CN102286528A - Application of banana ASR (ABA, stress, ripening induced protein) gene in plant stress resistance - Google Patents
Application of banana ASR (ABA, stress, ripening induced protein) gene in plant stress resistance Download PDFInfo
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Abstract
The invention discloses an application of the banana ASR (ABA, stress, ripening induced protein) gene in plant stress resistance. The invention provides a method for growing genetically modified plants, by which the encoded gene of the MaASR1 protein is transferred into a target plant to obtain a genetically modified plant with at least one of the following characteristics: (1) the stress resistance of the genetically modified plant is higher than the stress resistance of the target plant; (2) the leaf surface area of the genetically modified plant is smaller than the leaf surface area of the target plant; (3) the leaf thickness of the genetically modified plant is larger than the leaf thickness of the target plant; (4) the leafstalk length of the genetically modified plant is larger than the leafstalk length of the target plant; (5) the anthesis of the genetically modified plant is later than the anthesis of the target plant; and the amino acid sequence of the MaASR1 protein is sequence 2 in the sequence table. An experiment proves that by the method disclosed by the invention, when the MaASR1 gene of the banana fruit is transferred into the wild arabidopsis thaliana, a genetically modified plant with high stress resistance can be obtained, and the invention lays a foundation for plant breeding.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the application of a kind of banana ASR gene in plant stress-resistance.
Background technology
ASR (ABA, stress, ripening induced protein) gene is a functions peculiar gene in the class higher plant, exists with little gene family form in species such as tomato, apricot, grape and potato.This gene family member coding has the monomeric protein of a large amount of polare Aminosaerens, between these monomeric proteins with the non covalent bond form in conjunction with forming homodimer and discerning specific dna sequence dna, regulate the expression of corresponding gene.So the ASR gene is generally believed it is a kind of novel plant transcription factor.From 1993 were cloned into first Asr gene (Asr1) from tamato fruit after, follow-up research constantly began to concentrate on the molecular biology and genetics research of this gene and corresponding protein (Iusem, 1993).Rossi and Iusem (1995) have found second Asr gene (Asr2) and have thought that further these two genes belong to same gene family in tomato.Change promotor and the reporter gene fusion of Asr2 plants such as over to tomato, tobacco and pawpaw, find that this gene is to be subjected to the ABA inductive.Thereby illustrate that this gene is relevant with ABA.
In recent years, from potato (Solanum tubersum), corn (Zea mays), shaddock (Citrus maxima). pine tree (Pinus taeda), paddy rice (Oryza sativa), all be cloned into this gene in lily (Lilum longifera) and the grape plants such as (Vitis vinifera), but the surprisingly not existence of this gene in the Arabidopis thaliana.
The albumen of ASR genes encoding is that the molecule amount is little, and wetting ability is strong, has the albumen of high thermal stability.ASR gene in all plants all has the structural domain of two high conservatives: the one, be positioned at the Zn that depends on that contains a large amount of Histidines that N holds
2+Dna binding sequence row, the 2nd, be positioned at a nuclear localization signal of C end.Asr1 in the tomato of being cloned into is the earliest furtherd investigate discovery, and Asr1 albumen is positioned in the nucleus of tomato cell, can be with the form and the dna sequence dna generation combination of homodimer, and the bonded core parts are C
2-3(C/G) A.
But the ASR expression of gene mode in the different plant species is not quite similar.For example, ASR gene tomato, shaddock is expressed in the fruit of avocado and grape, also at the blade and the stem of root, paddy rice and the corn of the stem tuber of potato, pine tree, and expresses in the pollen of lily.This explanation, the ASR gene may participate in each process of development of plants.
Summary of the invention
An object of the present invention is to provide a kind of cultivation transgenic plant method.
Method provided by the invention for the proteic encoding gene of MaASR1 is imported the purpose plant, obtains to have following 1)-5) in the transgenic plant of at least a feature:
1) resistance of reverse of described transgenic plant is higher than described purpose plant;
2) the blade table area of described transgenic plant is less than described purpose plant;
3) vane thickness of described transgenic plant is greater than described purpose plant;
4) the petiole length of described transgenic plant is greater than described purpose plant;
5) be later than described purpose plant the flowering period of described transgenic plant;
The proteic aminoacid sequence of described MaASR1 is the sequence 2 in the sequence table.
The proteic encoding gene of described MaASR1 is the sequence 1 in the sequence table.
Described resistance of reverse is salt tolerance and/or drought tolerance.
Described resistance of reverse is by improving germination rate, increase root length and/or improving survival rate and embody.
The proteic encoding gene of described MaASR1 imports described purpose plant by expression vector.
Described expression vector is that the proteic encoding gene of described MaASR1 is inserted between the XbaI and Sac I restriction enzyme site of pBI121, expresses the proteic carrier of MaASR1.
Described purpose plant is dicotyledons or monocotyledons.
Described dicotyledons is an Arabidopis thaliana.
Another object of the present invention provides a kind of expression vector.
Expression vector provided by the invention is for inserting the proteic encoding gene of described MaASR1 among the pBI121 the proteic carrier of expression MaASR1 that obtains; The proteic encoding gene of described MaASR1 is the sequence 1 in the sequence table, and described expression vector is specially the proteic encoding gene of described MaASR1 is inserted between the Xba I and Sac I restriction enzyme site of pBI121, expresses the proteic carrier of MaASR1.
Described MaASR1 albumen, the proteic encoding gene of described MaASR1 and/or the application of described expression vector in plant breeding also are the scope of protection of the invention;
Or the application in cultivating the resistance of reverse transgenic plant of described MaASR1 albumen, the proteic encoding gene of described MaASR1 and/or described expression vector also is a scope of protection of the invention;
Or the application in improving plant stress tolerance of described MaASR1 albumen, the proteic encoding gene of described MaASR1 and/or described expression vector also is a scope of protection of the invention;
Described resistance of reverse is specially salt tolerance and/or drought tolerance; Described plant is specially dicotyledons or monocotyledons; Described dicotyledons especially is specially Arabidopis thaliana.
Of the present invention experimental results show that, the present invention is with the MaASR1 gene of banana (Musa acuminate L.AAA group cv.Brazilian), import in the wild-type Arabidopis thaliana, obtain transgenic plant, transgenic plant seed germination rate, root length, survival rate under high salt and/or drought stress condition all are higher than the wild-type Arabidopis thaliana, for plant breeding is laid a good foundation.
Description of drawings
Fig. 1 is the evaluation of MaASR1 plant expression vector
Fig. 2 is that the PCR of MaASR1 transgenic line detects
Fig. 3 is that the Southern blot of L14 and L38 strain system detects
Fig. 4 is the blade of the wild-type, L14 and the L38 that take from same area
Fig. 5 is wild-type, L14 and the L38 in 5 weeks of growth under the short day
Fig. 6 is wild-type, L14, the L38 percentage of germination on the substratum that contains different concns NaCl
Fig. 7 is wild-type, L14 and the L38 tolerance to different concns NaCl
Fig. 8 is wild-type, 35S::MaASR114,35S::MaASR138, abi4-1 and the gin1-3 percentage of germination on the substratum that contains different concns N.F,USP MANNITOL
Fig. 9 crosses the drought resistance of expressing plant for MaASR1
Figure 10 is wild-type and the survival rate of MaASR1 transfer-gen plant under drought stress
Figure 11 is the salt resistance of MaASR1 transfer-gen plant
Figure 12 is wild-type and the survival rate of MaASR1 transfer-gen plant under salt stress
Embodiment
Employed experimental technique is ordinary method if no special instructions among the following embodiment.
Used material, reagent etc. if no special instructions, all can obtain from commercial channels among the following embodiment.
The acquisition of embodiment 1, commentaries on classics MaASR1 gene Arabidopis thaliana
One, the discovery of MaASR1 gene
From the cDNA library of banana (Musa acuminate L.AAA group cv.Brazilian), obtained an ASR gene (called after MaASR1), the sequence 1 that its nucleotides sequence is classified as in the sequence table is found through sequence alignment and phylogenetic analysis, MaASR1 has the total conserved domain of this family gene, and promptly the N end depends on Zn
2+The DNA binding site and the nuclear localization signal and the homologous gene evolutionary distance in the monocotyledonss such as corn, paddy rice of C end nearer.
Two, the structure of MaASR1 plant expression vector
Be fundamental construction MaASR1 expression carrier with plant expression vector pBI121, specific as follows:
Extract the RNA of banana (Musa acumina te L.AAA group cv.Brazilian), reverse transcription obtains cDNA, with P1:5-CCTCTAGATCGGCCATTACGGCCGGGG-3 and P2:5-CCGAGCTCCTTATTTTTAAGGG-3 is primer, carries out pcr amplification, obtains the PCR product.The PCR product that obtains is sent to order-checking, and the result has the Nucleotide shown in the sequence 1 in the sequence table for this PCR product, and its gene is MaASR1, and the amino acid whose sequence of its encoded protein is the sequence 2 in the sequence table, this albumen called after MaASR1.Sequence 1 in the sequence table is made up of 683 Nucleotide, and the sequence 2 in the sequence table is made up of 143 amino acid.
Above-mentioned PCR product is cut with Xba I and Sac I enzyme, obtained enzyme and cut product, enzyme is cut product and the plant expression vector pBI121 (Biovector Co., LTD, Biovector008, the USA that cut through same enzyme.) connect, obtain connecting product, will connect product and change in the intestinal bacteria, obtain transformant.
Extract the plasmid of transformant, with P1:5-CCGAGGAGAAGCACCACCAC-3 and P2:5-GCCACCGCTGCAGCGATCTCCTC-3, for primer carries out pcr amplification, use Xba I and Sac I digested plasmid simultaneously, PCR and enzyme are cut evaluation as shown in Figure 1, wherein, and M, marker; 1, the plasmid cut of enzyme; 2, the PCR product 1, as can be seen, obtain the positive plasmid of 389bp, to cut through PCR and enzyme and be positive plasmid and send to order-checking, the result is, the carrier of this plasmid for obtaining between the XbaI that the sequence in the sequence table 1 inserted pBI121 and the Sac I restriction enzyme site, with this plasmid called after pBI121-MaASR1, sequence 1 is inserted is behind the 35S promoter between the multiple clone site before the no terminator.
Three, the acquisition of reorganization Agrobacterium
The pBI121-MaASR1 of above-mentioned acquisition is imported agrobacterium tumefaciens C58 (Agrobacterium tumefaciens, Sheng Changzhong, Wang Shufang, Zhang Xinping, Wang Yong, Tian Junying, the preliminary study of soil Agrobacterium C58 genetic transformation taxus chinensis in northeast, Nankai University's journal (natural science), 1999,32 (4): 27-32, the public can obtain from Chinese Academy of Tropical Agricultural Sciences's Haikou experiment centre.) in, obtain the bacterium of recombinating, extract the plasmid of reorganization bacterium, send to order-checking, the result will contain the Agrobacterium called after C58/pBI121-MaASR1 of this plasmid for this plasmid is pBI121-MaASR1.
Four, change the acquisition of MaASR1 Arabidopis thaliana
1, Arabidopis thaliana is cultivated
Will be through (the Arabidopsis thaliana Columbia ecotype of the wild-type Arabidopis thaliana behind 4 ℃ of dark processing 3d, Arabidopsis thaliana (L.), available from Arabidopsis Biological Resource Center (Ohio university, Ohio state, USA) seed broadcasting is in flowerpot, and, preserve moisture and treat seed germination in shop, surface layer of fine sand.After seedling grows true leaf, can transplant, cultivating in cultivating room and growth cabinet, is matrix with the vermiculite, and temperature is controlled at 21-23 ℃, every day vegetative growth phase, light application time was 10h, reproductive stage every day is that illumination is 16h, and intensity of illumination is 2000lx, and culturing room's relative humidity is 70%, preserve moisture after the transplanting about a week, grow young leaves until seedling.Water an amount of Arabidopis thaliana nutritive medium (2M KNO every 5d
3, 2M Ca (NO
3)
2, 2M MaSO
4.7H
2O, 1MNH
4NO
3, 1M KH
2PO
4, 0.02M FeSO
4.7H
2O, 0.02M Na
2EDTA, 0.05M H
3BO
3, 0.01MMnCl
2.4H
2O, 0.01M Na
2MOO
4), when treating that the flower tongue grows to 5-10cm and the extraction of side tongue, can be used for transforming, be the acceptor Arabidopis thaliana.
2, agriculture bacillus mediated infusion method arabidopsis thaliana transformation
Carry out Arabidopis thaliana with infusion method and transform, in 28 ℃, the 280r/min shaking culture is spent the night with the C58/pBI121-MaASR1 of above-mentioned acquisition.Centrifugal collection thalline, and it is resuspended to add an amount of conversion substratum, make the O.D value of Agrobacterium about 0.8, transform substratum for containing the 1/2MS of 50g/L Sucrose and 0.02% (V/V) tween 20, cut off existing fruit pod on the Arabidopis thaliana, acceptor Arabidopis thaliana flower tongue is immersed the flowerpot back-off and transformed in the substratum.Arabidopis thaliana after transforming is lain in the moisture-keeping container, take out behind dark culturing (23 ℃ of the culture temperature) 24h, continue to cultivate by culture condition before transforming; Arabidopis thaliana through transforming is treated to stop to water when the fruit pod begins flavescence after cultivating for 3 weeks.Bagging is collected seed during fully matured, dries standby.
3, Arabidopis thaliana seed resistance screening
With the air dried seed with 75% ethanol+0.02%Triton X100 solution soaking 10min; Remove ethanolic soln,, and seed blown and beaten on the aseptic filter paper with the dehydrated alcohol rinsing, air-dry; The air dried seed broadcasting is being selected on the substratum (0.7% Agra, 25 μ g/mL hygromycin B, PH 5.7 for 1/2MS, 1.5% Sucrose); Transfer to behind 4 ℃ of low temperature dark processing 3d and cultivate in the room, grow under 23 ℃ of conditions, the seedling replanting of selecting the energy normal growth behind the 10d is in flowerpot; Obtain 45 strain T1 for changeing the MaASR1 Arabidopis thaliana, calculate T1 for the ratio of seedling that can normal growth in the seed and deadly seedling 1: 80.
For changeing MaASR1 Arabidopis thaliana results seed, sowing obtains T2 for changeing the MaASR1 Arabidopis thaliana from T1.
4, T3 cultivates for changeing the MaASR1 Arabidopis thaliana
Select to separate than the T2 that meets 3: 1 and is transplanted in the culture medium, watered with an amount of 1/2 Arabidopis thaliana nutritive medium every 5 days after the week of preserving moisture, treat that it grows up for changeing the MaASR1 Arabidopis thaliana, the results seed, sowing continues to go down to posterity, and obtains 45 strain T3 for changeing the MaASR1 Arabidopis thaliana.
5, T3 is for the Molecular Detection of changeing the MaASR1 Arabidopis thaliana
1) PCR of MaASR1 transgenic arabidopsis detects
Extracting each strain with plant total DNA extraction test kit is that T3 is for changeing MaASR1 Arabidopsis leaf genomic dna, extracting method is with reference to the plant DNA extraction easy kit specification sheets of Omega company, with the genomic dna is template, with P1:5-CCGAGGAGAAGCACCACCAC-3 and P2:5-GCCACCGCTGCAGCGATCTCCTC-3 is primer, carrying out pcr amplification, is contrast with the wild-type Arabidopis thaliana, and the result as shown in Figure 2, wherein, Col: wild-type; L1-L12: the T3 that is numbered 1-12 is for changeing the MaASR1 Arabidopis thaliana; CK is the PCR product 1 of MaASR1, and the positive T3 of purpose band that obtains 389bp obtains the positive T3 of 34 strains for changeing the MaASR1 Arabidopis thaliana altogether for changeing the MaASR1 Arabidopis thaliana.
2) the Southern blot of MaASR1 transgenic arabidopsis detects
Extracting the positive T3 be numbered L14 and L38 respectively carries out the Southern trace for the complete genome DNA of the blade that changes the MaASR1 Arabidopis thaliana and detects, with EcoRI two transgenic lines are digested, (with the PBI121-MaASR1 plasmid is template through MaASR1 gene specific probe, with P1:5-CCGAGGAGAAGCACCACCAC-3 and P2:5-GCCACCGCTGCAGCGATCTCCTC-3 is the product that primer increases and obtains) hybridization, colour developing, with the wild-type Arabidopis thaliana is contrast, the result as shown in Figure 3, WT is the wild-type Arabidopis thaliana, L14 and L38 are that two positive T3 of independence are for changeing the MaASR1 Arabidopis thaliana, as seen from the figure, L14 and L38 all obtain 389bp purpose fragment, but the inserted mode of MaASR1 in two strain systems is inequality, and copy number is also different.
Adopt identical method, empty carrier pBI121 is imported in the wild-type Arabidopis thaliana, obtain T0, go down to posterity, up to obtaining T3 for changeing the empty carrier Arabidopis thaliana for changeing the empty carrier Arabidopis thaliana.Extract T3 for the DNA that changes the empty carrier Arabidopis thaliana, use P1:5-CCGAGGAGAAGCACCACCAC-3 and P2:5-GCCACCGCTGCAGCGATCTCCTC-3, carry out pcr amplification, do not obtain the purpose fragment, illustrate that it is that T3 is for changeing the empty carrier Arabidopis thaliana as primer.
The functional study of embodiment 2, commentaries on classics MaASR1 gene Arabidopis thaliana
One, phenotype
With wild-type Arabidopis thaliana (col), be numbered L14 and L38 positive T3 for changeing MaASR1 Arabidopis thaliana kind in vermiculite, at 23 ℃, 5 weeks of growth under the condition of 10h illumination, 12h dark.
Take pictures and observe as shown in Figure 4 and Figure 5, as seen from the figure, the blade table area of wild-type Arabidopis thaliana is big, and the edge is smooth, and blade is thinner relatively; L14 and L38 blade table area are little, crispatura in the edge, and vane thickness is bigger, and petiole is longer.And the blade edge amount of crimp of L14 is greater than L38.
The positive T3 that is numbered L14 and L38 delayed for the flowering period of changeing the MaASR1 Arabidopis thaliana, and lagging behind than wild-type, (the normal flowering time of wild-type (blooming from being seeded into) is 2 months about about 2 weeks.)。
Two, changeing MaASR1 Arabidopis thaliana resistance of reverse identifies
1, the MaASR1 transgenic seed is to the endurance of arid and salt stress
1) L14 and L38 seed are to the resistance test of NaCl
With wild-type Arabidopis thaliana (col), be numbered L14 (35S::MaASR114) and L38 (35S::MaASR138) positive T3 for the seed that changes the MaASR1 Arabidopis thaliana sow respectively contain 0,50,150, the substratum (1/2MS of 200mM NaCl, 1.5% Sucrose, 0.7% Agra, PH 5.7) go up and cultivated 15 days, make its sprouting, the statistics germination rate, the seed of each strain system is 50, the experiment triplicate, results averaged.Is contrast with T3 for changeing the empty carrier Arabidopis thaliana.With mutant gin1-3, abi4-1 is that (abi4-1 is an ABA non-sensitive type mutant strain in contrast, gin1-3 is an ABA responsive type mutant strain, in ABA susceptibility testing experiment, play the contrast effect, all available from Arabidopsis Biological Resource Center (Ohio university, Ohio state, USA).
The result as shown in Figure 6,
The wild-type Arabidopis thaliana contain 0,50,150, germination rate in the substratum of 200mM NaCl is respectively 100%, 90%, 55%, 8.67%;
The positive T3 that is numbered L14 for change the MaASR1 Arabidopis thaliana contain 0,50,150, germination rate in the substratum of 200mM NaCl is respectively 100%, 98%, 97%, 69.1%;
The positive T3 that is numbered L38 for change the MaASR1 Arabidopis thaliana contain 0,50,150, germination rate in the substratum of 200mM NaCl is respectively 100%, 93%, 82%, 51%;
Mutant gin1-3 contain 0,50,150, germination rate in the substratum of 200mM NaCl is respectively 100%, 92%, 91%, 90%;
Mutant abi4-1 contain 0,50,150, germination rate in the substratum of 200mM NaCl is respectively 100%, 96%, 92%, 91%;
T3 does not have significant difference for changeing empty carrier Arabidopis thaliana and wild-type Arabidopis thaliana result.
As can be seen, along with the rising of NaCl concentration, the germination rate of wild type seeds is subjected to severe inhibition, and in the substratum of 200mMNaCl, germination rate reduces to 8.67%.The sprouting of L14 and L38 seed also has been subjected to the inhibition of NaCl, but restraining effect a little less than.Wherein the salt resistance of L14 is higher than L38, and in the substratum of 200mMNaCl, germination rate also remains on 69.1%.
According to the method described above, with the wild-type Arabidopis thaliana, be numbered L14 and L38 positive T3 for the seed that changes the MaASR1 Arabidopis thaliana contain 0,100,150,175, on the substratum of 200mM NaCl during growth 12d, the statistics root is long, the experiment triplicate, results averaged, the seed of each strain system is 50.Is contrast with T3 for changeing the empty carrier Arabidopis thaliana.
The result as shown in Figure 7,
Wild-type Arabidopis thaliana (col) contains 0,100,150,175, the root length of the substratum of 200mM NaCl is respectively 2.7,2.2,1.25,0.3,0cm;
MaASR1 Arabidopis thaliana (35S::MaASR114) contains 0,100,150,175 to the positive T3 that is numbered L14 for changeing, the root length of the substratum of 200mM NaCl is respectively 2.6,2.6,2.2,1.2,0.5cm;
MaASR1 Arabidopis thaliana (35S::MaASR138) contains 0,100,150,175 to the positive T3 that is numbered L38 for changeing, the root length of the substratum of 200mM NaCl is respectively 2.55,2.55,2.35,1.3,0.5cm;
T3 does not have significant difference for changeing empty carrier Arabidopis thaliana and wild-type Arabidopis thaliana result.
As can be seen, when the concentration of NaCl in medium was lower than 100mM, the growth of each strain system all was not subjected to remarkably influenced.When NaCl concentration reached 150mM, wild-type began to show different upgrowth situations with the MaASR1 transfer-gen plant, and the root of wild-type shortens, and the root length of L14 and L38 is influenced not quite by NaCl.When growing 12d in the substratum at 200mMNaCl, wild type seeds is only stretched out radicle, and cotyledon launches as yet.And the cotyledon of existing two greens of MaASR1 transgenosis seedling, root reaches 0.5cm.
2) L14 and L38 seed are to the resistance test of nonionic osmotic stress
With the wild-type Arabidopis thaliana, be numbered L14 and L38 positive T3 for the seed that changes the MaASR1 Arabidopis thaliana sow respectively contain 0,100,200, the substratum (1/2MS of 300mM N.F,USP MANNITOL, 1.5% Sucrose, 0.7% Agra, PH5.7) go up cultivation 15 days, make its sprouting, the statistics germination rate, the seed of each strain system is 50, the experiment triplicate, results averaged.Is contrast with T3 for changeing the empty carrier Arabidopis thaliana.With mutant gin1-3, abi4-1 is that (abi4-1 is an ABA non-sensitive type mutant strain in contrast, gin1-3 is an ABA responsive type mutant strain, in ABA susceptibility testing experiment, play the contrast effect available from Arabidopsis Biological Resource Center (Ohio university, Ohio state, USA).
The result as shown in Figure 8,
The wild-type Arabidopis thaliana contain 0,100,200, germination rate in the substratum of 300mM N.F,USP MANNITOL is respectively 100%, 61%, 41%, 22.78%;
The positive T3 that is numbered L14 for change MaASR1 Arabidopis thaliana (35S::MaASR114) contain 0,100,200, germination rate in the substratum of 300mM N.F,USP MANNITOL is respectively 100%, 97%, 98%, 95.6%;
The positive T3 that is numbered L38 for change MaASR1 Arabidopis thaliana (35S::MaASR138) contain 0,100,200, germination rate in the substratum of 300mM N.F,USP MANNITOL is respectively 100%, 88%, 90%, 72%;
Mutant gin1-3 contain 0,100,200, germination rate in the substratum of 300mM N.F,USP MANNITOL is respectively 100%, 90%, 78%, 82%;
Mutant abi4-1 contain 0,100,200, germination rate in the substratum of 300mM N.F,USP MANNITOL is respectively 95%, 90%, 97%, 95%;
T3 does not have significant difference for changeing empty carrier Arabidopis thaliana and wild-type Arabidopis thaliana result.
As can be seen, the sprouting of L14 and L38 seed almost is not subjected to the inhibition of N.F,USP MANNITOL yet, and wherein L14 percentage of germination (being germination rate) in containing 300mM N.F,USP MANNITOL substratum also remains on 95.6%.But the sprouting of the wild type seeds under the same terms is subjected to severe inhibition, and in the substratum of 300mM N.F,USP MANNITOL, its percentage of germination reduces to 22.78%.
2, the MaASR1 transfer-gen plant is to the endurance of arid and salt stress
1) drought-resistant ability of L14 and L38
To 5 weeks of growth in seedling pan with the wild-type Arabidopis thaliana, be numbered L14 and L38 positive T3 for after changeing the MaASR1 Arabidopis thaliana and controlling water treatment (stop to water, choose 23 ℃, 8h illumination, 16h dark) 14d, rehydration 3d observes survival rate again.With 17 days plant of normal water supply always in contrast.Each strain is 50 strains, and experiment repeats 3 times, results averaged.Is contrast with T3 for changeing the empty carrier Arabidopis thaliana.
Take pictures as shown in Figure 9 behind control water treatment 14d, WT is the wild-type Arabidopis thaliana, L14 and L38 be respectively be numbered L14 and L38 positive T3 for changeing the MaASR1 Arabidopis thaliana, as can be seen, each no significant difference of strain system of 17 days contrast of normal water supply always; And after controlling water treatment 14d, the wild-type blade is whole chlorosis, and is withered dead, though the blade edge of L14 and L38 beginning flavescence also keeps strong vitality.
Survival rate is mapped as shown in figure 10,
Under the normal processing (contrast), wild-type Arabidopis thaliana, the positive T3 that is numbered L14 (35S::MaASR114) and L38 (35S::MaASR138) are respectively 100%, 100%, 100% for the survival rate of changeing the MaASR1 Arabidopis thaliana;
Behind the control water treatment 14d, wild-type Arabidopis thaliana, the positive T3 that is numbered L14 (35S::MaASR114) and L38 (35S::MaASR138) are respectively 18.2%, 83.1%, 77.5% for the survival rate of commentaries on classics MaASR1 Arabidopis thaliana;
Behind the rehydration 3d, wild-type Arabidopis thaliana, the positive T3 that is numbered L14 (35S::MaASR114) and L38 (35S::MaASR138) are respectively 22.2%, 87.3%, 80.4% for the survival rate of changeing the MaASR1 Arabidopis thaliana;
T3 does not have significant difference for changeing empty carrier Arabidopis thaliana and wild-type Arabidopis thaliana result.
As can be seen, after to Arabidopis thaliana control water 14d, the survival rate of wild-type has reduced to 18.2%, and the survival rate of L14 and L38 then is respectively 83.1% and 77.5%.Behind the rehydration 4d, the survival rate of wild-type rises to 22.2%, and the survival rate of L14 and L38 then is upgraded to 87.3% and 80.4% respectively.Proof MaASR1 overexpression plant has stronger drought-resistant ability than wild-type.
2) L14 and L38 resistance that high density NaCl is coerced
With the wild-type Arabidopis thaliana in 5 weeks of growth, be numbered L14 and L38 positive T3 for changeing the MaASR1 Arabidopis thaliana with nutritive medium (the 2M KNO that contains 300mMNaCl
3, 2M Ca (NO
3)
2, 2M MaSO
4.7H
2O, 1M NH
4NO
3, 1M KH
2PO
4, 0.02M FeSO
4.7H
2O, 0.02M Na
2EDTA, 0.05M H
3BO
3, 0.01M MnCl
2.4H
2O, 0.01M Na
2MOO
4) behind the pouring 24d, observe survival rate.With the plant of normal processing (with the nutritive medium pouring 24d that does not contain NaCl) in contrast.Each strain is 50 strains, and experiment repeats 3 times, results averaged.Is contrast with T3 for changeing the empty carrier Arabidopis thaliana.
Take pictures as shown in figure 11, WT is the wild-type Arabidopis thaliana, and L14 and L38 are respectively the positive T3 that is numbered L14 and L38 for changeing the MaASR1 Arabidopis thaliana, as can be seen, and each no significant difference of strain system of the contrast of normal processing; And handle with the nutritive medium that contains 300mMNaCl, the almost whole chlorosis of the blade of wild-type Arabidopis thaliana, withered dead, L14 and L38 then still keep strong vitality, the wherein blade edge flavescence of L38, partial blade begins to occur the wilting phenomenon, and the growth of L14 then is not subjected to tangible influence.
Survival rate is mapped as shown in figure 12,
Under the normal processing (contrast), wild-type Arabidopis thaliana (col), the positive T3 that is numbered L14 (35S::MaASR114) and L38 (35S::MaASR138) are respectively 100%, 100%, 100% for the survival rate of changeing the MaASR1 Arabidopis thaliana;
After 300mMNaCl handled 24d, wild-type Arabidopis thaliana (col), the positive T3 that is numbered L14 (35S::MaASR114) and L38 (35S::MaASR138) were respectively 10.2%, 83.1%, 77.5% for the survival rate of commentaries on classics MaASR1 Arabidopis thaliana;
As can be seen, after NaCl handled 24d, the survival rate of wild-type had reduced to 10.2%, and the survival rate of L14 and L38 then remains on 83.1% and 77.5% respectively.Proof MaASR1 transfer-gen plant has stronger salt resistance ability than wild-type.
T3 does not have significant difference for changeing empty carrier Arabidopis thaliana and wild-type Arabidopis thaliana result.
Claims (10)
1. cultivate the transgenic plant method for one kind,, obtain to have following 1 for the proteic encoding gene of MaASR1 is imported the purpose plant)-5) in the transgenic plant of at least a feature:
1) resistance of reverse of described transgenic plant is higher than described purpose plant;
2) the blade table area of described transgenic plant is less than described purpose plant;
3) vane thickness of described transgenic plant is greater than described purpose plant;
4) the petiole length of described transgenic plant is greater than described purpose plant;
5) be later than described purpose plant the flowering period of described transgenic plant;
The proteic aminoacid sequence of described MaASR1 is the sequence 2 in the sequence table.
2. method according to claim 1 is characterized in that: the proteic encoding gene of described MaASR1 is the sequence 1 in the sequence table.
3. method according to claim 1 and 2 is characterized in that: described resistance of reverse is salt tolerance and/or drought tolerance.
4. according to the arbitrary described method of claim 1-3, it is characterized in that: described resistance of reverse is by improving germination rate, increase root length and/or improving survival rate and embody.
5. according to the arbitrary described method of claim 1-4, it is characterized in that: the proteic encoding gene of described MaASR1 imports described purpose plant by expression vector.
6. according to the arbitrary described method of claim 1-5, it is characterized in that: described expression vector is for inserting the proteic encoding gene of described MaASR1 among the pBI121 the proteic carrier of expression MaASR1 that obtains.
7. according to the arbitrary described method of claim 1-6, it is characterized in that: described purpose plant is dicotyledons or monocotyledons.
8. according to the arbitrary described method of claim 1-7, it is characterized in that: described dicotyledons is an Arabidopis thaliana.
9. expression vector is for inserting the proteic encoding gene of described MaASR1 among the pBI121 the proteic carrier of expression MaASR1 that obtains; The proteic encoding gene of described MaASR1 is the sequence 1 in the sequence table.
10. described MaASR1 albumen, the proteic encoding gene of described MaASR1 and/or the application of the described expression vector of claim 9 in plant breeding;
Or described MaASR1 albumen, the proteic encoding gene of described MaASR1 and/or the described expression vector of claim 9 application in cultivating the resistance of reverse transgenic plant;
Or described MaASR1 albumen, the proteic encoding gene of described MaASR1 and/or the described expression vector of claim 9 application in improving plant stress tolerance;
Described resistance of reverse is specially salt tolerance and/or drought tolerance; Described plant is specially dicotyledons or monocotyledons; Described dicotyledons especially is specially Arabidopis thaliana.
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| CN103232533A (en) * | 2012-11-13 | 2013-08-07 | 华中科技大学 | Wheat drought-resistant gene TaASR1 and application thereof |
| CN107299103A (en) * | 2017-08-04 | 2017-10-27 | 中国科学院华南植物园 | Thick boisiana IpASR genes and its encoding proteins and application |
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| CN103232533A (en) * | 2012-11-13 | 2013-08-07 | 华中科技大学 | Wheat drought-resistant gene TaASR1 and application thereof |
| CN107299103A (en) * | 2017-08-04 | 2017-10-27 | 中国科学院华南植物园 | Thick boisiana IpASR genes and its encoding proteins and application |
| CN107299103B (en) * | 2017-08-04 | 2018-12-25 | 中国科学院华南植物园 | Thick boisiana IpASR gene and its coding albumen and application |
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