CN103060373B - Application of PtoeIF5Al and encoding gene thereof on controlling plant growth - Google Patents
Application of PtoeIF5Al and encoding gene thereof on controlling plant growth Download PDFInfo
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Abstract
The invention discloses an application of poplar protein PtoeIF5Al and an encoding gene thereof on controlling plant growth. The invention further provides a method for cultivating transgenic plants. The method comprises the following steps: expressing or enhancing protein shown in sequence 1 of an expressed sequence table in the target plant, and then obtaining the transgenic plants which have at least one phenotype in a)-b) compared with the target plant: a) the growth cycle is reduced or the growth speed is increased; and b) the stem length is increased. Experimental results show that compared with the wild arabidopsis thaliana, the homozygous transgenic strains obtained by the poplar protein PtoeIF5Al shown in sequence 1 of the excessive expressed sequence table of the arabidopsis thaliana and the homozygous transgenic strains obtained by the poplar protein PtoeIF5A3 shown in sequence 3 of the excessive expressed sequence table obviously bring the time from the moment of seeding to the moment of bolting forward under the same normal growing conditions, and the stem length is obviously increased while the seeds are mature. The invention has significance on research and application of controlling the growth and the development of plants.
Description
Technical field
The present invention relates to a kind of willow albumen PtoeIF5A1 and the application of encoding gene in regulating plant growth thereof.
Background technology
EIF5A is the class protein extensively existing in eukaryote, is unique protein that contains carboxylic putrescine Methionin (hypusine) residue, the high conservative between each species.EIF5A has participated in the multinomial physiological activity of cell, as cell proliferation, protein translation, cell aging and apoptosis etc.In yeast body, when eIF5A function is suppressed or lacks, Partial Protein is synthetic to be reduced.When the activity of eIF5A is suppressed, the delayed growth of cell or stagnation.At present, in plant, the biological function of eIF5A is also indefinite.
Summary of the invention
An object of the present invention is to provide the new purposes of willow albumen PtoeIF5A1, the aminoacid sequence of this protein is as shown in sequence table sequence 1, described new purposes is that albumen shown in sequence table sequence 1 can be used for regulating and controlling object plant following 1)-2) at least one proterties, or regulate material or the method for expressing quantity shown in sequence table sequence 1 to can be used for regulating and controlling object plant following 1)-2) at least one proterties:
1) growth cycle or the speed of growth;
2) plant height.
Another object of the present invention is to provide a kind of method of cultivating transgenic plant, be included in described object plant the step of expressing or strengthening albumen shown in expressed sequence table sequence 1, obtain comparing with object plant have following a)-b) in the transgenic plant of at least one phenotype:
A) growth cycle shortens or speed of growth increase;
B) plant height increases.
In aforesaid method, in described object plant, express or strengthen albumen shown in expressed sequence table sequence 1 and comprise the encoding gene of described albumen is imported to the step in described object plant.
In aforesaid method, described importing comprises the expression cassette of the encoding gene of described albumen imported in described object plant, and in described expression cassette, the encoding gene upstream of described albumen is containing the enhanser gene shown in sequence table sequence 5.
In aforesaid method, shown in described sequence table sequence 1, the encoding gene of albumen is following 1) or 2) or 3) gene:
1) its nucleotide sequence is the DNA molecular shown in sequence table sequence 2 4-483 positions;
2) with 1) 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 albumen shown in 99% identity and code sequence list sequence 1;
3) under stringent condition with 1) or 2) DNA molecular of albumen shown in the DNA sequence dna hybridization that limits and code sequence list sequence 1.
Described stringent condition can be as follows: 50 ℃, and at 7% sodium lauryl sulphate (SDS), 0.5M Na
3pO
4with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 2 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3pO
4with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 1 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3pO
4with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 0.5 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3pO
4with in the mixing solutions of 1mM EDTA, hybridize, at 50 ℃, 0.1 * SSC, rinsing in 0.1%SDS; Also can be: 50 ℃, at 7%SDS, 0.5M Na
3pO
4with in the mixing solutions of 1mM EDTA, hybridize, at 65 ℃, 0.1 * SSC, rinsing in 0.1%SDS; Also can be: at 6 * SSC, in the solution of 0.5%SDS, at 65 ℃, hybridization, then uses 2 * SSC, 0.1%SDS and 1 * SSC, 0.1%SDS respectively washes film once.
In aforesaid method, described importing realizes by following recombinant vectors: between the XhoI of carrier pBI121 and speI site, be connected into successively the DNA fragmentation with following sequence: the 4-483 bit sequence (being PtoeIF5A1 gene order) of sequence table sequence 5 the 1-74 bit sequence (being enhancer sequence), sequence table sequence 6 the 1-30 bit sequence (being Flag-tag coding gene sequence) and sequence table sequence 2.
The present invention also protects a kind of recombinant vectors; between the XhoI of carrier pBI121 and speI site, to be connected into successively the DNA fragmentation with following sequence: the 4-483 bit sequence of sequence table sequence 5 the 1-74 bit sequence, sequence table sequence 6 the 1-30 bit sequence and sequence table sequence 2, it can for cultivate compare with object plant have described in aforesaid method a)-b) in application in the transgenic plant of at least one phenotype.
Above-mentioned purpose plant can be monocotyledons or dicotyledons, and described dicotyledons specifically can be Arabidopis thaliana (Arabidopsis thaliana).
Experimental results show that, the Transgenic wheat line that willow albumen PtoeIF5A1 in Arabidopis thaliana shown in overexpression sequence table sequence 1 obtains, compare with the Transgenic wheat line and the wild-type Arabidopis thaliana that contrast the willow albumen PtoeIF5A3 acquisition shown in overexpression sequence table sequence 3, under identical normal growth condition, from be seeded into bolting time advance 11-12 days, from be seeded into the whole flavescence of plant time advance 17-21 days, and plant plant height during seed maturity has increased 6-8cm.The present invention is to the research of regulating growth of plants and apply significant.
Accompanying drawing explanation
Fig. 1 is the Western Blot detected result of transgenic arabidopsis plant.Wherein, WT represents wild-type Arabidopis thaliana, and 1-1 and 1-2 are respectively T
2for Arabidopis thaliana strain PtoeIF5A1-OE1 and the PtoeIF5A1-OE2 of the transgenosis PtoeIF5A1 that isozygotys, 3-1 is T
2arabidopis thaliana strain PtoeIF5A3-OE1 for the transgenosis PtoeIF5A3 that isozygotys; The detected result of the first behavior target protein, the whether consistent result of the second behavior red S staining examine in the beginning of spring applied sample amount.
Fig. 2 is the result that real-time fluorescence quantitative PCR detects goal gene relative expression quantity in transgenic arabidopsis plant.Wherein, PtoeIF5A1-OE1 and PtoeIF5A1-OE2 are T
2arabidopis thaliana strain for the transgenosis PtoeIF5A1 that isozygotys; Ptoe IF5A3-OE1 is T
2arabidopis thaliana strain for the transgenosis PtoeIF5A3 that isozygotys.
Fig. 3 is T
2for the phenotype of transgenic arabidopsis strain plant sowing after 35 days of isozygotying.Wherein, in figure A, front two row plant are T from left to right
2for the Arabidopis thaliana strain PtoeIF5A1-OE1 plant of the transgenosis PtoeIF5A1 that isozygotys, the not genetically modified wild-type Arabidopis thaliana of rear two behaviors WT plant, in figure B, front two row plant are T from left to right
2for the Arabidopis thaliana strain PtoeIF5A1-OE2 plant of the transgenosis PtoeIF5A1 that isozygotys, the not genetically modified wild-type Arabidopis thaliana of rear two behaviors WT plant, in figure C, front two row plant are T from left to right
2for the Arabidopis thaliana strain PtoeIF5A3-OE1 plant of the transgenosis PtoeIF5A3 that isozygotys, the not genetically modified wild-type Arabidopis thaliana of rear two behaviors WT plant.
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, willow albumen PtoeIF5A1 and encoding gene thereof
1, get turriform Cortex Populi Tomentosae BJHR01(populus tomentosa Carr.cv ' BJHR01 ') root of tissue cultured seedling, stem and leaf, extract total RNA, and the synthetic cDNA of reverse transcription, take this cDNA as template again, under the guiding of primer CDS1-PF and primer CDS1-PR, cDNA sequence with conventional PCR method amplification Cortex Populi Tomentosae eIF5A1 gene (being PtoeIF5A1), after reaction finishes, pcr amplification product is carried out to 1% agarose gel electrophoresis detection, reclaim the also DNA fragmentation of the about 500bp of purifying, be connected into carrier pGEM-Teasy(and be purchased from Promega company), obtain recombinant vectors pGEM-Teasy-PtoeIF5A1, through order-checking, confirm, this recombinant vectors pGEM-Teasy-PtoeIF5A1 is for having inserted the DNA fragmentation of 483bp shown in sequence table sequence 2 at the TA of carrier pGEM-Teasy cloning site place.The 4-483 bit sequence of sequence table sequence 2 is Cortex Populi Tomentosae PtoeIF5A1 gene order, coding there is the albumen being formed by 159 amino acid shown in sequence table sequence 1, by this albumen called after PtoeIF5A1, this albumen be for No. Genbank AEF15861.1.
The sequence of above-mentioned primer is as follows:
Primer CDS1-PF:5 '-
cATATGtCTGACGAGGAGCAT-3 ' (band underscore partial sequence is restriction enzyme NdeI recognition sequence);
Primer CDS1-PR:5 '-TTACTTGGGGCCAATGTCC-3 '.
2, get turriform Cortex Populi Tomentosae BJHR01(populus tomentosa Carr.cv ' BJHR01 ') root of tissue cultured seedling, stem and leaf, extract total RNA, and the synthetic cDNA of reverse transcription, take this cDNA as template again, under the guiding of primer CDS2-PF and primer CDS2-PR, cDNA sequence with conventional PCR method amplification Cortex Populi Tomentosae eIF5A3 gene (being PtoeIF5A3), after reaction finishes, pcr amplification product is carried out to 1% agarose gel electrophoresis detection, reclaim the also DNA fragmentation of the about 500bp of purifying, be connected into carrier pGEM-Teasy, obtain recombinant vectors pGEM-Teasy-PtoeIF5A3, through order-checking, confirm, this recombinant vectors pGEM-Teasy-PtoeIF5A3 is for having inserted the DNA fragmentation of 483bp shown in sequence table sequence 4 at the TA of carrier pGEM-Teasy cloning site place.The 4-483 bit sequence of sequence table sequence 4 is Cortex Populi Tomentosae PtoeIF5A3 gene order, coding there is the albumen being formed by 159 amino acid shown in sequence table sequence 3, by this albumen called after PtoeIF5A3, this albumen be for No. Genbank AEF15863.1.
The sequence of above-mentioned primer is as follows:
Primer CDS2-PF:5 '-
cATATGtCTGACGAGGAGCAG-3 ' (band underscore partial sequence is restriction enzyme NdeI recognition sequence);
Primer CDS2-PR:5 '-TCACTTCGGGCCAACGTC-3 '.
The structure of embodiment 2, plant recombination expression vector
1, get the recombinant vectors pGEM-Teasy-PtoeIF5A1 that embodiment 1 step 1 obtains, with after NdeI and SpeI double digestion, there is the carrier framework fragment of the intermediate carrier pBSKd of Flag-tag to be connected with the fusion through NdeI and SpeI double digestion, obtain carrier pBSK-Ω-Flag-PtoeIF5A1; Carrier pBSK-Ω-Flag-PtoeIF5A1 is used after XhoI and speI double digestion, with the carrier pBI121(China plasmid vector strain cell pnca gene preservation center through XhoI and speI double digestion) skeleton fragment be connected, acquisition recombinant vectors pBI121-Ω-Flag-PtoeIF5A1; Through order-checking, confirm, recombinant vectors pBI121-Ω-Flag-PtoeIF5A1 is connected into successively the DNA fragmentation with following sequence between the XhoI of carrier pBI121 and speI site: the 4-483 bit sequence (being PtoeIF5A1 gene order) of sequence table sequence 5 the 1-74 bit sequence (being enhancer sequence), sequence table sequence 6 the 1-30 bit sequence (being Flag-tag coding gene sequence), sequence table sequence 2.
2, get the recombinant vectors pGEM-Teasy-PtoeIF5A3 that embodiment 1 step 2 obtains, with after NdeI and SpeI double digestion, there is the intermediate carrier pBSK carrier framework fragment of Flag-tag to be connected with the fusion through NdeI and SpeI double digestion, obtain carrier pBSK-Ω-Flag-PtoeIF5A3; Carrier pBSK-Ω-Flag-PtoeIF5A3 is used after XhoI and speI double digestion, with the carrier pBI121(China plasmid vector strain cell pnca gene preservation center through XhoI and speI double digestion) skeleton fragment be connected, acquisition recombinant vectors pBI121-Ω-Flag-PtoeIF5A3; Through order-checking, confirm, recombinant vectors pBI121-Ω-Flag-PtoeIF5A3 is connected into successively the DNA fragmentation with following sequence between the XhoI of carrier pBI121 and speI site: the 4-483 bit sequence (being PtoeIF5A3 gene order) of sequence table sequence 5 the 1-74 bit sequence (being enhancer sequence), sequence table sequence 6 the 1-30 bit sequence (being Flag-tag coding gene sequence), sequence table sequence 4.
Embodiment 3, willow albumen PtoeIF5A1 and encoding gene thereof can Promoting plant growths
One, the acquisition of restructuring agrobacterium tumefaciens
Recombinant vectors pBI121-Ω-Flag-PtoeIF5A1 freeze-thaw method that embodiment 2 is obtained transforms agrobacterium tumefaciens GV3101(China's plasmid vector strain cell pnca gene preservation center), the agrobacterium tumefaciens GV3101 that acquisition contains recombinant vectors pBI121-Ω-Flag-PtoeIF5A1, by this restructuring Agrobacterium called after GV3101/pBI121-Ω--Flag--PtoeIF5A1;
Recombinant vectors pBI121-Ω-Flag-PtoeIF5A3 freeze-thaw method that embodiment 2 is obtained transforms agrobacterium tumefaciens GV3101, the agrobacterium tumefaciens GV3101 that acquisition contains recombinant vectors pBI121-Ω-Flag-PtoeIF5A3, by this restructuring Agrobacterium called after GV3101/pBI121-Ω-Flag-PtoeIF5A3.
Two, the acquisition of transgenic arabidopsis
Two kinds of restructuring Agrobacteriums that utilize step 1 to obtain, bud infusion method transforms Colombia environmental Arabidopis thaliana Col-0(Arabidopsis Biological Resource Center respectively, is called for short ABRC, network address: https: //abrc.osu.edu/), obtain T
2generation isozygoty 2 of the Arabidopis thaliana strains of transgenosis PtoeIF5A1, T
2generation isozygoty 2 of the Arabidopis thaliana strains of transgenosis PtoeIF5A3, concrete grammar is as follows:
Get in single bacterium colony access 5ml LB nutrient solution (containing kantlex Kan50 μ g/ml and gentamicin 25 μ g/ml) of restructuring Agrobacterium GV3101/pBI121-Ω-Flag-PtoeIF5A1 or GV3101/pBI121-Ω-Flag-PtoeIF5A3,28 ℃ of volume ratios of cultivating after a day with 1:100 are transferred to 200ml and continue to be cultured to OD containing in same antibiotic LB nutrient solution
600=1.0; The centrifugal 10min of 5000g room temperature, is resuspended in bacterial sediment in 200ml1/2MS (containing 50g/L sucrose and 200 μ l/LSilwet L-77), obtains bacteria suspension; To treat that genetically modified Arabidopis thaliana plant is inverted, and make inflorescence more than lotus throne leaf in bacteria suspension, soak 5min, during slightly rock 2～3 times; Plant is placed horizontally in 22 ℃ of environment, with lighttight plastics bag, seals basin alms bowl; After 16 hours, plant is taken out, vertically cultivate until results seed (is T
0for seed), seed is standby after drying at room temperature.
Get T
0for seed, carry out after surface sterilization, be placed in 4 ℃ of vernalization 2～3 days, then be evenly laid on 1/2MS agar plate (containing 10g/l sucrose, 50 μ g/ml kantlex, 8g/L agar, pH=5.7), move into 22 ℃ of incubators (16 hours every days illumination/8 hour dark, intensity of illumination is 80-100Lux) cultivate, after seed germination 6 days, the transgenosis seedling that selects anti-kantlex (shows as blade edge and root is longer, and not genetically modified wild-type Arabidopsis thaliana Seedlings can be sprouted, but seedling is yellow compared with contemporaneously transgenosis seedling leaves, root is very short), move into new not occurring after 4 leaves containing continuing to be cultured on the 1/2MS agar plate of kantlex, move in soil and grow again, results T
1for seed.T is screened in plantation after the same method
1for seed, transplant kalamycin resistance separated than being the T of 3:1
1for strain, and individual plant results T
1for the T that ties on each individual plant in strain
2for seed, get T
2for strain seed, carry out after the same method kalamycin resistance screening, obtain T
2in generation, no longer produces the Transgenic wheat line of kalamycin resistance separation.
T
0the contemporary seed of tying of conversion and the plant being grown up to by it are shown in representative; T
1t is shown in representative
0the seed producing for selfing and the plant being grown up to by it; T
2t is shown in representative
1the seed producing for selfing and the plant being grown up to by it.
Three, the Western-Blot of transfer-gen plant detects
Get the T that step 2 obtains
2for the Arabidopis thaliana strain of the transgenosis PtoeIF5A1 that isozygotys, T
2arabidopis thaliana strain and wild-type Arabidopis thaliana Col-0 plant (WT) for the transgenosis PtoeIF5A3 that isozygotys, extract total protein, carry out Western-Blot detection, partial results as shown in Figure 1, result shows, the plant of transgenosis PtoeIF5A1 and PtoeIF5A3 is all expressed corresponding fusion rotein (be the fusion rotein of Flag-tag and PtoeIF5A1 or PtoeIF5A3, be 19kD), and WT is without the expression of above-mentioned fusion rotein.
Above-mentioned Western-Blot identifies that the concrete grammar of transfer-gen plant is as follows:
Extract plant total protein to be measured and carry out Western-Blot detection, with mouse-anti Flag M2 monoclonal antibody (Sigma company, products catalogue is numbered F3165) as the primary antibodie that detects fusion rotein (be the fusion rotein of Flag-tag and PtoeIF5A1 or PtoeIF5A3, be 19kD); With the sheep anti-mouse igg (Beijing Bioisystech Co., Ltd of Zhong Shan Golden Bridge, products catalogue is numbered ZB-2305) of horseradish peroxidase, be two anti-; With RuBisCO(1,5-Ribulose Bisphosphate Carboxylase/Oxygenase) whether the applied sample amount of Protein Detection albumen is consistent, as Fig. 1 figure below is depicted as red S coloration result in the beginning of spring.
Four, the detection of the real-time fluorescence quantitative PCR of transfer-gen plant
Get the T that step 2 obtains
2for the Arabidopis thaliana strain of the transgenosis PtoeIF5A1 that isozygotys, T
2arabidopis thaliana strain and wild-type Arabidopis thaliana Col-0 plant (WT) for the transgenosis PtoeIF5A3 that isozygotys, extract respectively total RNA, reverse transcription obtains cDNA, take this cDNA as template, with special primer F1 and R1, the cDNA of gene PtoeIF5A1 is carried out to real-time fluorescence quantitative PCR amplification, with special primer F3 and R3, the cDNA of gene PtoeIF5A3 is carried out to real-time fluorescence quantitative PCR amplification, take PtoACT9 as internal reference, primer is PtoACT9-F and PtoACT9-R.Real-time fluorescence quantitative PCR is at StepOnePlus
tMon real-time fluorescence quantitative PCR instrument, carry out, 3 repetitions are established in a parallel test.Utilize the method for LivakKJ and Schmittgen TD (2001) report, 2
-Δ Δ CTcalculate relative expression quantity.
ΔΔG
T=（G
T.Target－C
T.Actin）
Timex－（G
T.Target－C
T.Actin）
Time0
Time x represents random time point, Time
0the target gene of expression 1 times of amount after PtoACT9 proofreaies and correct is expressed.
The sequence of above-mentioned primer (5 '-3 ') is as follows:
F1：5’-TGTCACTTCGTTGGGATTGA-3’；
R1：5’-CAAGATCTTTCCCCTCACCA-3’；
F3：5’-CTTGCAAGGTTGTGGAGGTT-3’；
R3：5’-TGCCATTCTCAGTCAGCAAG-3’；
PtoACT9-F：5’-TTGCTGACCGTATGAGCAAG-3’；
PtoACT9-R：5’-AATCCACATCTGCTGGAAGG-3’。
As shown in Figure 2, result shows result, not expressing gene PtoeIF5A1 and PtoeIF5A3 in WT plant; And in the Arabidopis thaliana strain of transgenosis PtoeIF5A1 in the Arabidopis thaliana strain of gene PtoeIF5A1 and transgenosis PtoeIF5A3 the expression amount of gene PtoeIF5A3 very high.
Five, the phenotypic evaluation of transgenic arabidopsis
Get the T that step 2 obtains
2for the Arabidopis thaliana strain (PtoeIF5A1-OE1, PtoeIF5A1-OE2) of the transgenosis PtoeIF5A1 that isozygotys, T
2arabidopis thaliana strain (PtoeIF5A3-OE1) and wild-type Arabidopis thaliana Col-0(WT for the transgenosis PtoeIF5A3 that isozygotys) seed, sterilizing is by 4 ℃ of vernalization 2-3 days, in 22-23 ℃ of temperature, illumination 16 hours every days, 8 hours dark and intensity of illumination, is after planting 120-150 μ mol/m
2under the condition of sec, normal water and fertilizer management, sow after 23 days, the Arabidopis thaliana strain plant of transgenosis PtoeIF5A1 has started bolting (when described bolting refers to that scape is extracted 0.1cm out), after sowing 35 days, the average plant height of the Arabidopis thaliana strain plant of transgenosis PtoeIF5A1 is 8cm, and Arabidopis thaliana strain plant development period of WT strain and transgenosis PtoeIF5A3 is consistent and equal boltings (as shown in Figure 3) not, add up each strain plant (each strain 30 strain) from being seeded into the bolting number of days of (when described bolting refers to that scape is extracted 0.1cm out), plant height when being seeded into the number of days of the whole flavescence of plant (comprising pod) and seed maturity, result is recorded in table 1 with the form of mean value.
The phenotypic evaluation result of table 1 transgenic arabidopsis
* represent to compare with WT at P ﹤ 0.05 significant difference.
The result of Fig. 3 and table 1 shows, willow PtoeIF5A1 overexpression can cause that plant-growth accelerates, thereby shortens growth cycle, and can make plant height increase.
Claims (7)
1. albumen shown in sequence table sequence 1 is at regulation and control object Arabidopis thaliana following 1)-2) in application at least one proterties:
1) growth cycle or the speed of growth; 2) plant height.
2. cultivate a method for transgenic arabidopsis, be included in object Arabidopis thaliana the step of expressing or strengthening albumen shown in expressed sequence table sequence 1, obtain comparing with object Arabidopis thaliana have following a)-b) in the transgenic arabidopsis of at least one phenotype:
A) growth cycle shortens or speed of growth increase;
B) plant height increases.
3. method according to claim 2, is characterized in that: in described object Arabidopis thaliana, express or strengthen albumen shown in expressed sequence table sequence 1 and comprise the encoding gene of described albumen is imported to the step in described object Arabidopis thaliana.
4. method according to claim 3, it is characterized in that: described importing comprises the expression cassette of the encoding gene of described albumen is imported in described object Arabidopis thaliana, in described expression cassette, the encoding gene upstream of described albumen is containing the enhanser shown in sequence table sequence 5.
5. according to the method described in claim 3 or 4, it is characterized in that: the encoding gene of albumen shown in described sequence table sequence 1 is the DNA molecular shown in sequence table sequence 2 4-483 positions.
6. method according to claim 5, is characterized in that: described importing realizes by following recombinant vectors: the DNA fragmentation that is connected into successively following sequence between the XhoI of carrier pBI121 and speI site: the 4-483 bit sequence of sequence table sequence 5 1-74 bit sequences, sequence table sequence 6 1-30 bit sequences and sequence table sequence 2.
A recombinant vectors cultivate compare with object Arabidopis thaliana have described in claim 2 a)-b) in application in the transgenic arabidopsis of at least one phenotype; Described recombinant vectors is between the XhoI of carrier pBI121 and speI site, to be connected into successively the DNA fragmentation of following sequence: the 4-483 bit sequence of sequence table sequence 5 1-74 bit sequences, sequence table sequence 6 1-30 bit sequences and sequence table sequence 2.
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| HQ529479;Wang,H.Z.等;《GenBank》;20110522 * |
| Wang,H.Z.等.HQ529479.《GenBank》.2011, |
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