CN102453083B - Plant stress tolerance related protein ZmPMP3 and coding gene thereof and application thereof - Google Patents
Plant stress tolerance related protein ZmPMP3 and coding gene thereof and application thereof Download PDFInfo
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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
Technical field
The present invention relates to a kind of and plant stress tolerance correlative protein ZmPMP3 and encoding gene 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 is all the major cause that causes crop failure, 50% of annual averaged occupation crop failure.Abiotic stress is by plant being produced to a series of morphology, the impact of Physiology and biochemistry state and molecular level, thereby the Growth and yield of the plant of impact.Plant, as a complicated biosystem, when response abiotic stress, often produces the variation of a series of biochemical moleculars and gene level.The inside and outside cytolemma of contact cell in plant materials, directly perception comes from the physical chemistry signal of outside, in this simultaneously, and the medium being connected with the external world as cell, when cell is subject to the external world and coerces, 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 has occurred, to defend external injury of coercing cell.Therefore think, some coerce relevant membranin in perception outer signals, and 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), in the plants such as flower of Stinkgrass (Puccinellia tenuiflora), clone has obtained coding and yeast (Saccharomyces cerevisiae) membrane protein gene that Pmp3p consistence is higher respectively.These genes are subject to arid, cold, and Salt Stress-induced, the low-molecular-weight membranin of coding one class.Yeast (Saccharomyces cerevisiae) Pmp3p is a kind of 55 amino acid whose acid hydrophobins that contain.The yeast mutants that has lacked PMP3 contrasts and compares with wild-type, has strengthened the susceptibility to sodium ion and hygromycin B, has also suppressed Trk1p and the dependency of Trk2p mutants which had growth to potassium ion simultaneously.Navarre and Goffeau think that Pmp3p plays and maintains cytolemma electromotive force as a kind of plasmalemma protein, 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 the P77240 with Escherichia coli, P34655, Q20516, Q22702, Q22701, Q22700 in nematode (Caenorhabditis elegans), and with plant in the albumen of some stress-inducings genes encoding of expressing there is high homology.
Also from corn, do not find at present similar albumen.
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 gene thereof.
Protein provided by the present invention, name be called ZmPMP3, synthetic, is following 1) or 2) or 3) protein:
1) protein that the aminoacid sequence shown in sequence 2 forms in sequence table;
2) protein that the aminoacid sequence shown in sequence 4 forms in sequence table;
3) by 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 to plant stress tolerance and/or moisture holding capacity by 1) derivative protein.
In above-mentioned sequence table, sequence 2 or sequence 4 form by 58 amino-acid residues.The replacement of described 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 encoding gene of the above-mentioned albumen relevant to plant stress tolerance also belongs to protection scope of the present invention.
The encoding gene of protein provided by the present invention is that described encoding gene is following 1)-8) in the gene shown in arbitrary:
1) DNA molecular shown in sequence 1 in sequence table;
2) in sequence table sequence 1 from the DNA molecular shown in the 87th-283 Nucleotide of 5 ' end;
3) in sequence table sequence 1 from the DNA molecular shown in the 88th-264 Nucleotide of 5 ' end;
4) DNA molecular shown in sequence 3 in sequence table;
5) in sequence table sequence 3 from the DNA molecular shown in the 88th-264 Nucleotide of 5 ' end;
6) in sequence table sequence 3 from the DNA molecular shown in the 88th-283 Nucleotide of 5 ' end;
7) under stringent condition with 1) or 2) or 3) or 4) or 5) or 6) DNA molecule hybridize that limits and the described DNA molecular with plant stress tolerance and/or moisture holding capacity related protein of coding;
8) with 1) or 2) or 3) or 4) or 5) or 6) DNA sequence dna that limits at least 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 at least have a DNA molecular of 99% homology and coding and described plant stress tolerance and/or moisture holding capacity related protein.
Wherein, in sequence table, sequence 1 is comprised of 389 deoxyribonucleotides, and its open reading frame (ORF) is from the 88th-264 bit bases of 5 ' end.
In sequence table, sequence 4 is comprised of 389 deoxyribonucleotides, and its open reading frame (ORF) is from the 88th-264 bit bases of 5 ' end.
Described stringent condition also can be at 6 * SSC, and in the solution of 0.5% SDS, at 65 ℃, hybridization, then uses 2 * SSC, 0.1%SDS and 1 * SSC, and 0.1% SDS respectively washes film once.
The recombinant expression vector that contains above-mentioned protein coding gene, recombinant bacterium, transgenic cell line or expression cassette also belong to protection scope of the present invention.
Between the Nco I that described recombinant expression vector specifically can be at pCAMBIA3301 and BstE II site, insert the pCAMBIA3301-ZmPMP3 that described encoding gene obtains.
The primer pair of above-mentioned arbitrary described encoding gene total length or its any fragment of increasing also belongs to protection scope of the present invention.
Another object of the present invention is to provide 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 gene is imported to the transgenic plant that object plant obtains, and the resistance of reverse of described transgenic plant and/or moisture holding capacity are higher than described object plant.
The excised leaf water retention that the moisture holding capacity of described transgenic plant is described transgenic plant higher than described object plant is higher than described object plant;
Described resistance of reverse is drought tolerance and/or salt tolerance.
Described encoding gene is to import in described object plant by described recombinant expression vector.
Described plant is dicotyledons or monocotyledons, and described dicotyledons is preferably Arabidopis thaliana.
Of the present invention experimental results show that, by clone's method, obtain ZmPMP3 gene, imported the transgenic arabidopsis obtaining in Arabidopis thaliana, compare with wild-type Arabidopis thaliana, the resistance of reverse of transgenic arabidopsis and/or water retention are higher than wild-type Arabidopis thaliana, resistance of reverse shows drought tolerance and/or salt tolerance, and water retention is mainly that the water retention of blade is higher than wild-type Arabidopis thaliana.The ZmPMP3 gene that this experiment obtains and transgenic arabidopsis have important meaning aspect plant genetics and breeding.
Accompanying drawing explanation
Fig. 1 is that ZmPMP3 gene crossing in yeast mutants expressed
Fig. 2 is ZmPMP3 Subcellular Localization.
Fig. 3 is the pcr amplification figure of ZmPMP3
Fig. 4 is that the enzyme of pCAMBIA3301-ZmPMP3 is cut evaluation figure.
Fig. 5 is the PCR evaluation figure of Agrobacterium GV3101/pCAMBIA3301-ZmPMP3.
Fig. 6 is the little transgenic arabidopsis genome of carrying of SDS method.
Fig. 7 is that transgenic arabidopsis Genomic PCR detects bar gene result.
Fig. 8 is that transgenic arabidopsis Genomic PCR detects ZmPMP3 gene result.
Fig. 9 is that statistics is counted in the sprouting of transgenic arabidopsis strain
Figure 10 is that the biology that is of transgenic arabidopsis strain is heavily added up
Figure 11 is the biology weight of the strain of transgenic arabidopsis strain
Figure 12 counts statistical graph for completing Arabidopis thaliana strain in the time of infertility
Figure 13 is the Arabidopis thaliana before 300mM NaCl processes
Figure 14 is that 300mM NaCl processes Arabidopis thaliana two days later
Figure 15 is that 300mM NaCl processed after two weeks, ck and strain 6 under control group and 300mM NaCl processing
Figure 16 is excised leaf water retention statistical graph
Embodiment
The experimental technique using in following embodiment if no special instructions, is ordinary method.
In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.
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 purchased from the white Bioisystech Co., Ltd in Yuanping City, Beijing; Intestinal bacteria competence TOP10, kantlex, taq enzyme, 2 * Pfu PCR Master Mix are purchased from TIANGEN Biotech (Beijing) Co., Ltd.; DNA marker is all purchased from Beijing Quanshijin Biotechnology Co., Ltd; PEG3000 (polyethylene glycol, polyoxyethylene glycol), carrier DNA, YPD substratum, LiAc damping fluid, TE damping fluid are purchased from general Jino (Beijing) Bioisystech Co., Ltd; Aureobasidin A (TaKaRa), pAUR123 carrier (TaKaRa), pMD18-T plasmid (TaKaRa) are purchased from logical (Beijing) bio tech ltd of the six directions; VITAMIN B4, glucose, dNTP mix are purchased from the biological limited liability company of 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, public Ke Cong Institute of Crop Science, Chinese Academy of Agricultural Science obtains.) be the mutant 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, public Ke Cong Institute of Crop Science, Chinese Academy of Agricultural Science obtains.) be the bacterial strain that contains yeast PMP3 gene, with this bacterial strain in contrast.
Transient expression carrier pBI221-GFP (Ying Fu for Subcellular Localization, 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, public Ke Cong Institute of Crop Science, Chinese Academy of Agricultural Science obtains.)。
Contain restriction enzyme site primer sequence:
Build pBI221-GFP Subcellular Localization carrier primer:
GFP-F：5`-TATCTAGAATGTCGGAGGGGACTGCCAACTGCG-3`
GFP-R：5`-ATGCCGCGGTAGTTGTTCTTGGTGATGGCGTAG-3`
Build 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, adjusts pH to 6.5, autoclaving
40% glucose that adds 5ml sterilizing.
YPDA solid medium (100ml): with YPDA liquid nutrient medium, just need add 2g agar, then autoclaving.
AbA: be the abbreviation of microbiotic Aureobasidin A
Key instrument
(concentrator 5301 for gene gun system (Bio-rad), vacuum rotating drying instrument, 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 corn (Zea mays L.) Inbred Lines CN165 (initiative of the king of Institute of Crop Science, Chinese Academy of Agricultural Science sky research institute), with M1-full-F/M1-full-R primer, carry out pcr amplification, obtain the fragment of 389bp, this fragment is connected to pMD18-T (TaKaRa) upper, obtains pMD18-T/ZmPMP3-1.
Also can artificial synthesized sequence 1, with M1-full-F/M1-full-R primer, carry out pcr amplification, this fragment is connected to pMD18-T (TaKaRa) upper, obtain equally pMD18-T/ZmPMP3-1.
M1-full-F：5`-AGCGAAAGGAGAGAAGGAATC-3`
M1-full-R：5`-CATGGGGTGGGTACGGTAG-3`
Take pMD18-T/ZmPMP3-1 as template, with M1-full-F and M1-full-R, as primer, carry out pcr amplification, obtain the PCR product of about 389bp.This PCR product is sent to order-checking, result has in sequence table sequence 1 from 5 ' end 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 sequence table sequence 1 from 5 ' end 88-264 position Nucleotide, the albumen called after ZmPMP3 of this genes encoding, the aminoacid sequence of this albumen is the sequence 2 in sequence table.Sequence 1 is comprised of 389 Nucleotide, and sequence 2 is comprised of 58 amino acid.
2, yeast genetic transformation:
A: the acquisition of Yeast expression carrier
Above-mentioned 1 pMD18-T/ZmPMP3-1 obtaining is carried out to double digestion with Kpn I and Sac I, the fragment obtaining is cut with the same enzyme of process the pAUR123 carrier segments obtaining and is connected, obtaining connecting product proceeds in intestinal bacteria competence Top10, obtain transformant, transformant is extracted to plasmid, send to order-checking, result is for this plasmid is for by sequence in sequence table 1, ' end 1-389 position Nucleotide is inserted into the Kpn I of pAUR123 carrier and the carrier that Sac I restriction enzyme site obtains from 5, called after pAUR123-ZmPMP3, in pAUR123-ZmPMP3, the coding region DNA fragmentation of ZmPMP3 is positioned at promotor PADH1 downstream, this recombinant vectors is yeast over-express 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.
After electrophoresis detection object band, purifying reclaims.
2) pAUR123-ZmPMP3-1 recombinant vectors
50 μ l systems: substep enzyme is cut:
Step 1) PCR product (50ng/ μ l) 25 μ l after purifying
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): 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 carrier large fragment.
Enzyme is cut to rear DNA fragmentation to be cut rear carrier large fragment with enzyme and is connected:
10 μ l systems:
Carrier (20ng/ μ l) 1 μ l
Fragment (50ng/ μ l) 7 μ l
T4 Buffer 1μl
16 ℃ of airbath 12h; Transform intestinal bacteria competence Top10, bacterium liquid PCR screening positive clone, obtains pAUR123-ZmPMP3.
Plasmid extraction: the little extraction reagent kit of plasmid (Tian Gen company):
(1) inoculate in the LB liquid nutrient medium that single bacterium colony hickie contains Amp resistance in 5ml, 37 ℃ of 200rpm shaking culture are spent the night.
(2) column equilibration step: adsorption column is put into collection tube, add 500 μ l balance liquids in adsorption column, the centrifugal 1min of 12000rpm, outwells the waste liquid in collection tube, relays adsorption column to reclaim in collector.(note: use the adsorption column of processing the same day)
(3) bacterium liquid is proceeded in Eppendorf pipe, the centrifugal 30sec of 12000rpm, Ex-all supernatant, collects thalline as far as possible.Add 250 μ l solution P1, resuspended thalline.(note: this step must thoroughly be hanged and open, otherwise can affect the cracking of thalline, cause plasmid extraction 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 now bacterium liquid should become limpid, as does not become limpid, illustrates that thalline is too much, and cracking is inabundant.(note: inviolent during mixing, in order to avoid interrupt genome of E.coli DNA, cause in the plasmid of extraction and be mixed with genome of E.coli DNA)
(5) add 350 μ l solution P3, upset mixes 6～8 times gently up and down immediately, now should occur white flocks.The centrifugal 10min of 12000rpm, precipitation is formed on centrifuge tube bottom.
(6) get in the adsorption column that supernatant processed in column equilibration, the centrifugal 30sec of 12000rpm, outwells the waste liquid in collection tube, and this step can repeatedly be collected.
(7) in adsorption column, add 700 μ l rinsing liquids, the centrifugal 30sec of 12000rpm, outwells the waste liquid in collection tube.
(8) in adsorption column, add 500 μ l rinsing liquids, the centrifugal 30sec of 12000rpm, outwells the waste liquid in collection tube.
(9) adsorption column is put back to collection tube, the centrifugal 2min of 12000rpm, to remove rinsing liquid as far as possible.
(10) room temperature is placed several minutes, removes as far as possible residual rinsing liquid.
(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 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 obtaining in (11) is rejoined to adsorption column 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 10mlYPDA liquid nutrient medium, 30 ℃ of 220rpm incubated overnight.
(2) get in right amount in 50ml liquid nutrient medium, it is 0.4 that 30 ℃ of 220rpm are cultured to OD600.
At (3) 25 ℃, the centrifugal 5min of 1000rpm collects bacterium.
(4) abandon supernatant, with the resuspended thalline of 10ml sterilized water.
At (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 each response preparation 2 μ g obtains, the carrier DNA 10 μ l of processing are in 1.5ml centrifuge tube.
Note: carrier DNA pre-treatment: boiling water boiling 10min, on ice 1min; Boiling water boiling 10min, on ice 1min again.
(8) from (6), get 200 μ l in (7).
(9) each reaction adds 600 μ l PEG solution.
(10) 30 ℃ of 200rpm shake after bacterium 30min, add 70 μ l DMSO to put upside down and mix gently, not vortex.
(11) 42 ℃ of thermal shock 15min, on ice 1min.
(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) the dull and stereotyped 30 ℃ of inversions of yeast are cultivated 3 days.
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 switching above bacterium liquid once after, shake bacterium 36hr, now OD
600be about 1.0, dilution bacterium liquid is to OD
600=0.3.(3) by 10 times, 500 times, 1000 times of the bacterium liquid difference redilution having diluted, and get this bacterium liquid having diluted 6 μ l,
Be inoculated in respectively the YPDA solid medium (YPDA+25mM NaCl+0.5 μ g/L AbA) that contains NaCl and AbA and in the YPDA solid medium (YPDA+0.5 μ g/L AbA) that contains AbA, be inverted for 30 ℃ and cultivate 48 hours, observe the upgrowth situation of bacterial strain.
Adopting uses the same method proceeds to paddy rice homologous gene OsLti6a in bacterial strain YR93-31, to obtain recombinant bacterium YR93-31/pAUR123-OsLti6a, there are some researches prove the function that OsLti6a can complementary bacterial strain YR93-31, using recombinant bacterium YR93-31/pAUR123-OsLti6a as positive control, for relatively weighing the supplementary degree of the function of ZmPMP3-1 gene pairs yeast mutants.
The YR93-1 (bacterial strain of pmp3 deletion mutant does not occur) of take is control strain 1, be seeded in the YPDA solid medium (YPDA+25mM NaCl) that does not contain the YPDA substratum (YR93-1 can not be grown in the YPDA substratum containing AbA resistance, only has the YR93-31 bacterial strain that has transformed recombinant plasmid can be grown in the YPDA substratum containing AbA resistance) of AbA resistance and contain NaCl.
With YR93-31 bacterial strain 2 in contrast, be inoculated in respectively the YPDA solid medium (YPDA+25mM NaCl+0.5 μ g/L AbA) that contains NaCl and AbA and in the YPDA solid medium (YPDA+0.5 μ g/L AbA) that contains AbA.
Result 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 in figure; Be respectively from left to right the bacterium liquid of 1000,500,10 and 1 times of dilutions.
As seen from the figure, in control medium (YPDA), four kinds of bacterial strain growing ways are basically identical, and the growing way of YR93-31 seems to be also better than other bacterial strains; And under 25mM NaCl processes, the growing way of four kinds of bacterial strains all weakens, but the growing way of YR93-31 bacterial strain is all lower than other 3 bacterial strains, therefore, infers that ZmPMP3-1 gene may be relevant to intracellular Na+ metabolism.
3, Subcellular Localization in 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 rear BI221-GFP carrier.
30 μ l system enzymes are cut object 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 rear fragment.
The pBI221-GFP that enzyme is cut after rear fragment is cut with enzyme is connected:
10 μ l systems:
Enzyme is cut rear pBI221-GFP (20ng/ μ l) 0.8 μ l
Enzyme is cut rear fragment (100ng/ μ l) 7.2 μ l
Buffer 1μl
16 ℃ of 12hr; Transform intestinal bacteria competence Top10, obtain transformant, extract the plasmid of transformant and send to order-checking, result is for this plasmid is for by sequence in sequence table 1, ' Xba I and Sac II that end 1-177 position Nucleotide is inserted into pBI221-GFP carrier cut the carrier that site obtains, called after pBI221-ZmPMP3-GFP from 5.In pBI221-ZmPMP3-GFP, the coding region of ZmPMP3 is positioned at promoter CaMV 35S downstream.
The preparation of onion material:
In Bechtop, after onion is peelled off to exterior skin, cut the bulb of third and fourth layer into about 4cm
2square, peel gently epidermis, entocuticle and MS solid medium are adjacent to, less as far as possible bubble, culture dish is standby with Parafilm sealing.
Portable particle gun bullet is made:
(1) prepare 1ml, 200 μ l, 20 μ l liquid-transfering guns, one bottle of dehydrated alcohol, timer, marking pen, paper handkerchief.
(2) claim 20mg (0.02g) PVP to put into the 1.5ml centrifuge tube of a dried and clean, add 1ml dehydrated alcohol, it is dissolved, the mother liquor that concentration is 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 1ml dehydrated alcohol carry out 3 cleanings (vortex, of short duration centrifugal, sucking-off.Attention: now have a small amount of bronze to be bonded on tube wall).
(5) add 100 μ l 0.05M spermidines, vortex mixes, ultrasonication 5～10sec (removal electric charge).
(6) add 25 μ l pBI221-ZmPMP3-GFP obtained above (1 μ g/ μ l) to mix.Vortex limit, limit adds 100 μ l 1M CaCl
2, the standing 10min of room temperature.
(7) with 1ml dehydrated alcohol, clean 5 times, method is the same.
(8) with 3ml 0.05mg/ml PVP, bronze is diluted in the large centrifuge tube of a 10ml.
(9) open N
2main valve, adjusts reducing valve, makes N
2pressure, in 0.4 left and right, is 99.997% N by purity
2drying tube 15min.Stop N
2air-flow, then cutting pipe, makes it be longer than platform 10cm.
(10) one end of the pipe of cutting is connected with syringe, vortex gold particle solution the rapid pipe that this solution is proceeded to this cutting, make its each end all leave the space of 6cm.
(11) pipe that rapidly upper step is filled to solution is placed on upholder and makes solution stop 3min at pipe, its position being full of of mark.With the slow mobile solution of syringe just through right side mark.Make to manage Rotate 180 °, solution is all poured out.
(12) make pipe rotation 30sec, open N
2at 0.35～0.4rpm, rotation 5min.
(13) pipe is taken out from supporting rack, stop N
2air-flow.Cut the pipe of sealing, bullet is put into the container that fills dry silica gel.
Particle gun bombardment onion epidermis:
(1) pack the bullet making into particle gun.
(2) particle gun is connected with helium tank, pressure is 150～300psi.
(3) open the pre-prepd culture dish that is placed with onion epidermis, particle gun is aimed to onion epidermis and bombard, every onion epidermis is made two rifles.
(4) 26 ℃, under dark condition, cultivate 24hr.
Take empty carrier pBI221-GFP as contrast.
Subcellular Localization fluorescent signal is observed:
The onion epidermis of cultivating 24h under taking-up dark condition, on slide glass, adds a water, and covered notes there is not bubble, is put on confocal microscope and observes green fluorescence signal, and wherein excitation wavelength is 488nm, and scanning wavelength is 500-535nm.
The results are shown in Figure shown in 2, wherein, A is the Subcellular Localization result before plasmolysis; B. the Subcellular Localization result after plasmolysis, C is for transforming the contrast of pBI221-GFP empty carrier.Nucleus, tenuigenin and cytolemma at the onion epidermis cell contrasting all can detect GFP fluorescence, show to compare with empty carrier pBI221-GFP, and ZmPMP3 is positioned in cytolemma.The result reality of ZmPMP3 Subcellular Localization, this gene is positioned at cytolemma and plays a role, and probably participates in the ion transport Relevant Physiological Courses of film.
Plant binary expression vector pCAMBIA3301 is (hereinafter to be referred as p3301, Canberra, Australia, in this article, the English professor of kingdom provides, 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., public Ke Cong Institute of Crop Science, Chinese Academy of Agricultural Science obtains.) 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., public Ke Cong Institute of Crop Science, Chinese Academy of Agricultural Science obtains.)
The preparation of conventional substratum and solution
YEB liquid nutrient medium (1L):
Yeast extract 1g
Extractum carnis 5g
Peptone 5g
Sucrose 5g
MgSO
4.7H
2O 0.5g
Adding distil water is settled to 1L, adjusts 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 YEB substratum after autoclaving, when temperature is down to 50 ℃ of left and right, add required microbiotic (Rif of 100mg/L, the Kan of 100mg/L), shake up.
The MS selective medium that contains PPT:
It is the PPT of 7mg/L that MS substratum after autoclaving adds final concentration when temperature is down to 50 ℃ of left and right, shakes up.
Bar gene primer:
Barp-F：5′-GCGGTCTGCACCATCGTC-3′
Barp-R：5′-GTACCGGCAGGCTGAAGTCCA-3′
With restriction enzyme site primer:
M1-3301-F1:5`-CATCGGCA
cCATGG(underscore is NcoI restriction enzyme site to CGGAGGGGACT-3`, and the former sequence in this site is T, but in this position, has introduced restriction enzyme site in the process of carrier construction, so made the T of this sequence into G in order to mate restriction enzyme site.)
M1-3301-R1：5`-ATGGGTCACCGTCGGCAAGGATGAC-3`
RT-PCR the primer:
ZmPMP3-1-RT-F：5`-CAACTGCGTGGACATCCTGA-3`
ZmPMP3-1-RT-R：5`-GGCGTAGATGGCGTAGATGA-3`
Ms substratum, D-mannitol (PEARLITOL 25C) are (sigma) purchased from Bioisystech Co., Ltd of Jing Ke HTC; NaCl is purchased from the biological limited liability company of Yu Ding state.
Arabidopis thaliana actin primer
Actin-F：5′-ATTCAGATGCCCAGAAGTCTTGT-3′
actin-R：5′-GAAACATTTTCTGTGAACGATTCC-3′
1, the acquisition of pCAMBIA3301-ZmPMP3 carrier
Take pMD18-T/ZmPMP3-1 as template, with M1-3301-F1 and M1-3301-R1, increase, the PCR product obtaining, through order-checking, this PCR product has in sequence sequence 3, and ' end 87-283 position Nucleotide, the coding region of this PCR product gene is that sequence 3 is from 5 ' end 88-264 position Nucleotide from 5.
Above-mentioned PCR product is cut to the pCAMBIA3301 carrier segments that obtain with BstEII double digestion with the same enzyme of process through NcoI to be connected, the connection product obtaining transforms intestinal bacteria competence Top10, obtain transformant, extract the plasmid of transformant and cut evaluation through PCR and enzyme, all positively send to order-checking, result is for this plasmid is for by sequence in sequence table 3, ' end 88-283 position Nucleotide is inserted into the NcoI of pCAMBIA3301 carrier (hereinafter to be referred as p3301) and the carrier that BstEII restriction enzyme site obtains, called after pCAMBIA3301-ZmPMP3 from 5.In pCAMBIA3301-ZmPMP3, the coding region of ZmPMP3 is positioned at promoter CaMV 35S downstream, is plant overexpression vector.
Specific as follows:
The coding region DNA of amplification ZmPMP3, introduces 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.
After electrophoresis detection object band, purifying reclaims, and obtains PCR product after purifying.The results are shown in Figure shown in 3, wherein swimming lane 1,2,3 is respectively the object band with restriction enzyme site that negative results of comparison, the pcr amplification of DNA maker, pcr amplification obtain, and arrow indication is object band, can find out the object fragment that obtains 177bp.
Build p3301-ZmPMP3:
50 μ l systems: substep enzyme is cut:
PCR product after purifying obtained above (50ng/ μ l) 25 μ l
Buffer3 5μl
ddH
2O 17.5μl
Nco I 1μl
37℃ 2h
Add 1ml dehydrated alcohol, at 4 ℃, the centrifugal 30min of 12000rpm; Outwell dehydrated 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 rear PCR product.
P3301 carrier enzyme is cut:
30 μ l systems (enzyme is cut three pipes simultaneously): substep enzyme is cut:
P3301 plasmid (50ng/ μ l) 10 μ l
Buffer3 3μl
ddH
2O 14.7μl
Nco I 1μl
37℃ 2h
Add 1ml dehydrated alcohol, at 4 ℃, the centrifugal 30min of 12000rpm; Outwell dehydrated alcohol, dry up.
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 rear p3301.
Connect:
9.8 μ l systems:
P3301 after enzyme is cut (20ng/ μ l) 0.8 μ l
Enzyme is cut rear PCR product (50ng/ μ l) 7 μ l
Buffer 1μl
16 ℃ of 12h; Transform intestinal bacteria 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 PCR positive colony.
Enzyme is cut detection:
PCR positive colony is extracted to NcoI and BstEII double digestion for plasmid, the results are shown in Figure shown in 4, swimming lane 1,2 is the electrophoresis result that enzyme is cut evaluation, swimming lane 3 is DNA marker, arrow indication is object band, as seen from the figure, obtain the fragment of 177bp, by this plasmid called after pCAMBIA3301-ZmPMP3.
2, turn 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, public Ke Cong Institute of Crop Science, Chinese Academy of Agricultural Science obtains.) seed first uses 75% alcohol disinfecting 1min, then use 0.5%NaClO (v/v) sterilization 10min, in super clean bench, use sterilizing ddH
2o rinsing 5 times, each 1min, dibbling is to MS solid medium, and 4 ℃ of vernalization 3d, are then positioned over illumination box, after growing approximately one week, transplant in vermiculite is housed under 22 ℃ of 16h illumination/8h dark conditions: in the capsule of Nutrition Soil (1: 1), continue to cultivate.
2), the preparation of Agrobacterium competent cell
(1) the mono-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 to OD600 be 0.5, 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) add the 20mM CaCl of 1ml precooling
2suspension cell, is used in ice bath 24h or is distributed into every pipe 200 μ l, and quick-frozen 1min in liquid nitrogen, puts-76 ℃ and save backup.
3), plant expression vector transforms the acquisition of Agrobacterium GV3101/pCAMBIA3301-ZmPMP3
(1) get 200 μ l GV3101 competent cells, the pCAMBIA3301-ZmPMP3 that adds 1 μ g to build, quick-frozen 1min in liquid nitrogen, 37 ℃ of water-bath 5min, then add 1ml not contain the YEB liquid nutrient medium of resistance, 28 ℃ of 140rpm, at a slow speed shaking culture 4h.
(2) the centrifugal 30sec of 10000rpm, abandons supernatant, adds 0.1ml YEB substratum Eddy diffusion cell, coats on the YEB flat board that contains 100 μ g/ml Kan and 125 μ g/ml Rif, cultivates approximately 36～48h for 28 ℃.
(3) the single bacterium colony growing on picking flat board, is inoculated in YEB nutrient solution (containing 100 μ g/ml Kan and 125 μ g/mlRif), and 28 ℃ of shaking culture are spent the night.
(4) carrying out bacterium liquid PCR identifies, primer is M1-3301-F1 and M1-3301-R1, the results are shown in Figure shown in 5, wherein, swimming lane 2-8 is the bacterium liquid PCR result of Agrobacterium GV3101, swimming lane 1 is DNA marker, swimming lane 9 is the negative contrast in PCR reflection, as seen from the figure, the fragment that obtains 177bp is PCR positive colony, and PCR is identified to positive colony extracts plasmid, sends to order-checking, result is for this plasmid is pCAMBIA3301-ZmPMP3, the positive colony called after GV3101/pCAMBIA3301-ZmPMP3 that contains this plasmid.
4), the conversion of Arabidopis thaliana
Be stained with colored method (floral dipping):
(1) with 1) the growth wild-type Arabidopis thaliana Columbia of about three weeks (Arabidopsis thaliana, ecotype Columbia) that obtains transforms, and water sufficient water in the day before yesterday transforming, before transforming, cut all fruit pods.
(2) in 1: 1000 ratio, the positive colony bacterium liquid GV3101/pCAMBIA3301-ZmPMP3 of activation is transferred and is had 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) with 200ml, infiltrate the resuspended thalline of damping fluid (1 * MS macroelement+5% sucrose), making OD600 is 0.8, adds sil-wet 40 μ l (0.2 ‰, volumn concentration).
(5) Arabidopis thaliana bud is immersed in (4), infect 1min.
(6) with preservative film or freshness protection package parcel plant, under 16 ℃ of dark conditions, place after 24hr, be put under normal condition and grow, obtain T0 for turning ZmPMP3 Arabidopis thaliana.
Results T0 is for the seed that turns ZmPMP3 Arabidopis thaliana, for T1 is for turning ZmPMP3 Arabidopis thaliana seed.
3, the screening of transgenic line and evaluation
1) ppt herbicide sprays is screened positive transgenic line:
(1) T1 is directly seeded in vermiculite is housed after 4 ℃ of vernalization treatment for turning ZmPMP3 Arabidopis thaliana seed: in the pallet of Nutrition Soil (1: 1), under normal condition, grow 20 days, ppt weedicide (Japanese Meiji Pharmaceutial Ltd. with 0.5 ‰ (volumn concentrations), draw 500 μ lppt stostes, be dissolved in 1L distilled water, the working fluid that acquisition volume percent is 0.5 ‰, sprays Arabidopsis thaliana Seedlings with this liquid.) spraying screening, continuous spraying is 3 days once a day, after one week, observations, because the ppt resistant gene on expression vector p3301 can be incorporated in Plant Genome with goal gene when transforming, so plant that can normal growth after weedicide ppt screening should be transgenic positive plant in theory, result is: obtain 35 strains growth normal plant, be ppt and screen positive T1 generation and turn ZmPMP3 Arabidopis thaliana.
Transplant ppt and screen positive T1 for turning ZmPMP3 Arabidopis thaliana plant continuation cultivation, every capsule kind 2 strains.Each ppt screens positive T1 generation and turns and when ZmPMP3 Arabidopis thaliana grows to 2 leaves, extract in a small amount DNA, carry out PCR evaluation, primer is M1-3301-F1 and M1-3301-R1, result is that the plant that obtains the fragment of 177bp is positive T1 generation to turn ZmPMP3 Arabidopis thaliana, obtains the positive T1 of 14 strains generation to turn ZmPMP3 Arabidopis thaliana.
2) by Genomic PCR, identify Arabidopis thaliana positive plant:
A:SDS method is extracted the positive T1 of 35 strain PPT Screening and Identification in a small amount for turning the total DNA of ZmPMP3 Arabidopis thaliana
SDS Extraction buffer (100ml):
Tris-HCl(1mol/L pH8.0) 10ml
EDTA(0.5mol/L pH8.0) 10ml
NaCl (1mol/L) 50ml (or 2.9g solid)
Sterilizing ddH
20 is settled to 100ml.
SDS lysis buffer:
SDS Extraction buffer: 20%SDS=15: 1 (V/V)
Step:
(1) get appropriate PPT and be accredited as positive T1 generation and turn ZmPMP3 Arabidopsis leaf and put into 1.5ml centrifuge tube, add the abundant grind into powder of liquid nitrogen (preferably only adding a liquid nitrogen goes to grind).
(2) add the SDS lysis buffer of 65 ℃ of preheatings of 800 μ l, after fully mixing, extracting 20min at 65 ℃, during mix again 2 times.
(3) add 250 μ l 5mol/L KAc, place 5min on ice.
(4) 12000rpm, centrifugal 10min, draws in the centrifuge tube that supernatant to is new.
(5) 12000rpm, centrifugal 5min, draws in the centrifuge tube that supernatant to is new.
(6) add 600 μ l Virahols to mix.
(7) 4 ℃ of 12000rpm, centrifugal 15min
(8) abandon supernatant, by 75% washing with alcohol, precipitate 2 times.
(9) the centrifugal 3min of 12000rpm, vacuum is drained or super clean bench dries up.
(10) add 20 μ l 1 * TE or sterilizing ddH
2o dissolving DNA precipitation ,-20 ℃ of preservations.
Purifying:
(1) in above-mentioned (10), add RNase 1 μ l, 37 ℃ of digestion 30min.
(2) mend sterilizing ddH
2o is to cumulative volume 600 μ l.
(3) add 300 μ l phenol, concussion mixes 5～10min; Add 300 μ l chloroforms, concussion mixes 5min.
(4) 12000rpm, centrifugal 10min, draws in the centrifuge tube that supernatant to is new.
(5) add 600 μ l chloroforms, concussion mixes 2min.
(6) 12000rpm, centrifugal 2min, draws in the centrifuge tube that supernatant to is new.
(7) add the NaAc of 1/10 volume to mix, then add the cold dehydrated alcohol of 2～3 times of volumes, 4 ℃ of standing 30min.
(8) 4 ℃ of 12000rpm, centrifugal 20min, abandons supernatant.
(9) 75% ethanol are washed precipitation 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 sterilizing ddH
2o dissolving DNA precipitation ,-20 ℃ save backup, obtain DNA.Through electrophoresis detection, result as shown in Figure 6, obtains genomic dna.
B: carry out PCR evaluation:
The genomic dna of above-mentioned acquisition of take is template, with primer Barp-F:5 '-GCGGTCTGCACCATCGTC-3 ' and Barp-R:5 '-GTACCGGCAGGCTGAAGTCCA-3 ', carries out PCR evaluation, detects bar gene.With primer M1-3301-F1 and M1-3301-R1, carry out PCR evaluation, detect 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
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 Genomic PCR, and swimming lane 1, for contrast, turns empty carrier arabidopsis gene group pcr amplification result; Swimming lane 2-13 is for turning ZmPMP3 Arabidopis thaliana strain Genomic PCR amplification; Swimming lane 14 is wild-type Arabidopis thaliana strain Genomic PCR amplification; Swimming lane 15 is positive control, 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 Genomic PCR, and swimming lane 1 is DNA marker (same Figure 10); Swimming lane 2, for for contrast, turns empty carrier arabidopsis gene group pcr amplification result; Swimming lane 2-12 is for turning pCAMBIA3301-ZmPMP3 gene Arabidopis thaliana strain Genomic PCR amplification; Swimming lane 13 is positive control, the pcr amplification result of plasmid pCAMBIA3301-ZmPMP3; Swimming lane 14 is wild-type Arabidopis thaliana strain Genomic PCR amplification.
As seen from the figure, pcr amplification goal gene and bar gene are identified, can amplify the object band of 230bp and the positive T1 of bar gene of 470bp for transgenic arabidopsis, obtain the positive T1 of 14 strains for transgenic arabidopsis, prove that ZmPMP3 gene has been incorporated in arabidopsis gene group.And contrast turn the bar band that empty carrier plant Arabidopis thaliana strain only has 470bp, in wild-type Arabidopis thaliana without this band.
3) from positive T1 generation, turning the seed that ZmPMP3 Arabidopis thaliana gathers in the crops is that T2 is for seed, by above-mentioned T2 for seed dibbling at the enterprising row filter of MS substratum that contains weedicide (7mg/L ppt), there is the positive plant that survives of four leaves to be transplanted to vermiculite is housed growth: in the capsule of Nutrition Soil (1: 1), to continue to cultivate, and gather in the crops seed and obtain T3 generation and turn ZmPMP3 Arabidopis thaliana.T3 shows as the strain of 100% resistance for turning ZmPMP3 Arabidopis thaliana through ppt screening, carry out again PCR evaluation, method is with 2), result obtains positive T3 for turning ZmPMP3 Arabidopis thaliana homozygous lines, from positive T3, for turning 12 strains of random choose ZmPMP3 Arabidopis thaliana homozygous lines, continue to breed, obtain T4 for turning ZmPMP3 Arabidopis thaliana seed.
In sowing T4 generation, turns ZmPMP3 Arabidopis thaliana seed, obtains T4 generation to turn ZmPMP3 Arabidopis thaliana homozygous lines, adopts 2) method, carry out PCR evaluation, obtain 12 T4 generations and turn ZmPMP3 Arabidopis thaliana homozygous lines.
Adopting uses the same method proceeds to wild-type Arabidopis thaliana Columbia (Arabidopsis thaliana by empty carrier pCAMBIA3301, ecotype Columbia) in, obtain turning empty carrier Arabidopis thaliana, through same PCR, identify, result is not for there is no goal gene ZmPMP3.
4) the total RNA of Arabidopis thaliana extracts and ThermoScript II
From 12 T4 generation, turn ZmPMP3 Arabidopis thaliana homozygous lines and choose 6 and be numbered 4,5,6,8,11 and 12 strains and carry out real time-PCR Molecular Detection, take wild-type Arabidopis thaliana and T4 generation and turn empty carrier Arabidopis thaliana as contrast.
Specific as follows: to use sky to take root in thing total RNA extraction reagent box and extract and be numbered 4,5,6,8,11 and 12 T4 generation and turn ZmPMP3 Arabidopis thaliana homozygous lines and the total RNA of contrast Arabidopsis leaf, and use invitrogen M-MlV ThermoScript II the first chain synthetic agent box to obtain cDNA.
By real time quantitative PCR method, use 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
Pcr amplification program:
94℃ 1min；
94℃ 5sec，
60℃ 5sec，
72℃ 31sec，；
40 circulations.
With actin gene in contrast.
Result is: with wild-type Arabidopis thaliana with turn empty carrier Arabidopis thaliana and compare, being numbered 4,5,6,8,11 and 12 T4 generation turns the expression (177bp fragment) that ZmPMP3 Arabidopis thaliana homozygous lines all can detect ZmPMP3-1, and wild-type and turning in empty carrier Arabidopis thaliana plant does not detect.Wild-type, turn empty carrier Arabidopis thaliana plant, be numbered 4,5,6,8,11 and 12 T4 generation and turn the expression (200bp fragment) that all has actin gene in 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 (PEARLITOL 25C) is processed for turning to T4, observes phenotype, specific as follows:
1) germination rate statistics: by the wild-type Arabidopis thaliana (CK) of sterilization, turn empty carrier Arabidopis thaliana and be numbered 6 and 11 T4 generation and turn seed dibbling respectively that 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, then moves to 22 ℃, 16hr illumination (100 μ E m
-2s
-1in the illumination box of)/8hr dark, sprout, in MS substratum, 150mM NaCl+MS substratum and 300mM D-mannitol+MS, 350mM D+MS substratum, statistics is cultivated the sprouting number after 3,4,5 days respectively.Each repeats 40 seeds, and each is processed independently 5 repetitions are set, and result is taken the mean.In statistics, take that to grow two green cotyledon be standard.
Result is as follows: 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 turns ZmPMP3 Arabidopis thaliana strain.
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 in No. 6 T4 generation, turns the germination rate sprouting 3,4,5 days of ZmPMP3 Arabidopis thaliana strain and is respectively 51.7%, 60%, 61.2%, apparently higher than the germination rate in 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 turns ZmPMP3 Arabidopis thaliana strain; And No. 11 T4 are respectively 85.6%, 95.6%, 96.7% for the germination rate sprouting 3,4,5 days that turns ZmPMP3 Arabidopis thaliana strain.
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 is respectively 80%, 86.7% for turning ZmPMP3 Arabidopis thaliana strain at the germination rate of sprouting 4,5 days; No. 11 T4 is respectively 76.9%, 85% for turning ZmPMP3 Arabidopis thaliana strain at the germination rate of sprouting 4,5 days.
Wild-type Arabidopis thaliana with turn empty carrier Arabidopis thaliana result without significant difference.Therefore, in 6, No. 11 T4 generation, turns ZmPMP3 Arabidopis thaliana strain germination rate in early days apparently higher than ck, ZmPMP3 be described except responding the coercing of salt, and also responds drought coerce at early germination.
2) biology is heavily added up:
By the wild-type Arabidopis thaliana (CK) of sterilization, turn empty carrier Arabidopis thaliana and be numbered 6,11,12 T4 generation turn seed that ZmPMP3 Arabidopis thaliana isozygotys according to 1) method process, 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;
The above growth of the statistics biology of respectively organizing each strain Arabidopsis thaliana Seedlings heavy (weight of whole strain seedling comprises overground part and root) of 2 weeks.
Each takes strain 20 strain Arabidopis thalianas, and test repeats 3 times, and result is taken the mean.
Result as shown in FIG. 10 and 11, wherein,
In MS substratum, in ck, No. 6 T4 generation, turns ZmPMP3 Arabidopis thaliana, No. 11 T4 generations and turns the biology that ZmPMP3 Arabidopis thaliana, No. 12 T4 generations turn ZmPMP3 Arabidopis thaliana and be heavily respectively 0.14225g, 0.14675g, 0.179g, 0.17875g;
In 150mM NaCl+MS substratum, in ck, No. 6 T4 generation, turns ZmPMP3 Arabidopis thaliana, No. 11 T4 generations and turns the biology that ZmPMP3 Arabidopis thaliana, No. 12 T4 generations turn ZmPMP3 Arabidopis thaliana and be heavily respectively 0.0835g, 0.10625g, 0.1215g, 0.09275g;
In 175mM NaCl+MS substratum, in ck, No. 6 T4 generation, turns ZmPMP3 Arabidopis thaliana, No. 11 T4 generations and turns the biology that ZmPMP3 Arabidopis thaliana, No. 12 T4 generations turn ZmPMP3 Arabidopis thaliana and be heavily respectively 0.067g, 0.0914g, 0.0947g, 0.0444g;
In 300mM D-mannitol+MS substratum, in ck, No. 6 T4 generation, turns ZmPMP3 Arabidopis thaliana, No. 11 T4 generations and turns the biology that ZmPMP3 Arabidopis thaliana, No. 12 T4 generations turn ZmPMP3 Arabidopis thaliana and heavily distinguish, 0.0467g, 0.0387g, 0.0554g, 0.053g.
Visible, the biology of 6, No. 11 strains focuses on and under salt stress, is all significantly higher than ck, and 11, No. 12 (preferably can be consistent with the strain of salt stress) strains biology under osmotic stress is heavy apparently higher than ck.
Wild-type Arabidopis thaliana and turn empty carrier Arabidopis thaliana result without significant difference.
3) evaluation in the time of infertility of ripe plant:
By the wild-type Arabidopis thaliana (CK in contrast) of sterilization, turn empty carrier Arabidopis thaliana and be numbered 4,5,6,11,12 T4 generation turn seed dibbling that ZmPMP3 Arabidopis thaliana isozygotys on 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 that forms of the Nutrition Soil-vermiculite of 1: 1, use plant nutrition liquid irrigates, and to Arabidopis thaliana, grows 4 weeks, is divided into two groups of processing:
Coerce group: to each strain pouring, contain the 300mM NaCl aqueous solution and carry out Stress treatment, pouring once, keeps ground moistening weekly;
Control group: to each strain pouring distilled water, pouring once, keeps ground moistening weekly;
Process (pouring 300mM NaCl is after two weeks) after two weeks and observe each strain phenotype, statistics survival rate.During transplanting, every basin is planted 5 strains, and every 3 basins (15 strain) are a repetition, and test arranges 3 repetitions.
Use plant nutrition liquid irrigates, to Arabidopis thaliana growth, in the time of 4 weeks, observe phenotype, result as shown in figure 13, when now Arabidopis thaliana enters generative growth phase, wild-type Arabidopis thaliana (contrast CK), is numbered 5,6,11 T4 generation and turns all boltings just of ZmPMP3 Arabidopis thaliana.Wild-type Arabidopis thaliana and turn empty carrier Arabidopis thaliana result without significant difference.Being numbered 4,12 T4 generation turns ZmPMP3 Arabidopis thaliana and is numbered 5,6,11 T4 generation and turns ZmPMP3 Arabidopis thaliana result without significant difference.
Process two days later, each strain phenotype of group is coerced in observation, result as shown in figure 14, through 300mM NaCl aqueous solution Stress treatment two days later, wild-type Arabidopis thaliana (CK in contrast) and be numbered 4,5,6,11,12 T4 generation and turn ZmPMP3 Arabidopis thaliana low side lotus throne leaf and all start flavescence.Wild-type Arabidopis thaliana and turn empty carrier Arabidopis thaliana result without significant difference.
Process after two weeks, observe phenotype, result as shown in figure 15, wherein NaCl processing ck is the wild-type Arabidopis thaliana through 300mM NaCl aqueous solution Stress treatment, contrast ck is the wild-type Arabidopis thaliana of processing with distilled water pouring, NaCl processes strain 6 and turns ZmPMP3 Arabidopis thaliana for 6 the T4 generation of being numbered through 300mM NaCl aqueous solution Stress treatment, and contrast strain 6 turns ZmPMP3 Arabidopis thaliana for 6 the T4 generation of being numbered of processing with distilled water pouring.Being numbered 5,11 T4 generation turns ZmPMP3 Arabidopis thaliana and is numbered 6 T4 generation and turns ZmPMP3 Arabidopis thaliana result without significant difference.Wild-type Arabidopis thaliana and turn empty carrier Arabidopis thaliana result without significant difference.
A kind of sedge of processing the Arabidopis thaliana part strain extraction after two weeks starts the flavescence of wilting, Arabidopis thaliana now can not further complete reproductive growth, shaky and start death, therefore at salt stress, process after two weeks, statistics can complete the time of infertility Arabidopis thaliana strain number of (plant that can gather in the crops seed), in triplicate, result is taken the mean in experiment.
Result is as follows:
After 300mM NaCl aqueous solution Stress treatment, wild-type Arabidopis thaliana (CK in contrast) the strain number that can complete the time of infertility is 2.67 strains;
After 300mM NaCl aqueous solution Stress treatment, in 4 the T4 generation of being numbered that can complete the time of infertility,, to turn ZmPMP3 Arabidopis thaliana strain number be 3.34 strains;
After 300mM NaCl aqueous solution Stress treatment, in 5 the T4 generation of being numbered that can complete the time of infertility,, to turn ZmPMP3 Arabidopis thaliana strain number be 5 strains;
After 300mM NaCl aqueous solution Stress treatment, in 6 the T4 generation of being numbered that can complete the time of infertility,, to turn ZmPMP3 Arabidopis thaliana strain number be 9.67 strains;
After 300mM NaCl aqueous solution Stress treatment, in 11 the T4 generation of being numbered that can complete the time of infertility,, to turn ZmPMP3 Arabidopis thaliana strain number be 3.34 strains;
After 300mM NaCl aqueous solution Stress treatment, in 12 the T4 generation of being numbered that can complete the time of infertility,, to turn ZmPMP3 Arabidopis thaliana strain number be 3.34 strains;
In the control group of processing through distilled water, wild-type Arabidopis thaliana (CK in contrast) the strain number that can complete the time of infertility is 5 strains;
In the control group that distilled water is processed, in 4 the T4 generation of being numbered that can complete the time of infertility,, to turn ZmPMP3 Arabidopis thaliana strain number be 5 strains;
In the control group that distilled water is processed, in 5 the T4 generation of being numbered that can complete the time of infertility,, to turn ZmPMP3 Arabidopis thaliana strain number be 5 strains;
In the control group that distilled water is processed, in 6 the T4 generation of being numbered that can complete the time of infertility,, to turn ZmPMP3 Arabidopis thaliana strain number be 5 strains;
In the control group that distilled water is processed, in 11 the T4 generation of being numbered that can complete the time of infertility,, to turn ZmPMP3 Arabidopis thaliana strain number be 5 strains;
In the control group that distilled water is processed, in 12 the T4 generation of being numbered that can complete the time of infertility,, to turn ZmPMP3 Arabidopis thaliana strain number be 5 strains; Partial results is done shown in Figure 12, can find out, the transgenic arabidopsis of excessively expressing ZmPMP3 gene has showed good salt tolerance at reproductive stage.
B, excised leaf moisture holding capacity:
By the wild-type Arabidopis thaliana (CK in contrast) of sterilization, turn empty carrier Arabidopis thaliana and be numbered 5,6 T4 generation turn seed dibbling that ZmPMP3 Arabidopis thaliana isozygotys on 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 that forms of the Nutrition Soil-vermiculite of 1: 1, use plant nutrition liquid irrigates, to Arabidopis thaliana, growing, (this tested in order to identify the hold facility of transgenic arabidopsis to moisture, was one of index of weighing the resistance to osmotic stress of plant in 6 weeks.), the whole strain of over-ground part of 6 weeks large Arabidopis thalianas is cut, take fresh weight a.Be placed in air and again take weight b after (23 ℃, humidity 40%) 5h.Then dry after 24h to constant weight with 90 ℃, take weight c.According to formula, calculate water retention.Test in triplicate results averaged.WRC(％)＝b-c/a-c×100
Result is as follows: the water retention of Ck is 75.49%; The water retention of strain 5 is 77.98%; The water retention of strain 6 is 79.02%.Wild-type Arabidopis thaliana and turn empty carrier Arabidopis thaliana result without significant difference.
Result mapping as shown in figure 16, can find out that the WRC of strain 5,6 is significantly higher than ck, illustrate that to be numbered 5,6 T4 higher than the blade moisture holding capacity of wild-type Arabidopis thaliana for turning ZmPMP3 Arabidopis thaliana wild-type Arabidopis thaliana.
Claims (2)
1. a method of cultivating the transgenic plant that resistance of reverse improves, be that the encoding gene of protein that the aminoacid sequence shown in sequence in sequence table 4 is formed imports object plant and obtains transgenic plant, the resistance of reverse of described transgenic plant is higher than described object plant;
Described resistance of reverse is drought tolerance and/or salt tolerance; Described object plant is Arabidopis thaliana;
Described encoding gene is following 1)-3) in the gene shown in arbitrary:
1) DNA molecular shown in sequence 3 in sequence table;
2) in sequence table sequence 3 from the DNA molecular shown in the 88th-264 Nucleotide of 5 ' end;
3) in sequence table sequence 3 from the DNA molecular shown in the 88th-283 Nucleotide of 5 ' end;
The encoding gene of the protein that the aminoacid sequence shown in sequence 4 forms in sequence table is to import in described object plant by recombinant expression vector; The recombinant expression vector that the multiple clone site of the encoding gene insertion vector pCAMBIA3301 that described recombinant expression vector is protein that the aminoacid sequence shown in sequence in sequence table 4 is formed obtains.
2. a method of cultivating the transgenic plant that moisture holding capacity improves, be that the encoding gene of protein that the aminoacid sequence shown in sequence in sequence table 4 is formed imports object plant and obtains transgenic plant, the moisture holding capacity of described transgenic plant is higher than described object plant; The blade water retention that the moisture holding capacity of described transgenic plant is described transgenic plant higher than described object plant is higher than described object plant;
Described object plant is Arabidopis thaliana;
Described encoding gene is following 1)-3) in the gene shown in arbitrary:
1) DNA molecular shown in sequence 3 in sequence table;
2) in sequence table sequence 3 from the DNA molecular shown in the 88th-264 Nucleotide of 5 ' end;
3) in sequence table sequence 3 from the DNA molecular shown in the 88th-283 Nucleotide of 5 ' end;
The encoding gene of the protein that the aminoacid sequence shown in sequence 4 forms in sequence table is to import in described object plant by recombinant expression vector; The recombinant expression vector that the multiple clone site of the encoding gene insertion vector pCAMBIA3301 that described recombinant expression vector is protein that the aminoacid sequence shown in sequence in sequence table 4 is formed obtains.
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| CN100999549A (en) * | 2006-12-30 | 2007-07-18 | 中国科学技术大学 | Plant conversion-resisting resisting related protein and its coding gene and application |
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| CN100999549A (en) * | 2006-12-30 | 2007-07-18 | 中国科学技术大学 | Plant conversion-resisting resisting related protein and its coding gene and application |
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