9) open N ₂Main valve, the adjustment reducing valve makes N ₂Pressure is about 0.4, and using purity is 99.997% N ₂Drying tube 15min.Stop N ₂Air-flow, the cutting pipe makes it be longer than platform 10cm then.

One end of the pipe that (10) will cut links to each other with syringe, and vortex gold particle solution also changes this solution over to the pipe of this cutting rapidly, makes its each end all leave the space of 6cm.

(11) will go up pipe that the step fills solution rapidly and place on the upholder and make solution stop 3min, its position that is full of of mark at pipe.Slowly move solution just through the right side mark with syringe.Make pipe Rotate 180 °, solution is all poured out.

(12) make pipe rotation 30sec, open N ₂At 0.35～0.4rpm, rotation 5min.

(13) will manage and take out, stop N from supporting rack ₂Air-flow.Cut the pipe of sealing, bullet is put into the container that fills dried silica gel.

Particle gun bombardment onion epidermis:

(1) with the bullet that the makes particle gun of packing into.

(2) particle gun is linked to each other with the helium jar, pressure is 150～300psi.

(3) open the pre-prepd petridish that is placed with onion epidermis, particle gun is aimed at onion epidermis bombard, every onion epidermis is made two rifles.

(4) 26 ℃, dark condition is cultivated 24hr down.

With empty carrier pBI221-GFP is contrast.

The Subcellular Localization fluorescent signal is observed:

The onion epidermis that takes out cultivation 24h under the dark condition adds a water on slide glass, covered notes that bubble is not arranged, and is put in to observe the green fluorescence signal on the fluorescence co-focusing microscope, and wherein excitation wavelength is 488nm, and scanning wavelength is 500-535nm.

The result sees shown in Figure 2, and wherein, A is the Subcellular Localization result before the plasmolysis; B. the Subcellular Localization result after the plasmolysis, C is for transforming the contrast of pBI221-GFP empty carrier.Nucleus, tenuigenin and cytolemma at the onion epidermis cell that contrasts all can detect GFP fluorescence, show with empty carrier pBI221-GFP and compare that ZmPMP3 is positioned in the cytolemma.Result's reality of ZmPMP3 Subcellular Localization, this gene is positioned at cytolemma and plays a role, and participates in the ion transport relevant physiological process of film probably.

The acquisition and the functional study of embodiment 2, commentaries on classics ZmPMP3 Arabidopis thaliana

Plant binary expression vector pCAMBIA3301 (hereinafter to be referred as p3301, Canberra, Australia is provided by the English professor of kingdom in this article; Zhao J., Sun Z., Zheng J., Guo X.; Dong Z., Huai J., Gou M.; He J., Y.Jin, Wang J.and Wang G.; Cloning and characterization of a novel CBL-interacting protein kinase from maize, Plant Mol Biol 69 (2009): 661-674., the public can obtain from crop science institute of the Chinese Academy of Agricultural Sciences.) and agrobacterium tumefaciens GV3101 (Zhao J., Sun Z., Zheng J., Guo X.; Dong Z., Huai J., Gou M.; He J., Y.Jin, Wang J.and Wang G.. (2009) Cloning and characterization of a novel CBL-interacting protein kinase from maize; Plant Molecular Biology, 69:661-674., the public can obtain from crop science institute of the Chinese Academy of Agricultural Sciences.)

The preparation of substratum commonly used and solution

YEB liquid nutrient medium (1L):

Yeast extract 1g

Carnis Bovis seu Bubali cream 5g

Peptone 5g

Sucrose 5g

MgSO ₄.7H ₂O 0.5g

Adding distil water is settled to 1L, transfers PH to 7.0, autoclaving.

YEB solid medium: add 15g agar powder, autoclaving in every liter of liquid nutrient medium.

Contain antibiotic YEB substratum:

In the YEB substratum behind autoclaving, when temperature is reduced to 50 ℃ of left and right sides, add required microbiotic (Rif of 100mg/L, the Kan of 100mg/L), shake up.

The MS selective medium that contains PPT:

MS substratum behind the autoclaving adds final concentration when temperature is reduced to 50 ℃ of left and right sides be the PPT of 7mg/L, shakes up.

The bar gene primer:

Barp-F：5′-GCGGTCTGCACCATCGTC-3′

Barp-R：5′-GTACCGGCAGGCTGAAGTCCA-3′

Have the restriction enzyme site primer:

M1-3301-F1:5`-CATCGGCA CCATGG(underscore is the NcoI restriction enzyme site to CGGAGGGGACT-3`, and the former sequence in this site is T, but in the process of carrier construction, has introduced restriction enzyme site in this position, so made the T of this sequence into G in order to mate restriction enzyme site.)

M1-3301-R1：5`-ATGGGTCACCGTCGGCAAGGATGAC-3`

The RT-PCR the primer:

ZmPMP3-1-RT-F：5`-CAACTGCGTGGACATCCTGA-3`

ZmPMP3-1-RT-R：5`-GGCGTAGATGGCGTAGATGA-3`

Ms substratum, D-mannitol (D-N.F,USP MANNITOL) are (sigma) available from through Bioisystech Co., Ltd of HTC of section; NaCl is available from the biological Ltd of ancient cooking vessel state.

Arabidopis thaliana actin primer

Actin-F：5′-ATTCAGATGCCCAGAAGTCTTGT-3′

actin-R：5′-GAAACATTTTCTGTGAACGATTCC-3′

1, the acquisition of pCAMBIA3301-ZmPMP3 carrier

With pMD18-T/ZmPMP3-1 is template; Increase with M1-3301-F1 and M1-3301-R1; The PCR product that obtains; Through order-checking, this PCR product has in the sequence sequence 3, and ' terminal 87-283 position Nucleotide, the coding region of this PCR product gene are that sequence 3 is from 5 ' terminal 88-264 position Nucleotide from 5.

Above-mentioned PCR product is cut the pCAMBIA3301 carrier segments that obtain with the BstEII double digestion with the same enzyme of process through NcoI to be connected; The connection product transformed into escherichia coli competence Top10 that obtains; Obtain transformant; Extract the plasmid of transformant and cut evaluation through PCR and enzyme; Be male and send to order-checking, the result for this plasmid for ' terminal 88-283 position Nucleotide is inserted into the NcoI of pCAMBIA3301 carrier (hereinafter to be referred as p3301) and the carrier that the BstEII restriction enzyme site obtains, called after pCAMBIA3301-ZmPMP3 from 5 with sequence in the sequence table 3.The coding region of ZmPMP3 is positioned at promotor CaMV35S downstream among the pCAMBIA3301-ZmPMP3, is plant overexpression vector.

Specific as follows:

The coding region DNA of amplification ZmPMP3, introduce restriction enzyme site:

50 μ l systems: recombinant plasmid pMD18-T/ZmPMP3-1 0.85 μ l

M1-3301-F1 2.5μl(10μM)

M1-3301-R1 2.5μl(10μM)

2×Pfu?PCR?Master?Mix 25μl

ddH ₂O 19.15μl

Pcr amplification program: 94 ℃ of 10min;

94℃?30sec；

68℃?30sec；

72℃?1min；

72℃?10min；

4 ℃ of insulations, 35 circulations.

Purifying and recovering behind the electrophoresis detection purpose band obtains PCR product behind the purifying.The result sees shown in Figure 3, and wherein

swimming lane

1,2,3 is respectively the purpose band that has restriction enzyme site that negative results of comparison, the pcr amplification of DNA maker, pcr amplification obtain, and the arrow indication is the purpose band, can find out the purpose fragment that obtains 177bp.

Make up p3301-ZmPMP3:

50 μ l systems: the substep enzyme is cut:

PCR product behind the above-mentioned purifying that obtains (50ng/ μ l) 25 μ l

Buffer3 5μl

ddH ₂O 17.5μl

Nco?I 1μl

37℃ 2h

Add the 1ml absolute ethyl alcohol, under 4 ℃, the centrifugal 30min of 12000rpm; Outwell absolute ethyl alcohol, dry up.

Add: Buffer3 5 μ l

ddH ₂O 43.5μl

BSA 0.5μl

BstE?II 1μl

60 ℃ of 2h; Run 1% sepharose, cut glue and reclaim, obtain enzyme and cut back PCR product.

P3301 carrier enzyme is cut:

30 μ l systems (enzyme is cut three pipes simultaneously): the substep enzyme is cut:

P3301 plasmid (50ng/ μ l) 10 μ l

Buffer3 3μl

ddH ₂O 14.7μl

Nco?I 1μl

37℃?2h

Add: Buffer3 3 μ l

ddH ₂O 25.7μl

BSA 0.3μl

BstE?II 1μl

60 ℃ of 2h; Run 1% sepharose, the time of race will be grown as far as possible, cuts glue and reclaims, and obtains enzyme and cuts back p3301.

Connect:

9.8 μ l system:

P3301 after enzyme is cut (20ng/ μ l) 0.8 μ l

Enzyme is cut back PCR product (50ng/ μ l) 7 μ l

Buffer 1μl

T4 ligase enzyme 1 μ l

16 ℃ of 12h; Transformed into escherichia coli competence Top10, bacterium liquid PCR screening positive clone.

Bacterium liquid PCR screening positive clone:

10 μ l systems: fresh bacterium liquid 1 μ l

M1-3301-F1 and M1-3301-R1 (10 μ M) 2 * 0.5 μ l

Taq 0.5μl

Buffer 1μl

dNTP 1μl

ddH ₂O 5.5μl

Use 1% agarose gel electrophoresis, detect PCR result, the fragment that obtains 177bp is the PCR positive colony.

Enzyme is cut detection:

The PCR positive colony is extracted plasmid with NcoI and BstEII double digestion; The result sees shown in Figure 4, and

swimming lane

1,2 is the electrophoresis result that enzyme is cut evaluation, and swimming lane 3 is DNA marker; The arrow indication is the purpose band; From figure, find out, obtain the fragment of 177bp, this plasmid called after pCAMBIA3301-ZmPMP3.

2, change the acquisition of ZmPMP3 Arabidopis thaliana

1) Arabidopis thaliana

Wild-type Arabidopis thaliana Columbia (Arabidopsis thaliana; Ecotype Columbia) (DIRK VALVEKENS; MARC VAN MONTAGU; MIEKE VAN LIJSEBETTENS.Agrobacterium tumefaciens-mediated transformation of Arabidopsis thaliana root explants by using kanamycin selection.Proc.Natl.Acad.Sci.USA Vol.85; Pp.5536-5540, the public can obtain from crop science institute of the Chinese Academy of Agricultural Sciences.) seed earlier with 75% alcohol disinfecting 1min, uses 0.5%NaClO (v/v) sterilization 10min, in the super clean bench, with sterilization ddH again ₂O rinsing 5 times, each 1min, dibbling is to the MS solid medium, and 4 ℃ of vernalization 3d are positioned over illumination box then, and 22 ℃ of 16h illumination/8h dark conditions are transplanted in vermiculite is housed after the about week of growth down: continue in the capsule of nutrition soil (1: 1) to cultivate.

2), the preparation of Agrobacterium competent cell

(1) the single bacterium colony of picking agrobacterium tumefaciens GV3101 (contains Rif in 3ml YEB liquid nutrient medium ⁺100 μ g/ml), 28 ℃ of shaking culture are spent the night.

(2) get incubated overnight bacterium liquid 1% and be inoculated in YEB liquid nutrient medium (Rif ⁺100 μ g/ml) in, 28 ℃ of shaking culture are 0.5 to OD600, need 4h.

(3) ice bath 30min, 4 ℃ of centrifugal 5min of 5000rpm abandon supernatant.

(4) add 10ml 0.15M NaCl suspension agrobatcerium cell, 4 ℃ of centrifugal 5min of 5000rpm remove supernatant.

(5) the 20mM CaCl of adding 1ml precooling ₂Suspension cell uses in the ice bath 24h or is distributed into every pipe 200 μ l, quick-frozen 1min in the liquid nitrogen, and it is subsequent use to put-76 ℃ of preservations.

3), plant expression vector transforms the acquisition of Agrobacterium GV3101/pCAMBIA3301-ZmPMP3

(1) get 200 μ l GV3101 competent cells, add the pCAMBIA3301-ZmPMP3 that 1 μ g builds, quick-frozen 1min in the liquid nitrogen, 37 ℃ of water-bath 5min add the YEB liquid nutrient medium that 1ml does not contain resistance then, 28 ℃ of 140rpm, shaking culture 4h at a slow speed.

(2) the centrifugal 30sec of 10000rpm abandons supernatant, adds 0.1ml YEB substratum suspension cell again, coats on the YEB flat board that contains 100 μ g/ml Kan and 125 μ g/ml Rif, cultivates about 36～48h for 28 ℃.

(3) the single bacterium colony that grows on the picking flat board is inoculated in the YEB nutrient solution (containing 100 μ g/ml Kan and 125 μ g/mlRif), and 28 ℃ of shaking culture are spent the night.

(4) carry out bacterium liquid PCR and identify that primer is M1-3301-F1 and M1-3301-R1, the result sees shown in Figure 5; Wherein, swimming lane 2-8 is the bacterium liquid PCR result of Agrobacterium GV3101, and swimming lane 1 is DNA marker; Swimming lane 9 finds out that the fragment that obtains 177bp is the PCR positive colony for the negative contrast in the PCR reflection from figure; PCR is identified that positive colony extracts plasmid; Send to order-checking, the result contains the positive colony called after GV3101/pCAMBIA3301-ZmPMP3 of this plasmid for this plasmid is pCAMBIA3301-ZmPMP3.

4), the conversion of Arabidopis thaliana

Be stained with colored method (floral dipping):

(1) with 1) the wild-type Arabidopis thaliana Columbia (Arabidopsis thaliana, ecotype Columbia) in about three weeks of the growth that obtains transform, and water sufficient water in the previous day that transforms, cut all fruit pods before transforming.

(2) in 1: 1000 ratio activatory positive colony bacterium liquid GV3101/pCAMBIA3301-ZmPMP3 is transferred and has in the YEB liquid nutrient medium of kalamycin resistance in 500ml, 28 ℃ of shaking culture to OD600 be 1.2 (cultivating 24h).

(3) the 4 ℃ of centrifugal 15min collection of 4000rpm bacterium.

(4) infiltrate the resuspended thalline of damping fluid (1 * MS macroelement+5% sucrose) with 200ml, making OD600 is 0.8, adds sil-wet 40 μ l (0.2 ‰, volumn concentration).

(5) the Arabidopis thaliana bud is immersed in (4), infect 1min.

(6) with preservative film or freshness protection package parcel plant, behind 16 ℃ of dark condition held 24hr, be put under the normal condition and grow, obtain T0 for changeing the ZmPMP3 Arabidopis thaliana.

Results T0 is for the seed that changes the ZmPMP3 Arabidopis thaliana, for T1 for changeing ZmPMP3 Arabidopis thaliana seed.

3, the screening of transgenic line and evaluation

1) the ppt herbicide sprays is screened positive transgenic line:

(1) T1 directly is seeded in through 4 ℃ of vernalization treatment after vermiculite is housed for changeing ZmPMP3 Arabidopis thaliana seed: in the pallet of nutrition native (1: 1); Growth is 20 days under the normal condition; (Japanese Meiji Pharmaceutial Ltd. draws 500 μ lppt stostes, is dissolved in the 1L zero(ppm) water with the ppt weedicide of 0.5 ‰ (volumn concentrations); The acquisition volume percent is 0.5 ‰ working fluid, sprays the Arabidopis thaliana seedling with this liquid.) the spraying screening; Sprayed continuously once a day 3 days, after the week, observations; Because the ppt resistant gene on the expression vector p3301 can be incorporated in the Plant Genome with goal gene when transforming; So plant that can normal growth after weedicide ppt screening should be the transgenic positive plant in theory, the result is: obtain 35 strains growth normal plant, be ppt and screen positive T1 for changeing the ZmPMP3 Arabidopis thaliana.

Transplant ppt and screen positive T1 for changeing ZmPMP3 Arabidopis thaliana plant continuation cultivation, every capsule kind 2 strains.Each ppt screens positive T1 and extracts DNA when the ZmPMP3 Arabidopis thaliana grows to 2 leaves in a small amount for changeing; Carrying out PCR identifies; Primer is M1-3301-F1 and M1-3301-R1; The result be the segmental plant that obtains 177bp be positive T1 for changeing the ZmPMP3 Arabidopis thaliana, obtain the positive T1 of 14 strains for changeing the ZmPMP3 Arabidopis thaliana.

2) identify the Arabidopis thaliana positive plant through genome PCR:

The A:SDS method is extracted the positive T1 of 35 strain PPT Screening and Identification in a small amount for changeing the total DNA of ZmPMP3 Arabidopis thaliana

SDS extracts damping fluid (100ml):

Tris-HCl(1mol/L?pH8.0) 10ml

EDTA(0.5mol/L?pH8.0) 10ml

NaCl (1mol/L) 50ml (or 2.9g solid)

Sterilization ddH

₂0 is settled to 100ml.

The SDS lysis buffer:

SDS extracts damping fluid: 20%SDS=15: 1 (V/V)

Step:

(1) gets an amount of PPT and be accredited as positive T1 and put into the 1.5ml centrifuge tube, add the abundant grind into powder of liquid nitrogen (preferably only adding a liquid nitrogen goes to grind) for changeing the ZmPMP3 Arabidopsis leaf.

(2) add the SDS lysis buffer of 65 ℃ of preheatings of 800 μ l, fully behind the mixing, 65 ℃ of following extracting 20min, during mixing 2 times again.

(3) add 250 μ l 5mol/L KAc, place 5min on ice.

(4) 12000rpm, centrifugal 10min draws in the new centrifuge tube of supernatant to.

(5) 12000rpm, centrifugal 5min draws in the new centrifuge tube of supernatant to.

(6) add 600 μ l Virahol mixings.

(7) 4 ℃ of 12000rpm, centrifugal 15min

(8) abandon supernatant, precipitate 2 times with 75% washing with alcohol.

(9) the centrifugal 3min of 12000rpm, vacuum is drained or super clean bench dries up.

(10) add 20 μ l 1 * TE or sterilization ddH ₂O dissolving DNA deposition ,-20 ℃ of preservations.

Purifying:

(1) adds RNase 1 μ l in above-mentioned (10), 37 ℃ of digestion 30min.

(2) mend sterilization ddH ₂O is to TV 600 μ l.

(3) add 300 μ l phenol, concussion mixing 5～10min; Add 300 μ l chloroforms, concussion mixing 5min.

(5) add 600 μ l chloroforms, concussion mixing 2min.

(6) 12000rpm, centrifugal 2min draws in the new centrifuge tube of supernatant to.

(7) add the NaAc mixing of 1/10 volume, add the cold absolute ethyl alcohol of 2～3 times of volumes again, 4 ℃ leave standstill 30min.

(8) 4 ℃ of 12000rpm, centrifugal 20min abandons supernatant.

(9) 75% ethanol are washed deposition 2 times.

(10) the centrifugal 3min of 12000rpm, vacuum is drained or super clean bench dries up.

(11) add 20 μ l 1 * TE or sterilization ddH ₂O dissolving DNA deposition ,-20 ℃ of preservations are subsequent use, obtain DNA.Through electrophoresis detection, the result is as shown in Figure 6, obtains genomic dna.

B: carry out PCR and identify:

Genomic dna with above-mentioned acquisition is a template, carries out PCR with primer Barp-F:5 '-GCGGTCTGCACCATCGTC-3 ' and Barp-R:5 '-GTACCGGCAGGCTGAAGTCCA-3 ' and identifies, detects the bar gene.With primer M1-3301-F1 and M1-3301-R1, carry out PCR and identify, detect the ZmPMP3 gene.

20 μ l reaction systems:

10×PCR?buffer 2μl

dNTP?mix 0.4μl

Each 0.5 μ l of Barp-F and Barp-R (or M1-3301-F1 and M1-3301-R1) (10 μ M)

Positive T1 is for plant DNA 1.5 μ l

Taq?polymerase(2.5U/μl) 0.6μl

ddH ₂O 13.5μl

The pcr amplification program:

94℃?10min；

94℃?30sec；

63 ℃ (bar gene)/58 ℃ (ZmPMP3-1 gene);

30sec, 72 ℃ of 2min, 33 circulations;

72 ℃ of 10min; 4 ℃ of insulations.

1% agarose gel electrophoresis detected result, the positive T1 of Fig. 7 detects bar gene result for transgenic arabidopsis genome PCR, and swimming lane 1 is contrast, changes empty carrier arabidopsis gene group pcr amplification result; Swimming lane 2-13 is genome pcr amplification result for changeing the strain of ZmPMP3 Arabidopis thaliana; Swimming lane 14 is genome pcr amplification result for the strain of wild-type Arabidopis thaliana; Swimming lane 15 is over against photograph, the pcr amplification result of plasmid pCAMBIA3301-ZmPMP3; Swimming lane 16 is DNA marker.

Fig. 8 is that T1 detects ZmPMP3 result for transgenic arabidopsis genome PCR, and swimming lane 1 is DNA marker (same Figure 10); Swimming lane 2 changes empty carrier arabidopsis gene group pcr amplification result for being contrast; Swimming lane 2-12 is genome pcr amplification result for changeing the strain of pCAMBIA3301-ZmPMP3 gene Arabidopis thaliana; Swimming lane 13 is over against photograph, the pcr amplification result of plasmid pCAMBIA3301-ZmPMP3; Swimming lane 14 is genome pcr amplification result for the strain of wild-type Arabidopis thaliana.

From figure, find out; Pcr amplification goal gene and bar gene are identified; The positive T1 of bar gene of purpose band and 470bp that can amplify 230bp obtains the positive T1 of 14 strains for transgenic arabidopsis for transgenic arabidopsis, proves that the ZmPMP3 gene has been incorporated in the arabidopsis gene group.And the commentaries on classics empty carrier plant Arabidopis thaliana strain of contrast system has only the bar band of 470bp, does not have this band in the wild-type Arabidopis thaliana.

3) seed of gathering in the crops for commentaries on classics ZmPMP3 Arabidopis thaliana from positive T1 is that T2 is for seed; With above-mentioned T2 for the seed dibbling at the enterprising row filter of MS substratum that contains weedicide (7mg/L ppt); There is the one-tenth of the four leaves positive plant of living to be transplanted to vermiculite is housed growth: continue in the capsule of nutrition soil (1: 1) to cultivate, and the results seed to obtain T3 for changeing the ZmPMP3 Arabidopis thaliana.T3 for changeing the ZmPMP3 Arabidopis thaliana through the strain that the ppt screening shows as 100% resistance is; Carrying out PCR again identifies; Method is with 2); The result obtains positive T3 for changeing ZmPMP3 Arabidopis thaliana homozygous lines, and 12 strain systems of random choose continue to breed the ZmPMP3 Arabidopis thaliana homozygous lines for changeing from positive T3, obtain T4 for changeing ZmPMP3 Arabidopis thaliana seed.

Sowing T4 obtains T4 for changeing ZmPMP3 Arabidopis thaliana homozygous lines for changeing ZmPMP3 Arabidopis thaliana seed, adopts 2) method, carry out PCR and identify, obtain 12 T4 for changeing ZmPMP3 Arabidopis thaliana homozygous lines.

Adopting uses the same method changes empty carrier pCAMBIA3301 over to wild-type Arabidopis thaliana Columbia (Arabidopsis thaliana; Ecotype Columbia) in; Obtain changeing the empty carrier Arabidopis thaliana, identify that through same PCR the result is not for there being goal gene ZmPMP3.

4) the total RNA of Arabidopis thaliana extracts and ThermoScript II

Choosing 6 and be numbered 4,5,6,8,11 and 12 strains system and carry out real time-PCR Molecular Detection for changeing ZmPMP3 Arabidopis thaliana homozygous lines from 12 T4, is contrast with T4 for commentaries on classics empty carrier Arabidopis thaliana with the wild-type Arabidopis thaliana.

Specific as follows: as to use the sky to take root in thing total RNA extraction reagent box and extract and be numbered 4,5,6,8,11 and 12 T4 for changeing ZmPMP3 Arabidopis thaliana homozygous lines and the total RNA of contrast Arabidopsis leaf, and use the invitrogen M-MlV ThermoScript II first chain synthetic agent box to obtain cDNA.

Through real time quantitative PCR method, use the Sybergreen dyestuff.

20 μ l reaction systems:

ZmPMP3-1-RT-F and ZmPMP3-1-RT-R (10 μ M) 2 * 0.4 μ l

T4 is for plant 1 μ l

2×Pfu?PCR?subygreen?Mix 10μl

ddH ₂O 6.8μl

The pcr amplification program:

94℃?1min；

94℃?5sec，

60℃?5sec，

72℃?31sec，；

40 circulations.

With the actin gene as contrast.

The result is: compare with commentaries on classics empty carrier Arabidopis thaliana with the wild-type Arabidopis thaliana; Be numbered 4,5,6,8,11 and 12 T4 and all can detect the expression (177bp fragment) of ZmPMP3-1, and do not detect in wild-type and the commentaries on classics empty carrier Arabidopis thaliana plant for changeing ZmPMP3 Arabidopis thaliana homozygous lines.Wild-type, change empty carrier Arabidopis thaliana plant, be numbered 4,5,6,8,11 and 12 T4 and actin expression of gene (200bp fragment) all arranged for changeing in the ZmPMP3 Arabidopis thaliana homozygous lines.

4, the phenotypic evaluation of transgenic line

A: ZmPMP3 Arabidopis thaliana homozygous lines is carried out salt stress (NaCl) and drought stress (D-N.F,USP MANNITOL) is handled for changeing to T4, observes phenotype, and is specific as follows:

1) germination rate statistics: with disinfectant wild-type Arabidopis thaliana (CK), change the empty carrier Arabidopis thaliana and be numbered 6 and 11 T4 for changeing seed dibbling respectively that the ZmPMP3 Arabidopis thaliana isozygotys to containing different concns NaCl (0; 150mM) and on the MS substratum of different concns D-mannitol (0,300 and 350mM); 4 ℃ of dark condition vernalization 3 days moves to 22 ℃, 16hr illumination (100 μ E m then ^-2s ^-1Sprout in the illumination box of)/8hr dark, statistics is cultivated the sprouting number after 3,4,5 days in MS substratum, 150mM NaCl+MS substratum and 300mM D-mannitol+MS, 350mM D+MS substratum respectively.Each repeats 40 seeds, and each is handled independently 5 repetitions are set, and the result takes the mean.In the statistics, be standard to grow two green cotyledon.

The result is following: as shown in Figure 9,

In MS substratum (A), wild-type (ck) is respectively 99%, 100%, 100% at the germination rate of sprouting 3,4,5 days; And No. 6 T4 are respectively 98.5%, 99%, 99% for the germination rate sprouting 3,4,5 days that changes ZmPMP3 Arabidopis thaliana strain system.

In 150mM NaCl+MS substratum (B), wild-type (ck) is respectively 20%, 22.5%, 25% at the germination rate of sprouting 3,4,5 days; And No. 6 T4 are respectively 51.7%, 60%, 61.2% for the germination rate sprouting 3,4,5 days that changes ZmPMP3 Arabidopis thaliana strain system, the germination rate in the ck.

In 300mM D-mannitol+MS substratum (C), wild-type (ck) is respectively 24.2%, 90.8%, 96.7% at the germination rate of sprouting 3,4,5 days; And No. 6 T4 are respectively 60%, 84.4%, 90% for the germination rate sprouting 3,4,5 days that changes ZmPMP3 Arabidopis thaliana strain system; And No. 11 T4 are respectively 85.6%, 95.6%, 96.7% for the germination rate sprouting 3,4,5 days that changes ZmPMP3 Arabidopis thaliana strain system.

In 350mM D-mannitol+MS substratum (D), wild-type (ck) is respectively 47.5%, 82.5% at the germination rate of sprouting 4,5 days; No. 6 T4 tie up to the germination rate of sprouting 4,5 days for the strain of commentaries on classics ZmPMP3 Arabidopis thaliana and are respectively 80%, 86.7%; No. 11 T4 tie up to the germination rate of sprouting 4,5 days for the strain of commentaries on classics ZmPMP3 Arabidopis thaliana and are respectively 76.9%, 85%.

The wild-type Arabidopis thaliana does not have significant difference with commentaries on classics empty carrier Arabidopis thaliana result.Therefore, 6, No. 11 T4 tie up to early stage germination rate apparently higher than ck for changeing the strain of ZmPMP3 Arabidopis thaliana, explains that ZmPMP3 except that the coercing of response salt, also responds drought in early days in sprouting and coerces.

2) biology is heavily added up:

With disinfectant wild-type Arabidopis thaliana (CK), change the empty carrier Arabidopis thaliana and be numbered 6,11,12 T4 for changeing seed that the ZmPMP3 Arabidopis thaliana isozygotys according to 1) method handle, different is in following substratum, to cultivate respectively:

1) MS substratum; 2) 150mMNaCl+MS substratum; 3) 175mMNaCl+MS substratum;

4) 300mM D-mannitol+MS substratum;

Above growth 2 all respectively organizing biologies heavy (weight of whole strain seedling comprises overground part and root) that each strain is the Arabidopis thaliana seedling of statistics.

Each takes by weighing strain is 20 strain Arabidopis thalianas, test repetition 3 times, and the result takes the mean.

The result shown in Figure 10 and 11, wherein,

In the MS substratum, ck, No. 6 T4 heavily are respectively 0.14225g, 0.14675g, 0.179g, 0.17875g for commentaries on classics ZmPMP3 Arabidopis thaliana, No. 12 T4 for the biology that changes the ZmPMP3 Arabidopis thaliana for commentaries on classics ZmPMP3 Arabidopis thaliana, No. 11 T4;

In 150mM NaCl+MS substratum, ck, No. 6 T4 heavily are respectively 0.0835g, 0.10625g, 0.1215g, 0.09275g for commentaries on classics ZmPMP3 Arabidopis thaliana, No. 12 T4 for the biology that changes the ZmPMP3 Arabidopis thaliana for commentaries on classics ZmPMP3 Arabidopis thaliana, No. 11 T4;

In 175mM NaCl+MS substratum, ck, No. 6 T4 heavily are respectively 0.067g, 0.0914g, 0.0947g, 0.0444g for commentaries on classics ZmPMP3 Arabidopis thaliana, No. 12 T4 for the biology that changes the ZmPMP3 Arabidopis thaliana for commentaries on classics ZmPMP3 Arabidopis thaliana, No. 11 T4;

In 300mM D-mannitol+MS substratum, ck, No. 6 T4 heavily distinguish 0.0467g, 0.0387g, 0.0554g, 0.053g for commentaries on classics ZmPMP3 Arabidopis thaliana, No. 12 T4 for the biology that changes the ZmPMP3 Arabidopis thaliana for commentaries on classics ZmPMP3 Arabidopis thaliana, No. 11 T4.

It is thus clear that the biology of 6, No. 11 strains system focuses on and all is significantly higher than ck under the salt stress, and the biology that 11, No. 12 (preferably can be consistent with the strain system of salt stress) strains tie up under the osmotic stress is heavy apparently higher than ck.

The wild-type Arabidopis thaliana does not have significant difference with commentaries on classics empty carrier Arabidopis thaliana result.

3) evaluation in the time of infertility of ripe plant:

With disinfectant wild-type Arabidopis thaliana (as contrast CK), change the empty carrier Arabidopis thaliana and be numbered 4,5,6,11,12 T4 for change seed dibbling that the ZmPMP3 Arabidopis thaliana isozygotys to the MS solid medium under 4 ℃ of dark conditions vernalization 3d, move to 22 ℃, 16hr illumination every day (100 μ E m ^-2s ^-1The vertical growth 5d that places in the illumination box of)/8hr dark; Then seedling replanting is continued to cultivate to containing in the little basin that weight ratio is the composite soil formed of 1: 1 nutrition soil-vermiculite; The use plant nutrition liquid irrigates, and grows for 4 weeks to Arabidopis thaliana, is divided into two groups of processing:

Coerce group: contain the 300mM NaCl aqueous solution to each strain system pouring and coerce processing, pouring once keeps ground moistening weekly;

Control group: to each strain system pouring zero(ppm) water, pouring once keeps ground moistening weekly;

It is phenotype that each strain is observed in two week of processing back (after two weeks of pouring 300mM NaCl), the statistics survival rate.During transplanting, every basin is planted 5 strains, and per 3 basins (15 strain) are a repetition, and test is provided with 3 repetitions.

The use plant nutrition liquid irrigates, and observes phenotype to Arabidopis thaliana growth during 4 weeks, and the result is shown in figure 13, when this moment, Arabidopis thaliana got into generative growth phase, wild-type Arabidopis thaliana (contrast CK), is numbered 5,6,11 T4 for changeing all boltings just of ZmPMP3 Arabidopis thaliana.The wild-type Arabidopis thaliana does not have significant difference with commentaries on classics empty carrier Arabidopis thaliana result.Be numbered 4,12 T4 and do not have significant difference with the T4 that is numbered 5,6,11 for changeing ZmPMP3 Arabidopis thaliana result for changeing the ZmPMP3 Arabidopis thaliana.

Handle two days later; It is phenotype that each strain of group is coerced in observation; The result is shown in figure 14, coerces processing two days later through the 300mM NaCl aqueous solution, wild-type Arabidopis thaliana (as contrast CK) be numbered 4,5,6,11,12 T4 and all begin flavescence for commentaries on classics ZmPMP3 Arabidopis thaliana low side lotus throne leaf.The wild-type Arabidopis thaliana does not have significant difference with commentaries on classics empty carrier Arabidopis thaliana result.

After handling for two weeks; Observe phenotype; The result is shown in figure 15, and wherein NaCl handles ck for coerce the wild-type Arabidopis thaliana of processing through the 300mM NaCl aqueous solution, and contrast ck is the wild-type Arabidopis thaliana of handling with the zero(ppm) water pouring; It is 6 to be numbered 6 T4 for changeing the ZmPMP3 Arabidopis thaliana for what coerce processing through the 300mM NaCl aqueous solution that NaCl handles strain, and the contrast strain is 6 to be numbered 6 T4 for changeing the ZmPMP3 Arabidopis thaliana for what handle with the zero(ppm) water pouring.Be numbered 5,11 T4 and do not have significant difference with the T4 that is numbered 6 for changeing ZmPMP3 Arabidopis thaliana result for changeing the ZmPMP3 Arabidopis thaliana.The wild-type Arabidopis thaliana does not have significant difference with commentaries on classics empty carrier Arabidopis thaliana result.

Handle a kind of sedge that the Arabidopis thaliana part strain system after two weeks extracts out and begin the flavescence of wilting; The Arabidopis thaliana of this moment can not further be accomplished reproductive growth; Shaky and begin death, therefore after salt stress handled for two weeks, statistics can be accomplished the time of infertility Arabidopis thaliana strain number of (plant that promptly can gather in the crops seed); The experiment triplicate, the result takes the mean.

The result is following:

After the 300mM NaCl aqueous solution was coerced processing, wild-type Arabidopis thaliana (as contrast CK) the strain number that can accomplish the time of infertility was 2.67 strains;

After the 300mM NaCl aqueous solution was coerced processing, 4 the T4 of being numbered that can accomplish the time of infertility was 3.34 strains for changeing ZmPMP3 Arabidopis thaliana strain number;

After the 300mM NaCl aqueous solution was coerced processing, 5 the T4 of being numbered that can accomplish the time of infertility was 5 strains for changeing ZmPMP3 Arabidopis thaliana strain number;

After the 300mM NaCl aqueous solution was coerced processing, 6 the T4 of being numbered that can accomplish the time of infertility was 9.67 strains for changeing ZmPMP3 Arabidopis thaliana strain number;

After the 300mM NaCl aqueous solution was coerced processing, 11 the T4 of being numbered that can accomplish the time of infertility was 3.34 strains for changeing ZmPMP3 Arabidopis thaliana strain number;

After the 300mM NaCl aqueous solution was coerced processing, 12 the T4 of being numbered that can accomplish the time of infertility was 3.34 strains for changeing ZmPMP3 Arabidopis thaliana strain number;

In the control group that zero(ppm) water is handled, wild-type Arabidopis thaliana (as contrast CK) the strain number that can accomplish the time of infertility is 5 strains;

In the control group that zero(ppm) water is handled, 4 the T4 of being numbered that can accomplish the time of infertility is 5 strains for changeing ZmPMP3 Arabidopis thaliana strain number;

In the control group that zero(ppm) water is handled, 5 the T4 of being numbered that can accomplish the time of infertility is 5 strains for changeing ZmPMP3 Arabidopis thaliana strain number;

In the control group that zero(ppm) water is handled, 6 the T4 of being numbered that can accomplish the time of infertility is 5 strains for changeing ZmPMP3 Arabidopis thaliana strain number;

In the control group that zero(ppm) water is handled, 11 the T4 of being numbered that can accomplish the time of infertility is 5 strains for changeing ZmPMP3 Arabidopis thaliana strain number;

In the control group that zero(ppm) water is handled, 12 the T4 of being numbered that can accomplish the time of infertility is 5 strains for changeing ZmPMP3 Arabidopis thaliana strain number; Partial results is done shown in Figure 12, can find out, crosses the transgenic arabidopsis of expressing the ZmPMP3 gene and has showed good salt tolerance at reproductive stage.

B, excised leaf moisture holding capacity:

With disinfectant wild-type Arabidopis thaliana (as contrast CK), change the empty carrier Arabidopis thaliana and be numbered 5,6 T4 for change seed dibbling that the ZmPMP3 Arabidopis thaliana isozygotys to the MS solid medium under 4 ℃ of dark conditions vernalization 3d, move to 22 ℃, 16hr illumination every day (100 μ E m ^-2s ^-1The vertical growth 5d that places in the illumination box of)/8hr dark; Then seedling replanting is continued to cultivate to containing in the little basin that weight ratio is the composite soil formed of 1: 1 nutrition soil-vermiculite; The use plant nutrition liquid irrigates; Growing for 6 weeks to Arabidopis thaliana, (this tests in order to identify the hold facility of transgenic arabidopsis to moisture, is one of index of weighing the anti-osmotic stress of plant.), the whole strain of over-ground part of big Arabidopis thaliana is cut with 6 weeks, takes by weighing fresh weight a.Be placed in the air and take by weighing weight b once more behind (23 ℃, humidity 40%) 5h.Then with 90 ℃ the oven dry 24h after to constant weight, take by weighing weight c.Calculate water retention according to formula.The experiment triplicate, results averaged.WRC(％)＝b-c/a-c×100

The result is following: the water retention of Ck is 75.49%; Strain is that 5 water retention is 77.98%; Strain is that 6 water retention is 79.02%.The wild-type Arabidopis thaliana does not have significant difference with commentaries on classics empty carrier Arabidopis thaliana result.

The result maps shown in figure 16, can find out that strain is that 5,6 WRC is significantly higher than ck, explains that to be numbered 5,6 T4 higher than the blade moisture holding capacity of wild-type Arabidopis thaliana for changeing ZmPMP3 Arabidopis thaliana wild-type Arabidopis thaliana.

Claims

1. a protein is following 1) or 2) or 3) protein:

2. the said proteinic encoding sox of claim 1.

3. encoding sox according to claim 2 is characterized in that: said encoding sox is following 1)-8) in the gene shown in arbitrary:

1) dna molecular shown in the sequence 1 in the sequence table;

4) dna molecular shown in the sequence 3 in the sequence table;

4. the recombinant expression vector, reorganization bacterium, transgenic cell line or the expression cassette that contain claim 2 or 3 said encoding soxs.

5. recombinant expression vector according to claim 4 is characterized in that: the recombinant expression vector that said recombinant expression vector obtains for the MCS with claim 2 or 3 described encoding soxs insertion carrier pCAMBIA3301.

6. a method of cultivating the transgenic plant of resistance of reverse and/or moisture holding capacity raising is that claim 2 or 3 described encoding soxs importing purpose plants are obtained transgenic plant, and resistance of reverse of said transgenic plant and/or moisture holding capacity are higher than said purpose plant.

7. method according to claim 6 is characterized in that:

The blade water retention that it is said transgenic plant that the moisture holding capacity of said transgenic plant is higher than said purpose plant is higher than said purpose plant;

Said resistance of reverse is drought tolerance and/or salt tolerance.

8. according to claim 6 or 7 described methods, it is characterized in that:

Claim 2 or 3 said encoding soxs are to import in the said purpose plant through claim 4 or 5 said recombinant expression vectors.

9. according to the arbitrary described method of claim 6-8, it is characterized in that: said purpose plant is dicotyledons or monocotyledons, and said dicotyledons is preferably Arabidopis thaliana.