CN107129529A - Applications of the soybean transcription factor GmAREB3 in vegetable fat metabolic regulation - Google Patents
Applications of the soybean transcription factor GmAREB3 in vegetable fat metabolic regulation Download PDFInfo
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Abstract
The invention discloses applications of the soybean transcription factor GmAREB3 in vegetable fat metabolic regulation.The present invention provides following 1) -3) in application of any material in the regulation and control total fat content of plant tissue and/or regulation and control plant tissue in content of fatty acid：1) Protein G mAREB3；2) encoding proteins GmAREB3 DNA molecular；3) recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium of the DNA molecular containing encoding proteins GmAREB3；The amino acid sequence of the Protein G mAREB3 is the sequence 2 in sequence table.The experiment proves that, transcription factor GmAREB3 and its encoding gene can regulate and control to improve fat content in vegetable seeds after fat content in vegetable seeds, overexpression.The gene pairs is improved with Crop Improvement grease composition, especially for grease composition in the oilseed plant seeds such as soybean is improved, and cultivating high grease kind has important theory and realistic meaning.
Description
Technical field
It is metabolized the present invention relates to biological technical field, more particularly to a kind of soybean transcription factor GmAREB3 in vegetable fat
Application in regulation and control.
Background technology
71% grease comes from plant in human diet.In several main oil-producing crops in the world, soybean total yield
Oil mass accounts for 30%, occupies first (table 1) of cosmopolitian plant oil yield.
Table 1 is oil-producing crop main in the world
The synthesis of aliphatic acid is one of most important metabolic pathway in plant, it be present in plant any one is thin
It is necessary to growing in born of the same parents.The death of cell can be caused to its blocking, thus up to now it has not been found that one
Block the plant mutant of aliphatic acid synthesis.
Plant has very big difference with other eucaryotes on the enzyme for participating in fatty acid synthesis pathway.From acetyl-CoA and third
The aliphatic acid that two acyl CoA synthesize 16 or 18 carbon atoms at least needs the reaction of 30 different enzymatics to complete this mistake
Journey, and in animal, fungi and some bacteriums, above reaction is completed by a multienzyme complex being present in kytoplasm
's.In plant, the enzyme for participating in aliphatic acid synthesis is present in the kytoplasm of plastid in solvable form.
In most plants, grease is all stored in the form of triacylglycerol (Triacylglycerols, TAG), and it contains
Amount is a very important economical character, and TAG biosynthesis is referred to as Kennedy approach, as synthesized in eucaryote
1 and 2 of glycerol 3-phosphate is transferred to after the approach of film glyceride, aliphatic acid removal CoA, intermediate product PA is formed.PA is gone
Phosphorylation produces DAG.The final step synthesized in TAG, the 3rd fatty acid molecule is transferred to the-OH positions of DAG 3 ' of sky,
This single step reaction is catalyzed by diacylglycerol transacetylase (diacylglycerol acyltransferase, DGAT),
This reaction is considered as unique rate-limiting step in TAG biosynthesis.People have cognition to lipid route of synthesis, and
Through the enzyme gene for having cloned many participation lipid synthesis.However, in plant, the Regulation Mechanism and its related gene synthesized to lipid
Still know little about it.
In plant, bZIP class transcription factors are an extended familys, and its member is up to more than 100 in plant, can be divided into again
Several subtribes, its function is quite different, is played an important role in the growth, development in plant.Applicant laboratory early stage clusters
131 bZIP class transcription factors in soybean gene group, and then find, tri- bases of GmbZIP44, GmbZIP62 and GmbZIP78
Because of response abiotic stress, and participate in salt tolerant, low temperature resistant regulation and control.Their overexpression improves the salt tolerant of transfer-gen plant and resistance to
Low temperature.Afterwards, applicant has cloned bZIP class transcription factor genes GmbZIP123 encoding gene GmbZIP123 in soybean,
Homologous clustering finds that GmbZIP123 genes and the affinity of arabidopsis S1 subtribe bZIP class transcription factors are nearest, therefore
Belong to S races.It is reported that many A subtribes genes in bZIP families participate in plant for ABA response, such as ABI5, AREB1/
2 etc., participate in seed maturity drying, sprouting, dormancy and environment-stress reaction.AREB3 encodes ABA response element knots in arabidopsis
Hop protein, is the insensitive albuminoids of ABA, is positioned in nucleus, not yet report that it is related to fat metabolic.In view of soybean gene
Group sequence has been completed, and GmAREB3 gene orders are consistent with Glyma06g47220.2, and promoter Analysis shows that it is responded for ABA
Element binding factor, but do not have functional description.Soybean GmAREB3 and arabidopsis AREB3 protein sequence similitude is small
In 20%, therefore soybean GmAREB3 function is still unknown.
The content of the invention
It is an object of the present invention to provide following 1) -3) in any material new application.
The present invention provide following 1) -3) in any material planted in the regulation and control total fat content of plant tissue and/or regulation and control
Application in thing tissue in content of fatty acid or the application in the plant that grease and/or content of fatty acid are improved is cultivated：
1) Protein G mAREB3；
2) encoding proteins GmAREB3 DNA molecular；
3) recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium of the DNA molecular containing encoding proteins GmAREB3；
The Protein G mAREB3 is following (1) or (2)：
(1) protein being made up of the amino acid sequence shown in sequence in sequence table 2；
(2) by the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues substitution and/or
Missing and/or addition and the protein as derived from (1) with identical function.
In above-mentioned application, the DNA molecular of the encoding proteins GmAREB3 is following 1) to any described DNA points in 4)
Son：
1) DNA molecular in sequence table shown in sequence 1；
2) DNA molecular in sequence table shown in sequence 1 1-948；
1) or 2) 3) under strict conditions with the DNA molecular hybridization that is limited and encoding as shown in sequence in sequence table 2
The protein DNA molecule of amino acid sequence composition；
1) or 2) 4) at least have 70% with the DNA molecular that is limited, at least have 75%, at least having 80%, at least
With 85%, at least have 90%, at least have 95%, at least have 96%, at least having 97%, at least having 98% or extremely
There is the protein DNA molecule that 99% homology and coding are made up of the amino acid sequence shown in sequence in sequence table 2 less.
The recombinant vector of the DNA molecular containing encoding proteins GmAREB3 is by the encoding proteins GmAREB3's
In DNA molecular insertion expression vector, expressing protein GmAREB3 recombinant vector is obtained.In an embodiment of the present invention, expression is carried
Body is pGWB412, and recombinant vector is, by the nucleotides inserted carrier pGWB412 shown in sequence in sequence table 1, to obtain restructuring and carry
Body；Inserted by the way of homologous recombination, specific method is shown in embodiment.
In above-mentioned application, the total fat content of the regulation and control plant tissue is the raising total fat content of plant tissue；
Content of fatty acid is content of fatty acid in raising plant tissue in the regulation and control plant tissue；
The aliphatic acid is palmitic acid, oleic acid, linoleic acid and/or tribute polyacid.
Above-mentioned total fat content is embodied by the percentage of w lipid in seed and seed weight.
It is described to be organized as seed in above-mentioned application；The plant is monocotyledon or dicotyledon.It is described dicotyledonous
Plant is specially legume or crucifer.In an embodiment of the present invention, dicotyledon is arabidopsis.
It is a further object to provide a kind of genetically modified plants for cultivating grease and/or content of fatty acid raising
Method.
The method that the present invention is provided, for encoding proteins GmAREB3 DNA molecular is imported in purpose plant, obtains turning base
Because of plant；Total fat content and/or content of fatty acid are higher than the purpose plant in the Transgenic plant tissue；
The Protein G mAREB3 is following (1) or (2)：
(1) protein being made up of the amino acid sequence shown in sequence in sequence table 2；
(2) by the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues substitution and/or
Missing and/or addition and the protein as derived from (1) with identical function.
Above-mentioned total fat content is embodied by the percentage of w lipid in seed and seed weight.
In the above method, the aliphatic acid is palmitic acid, oleic acid, linoleic acid, leukotrienes and/or tribute polyacid；
The DNA molecular of the encoding proteins GmAREB3 is imported in the purpose plant by recombinant vector；
The DNA molecular of the encoding proteins GmAREB3 is following 1) to any described DNA molecular in 4)：
1) DNA molecular in sequence table shown in sequence 1；
2) DNA molecular in sequence table shown in sequence 1 1-948；
1) or 2) 3) under strict conditions with the DNA molecular hybridization that is limited and encoding as shown in sequence in sequence table 2
The protein DNA molecule of amino acid sequence composition；
1) or 2) 4) at least have 70% with the DNA molecular that is limited, at least have 75%, at least having 80%, at least
With 85%, at least have 90%, at least have 95%, at least have 96%, at least having 97%, at least having 98% or extremely
There is the protein DNA molecule that 99% homology and coding are made up of the amino acid sequence shown in sequence in sequence table 2 less.
The recombinant vector is to insert the DNA molecular of the encoding proteins GmAREB3 in expression vector, obtains expressing egg
White GmAREB3 recombinant vector.In an embodiment of the present invention, expression vector is pGWB412, and recombinant vector is by sequence table
Nucleotides inserted carrier pGWB412 shown in sequence 1, obtains recombinant vector；Inserted by the way of homologous recombination, specific method
See embodiment.In an embodiment of the present invention, the Gateway systems produced using Invitrogen companies/GW/TA Cloning kits, entry vector TOPO and purpose carrier pGWB412 are marked with Spectinomycin resistance, can
High frequency zone Escherichia coli, the two has homologous recombination site attL1 and attL2, is connected with the carrier TOPO of target gene with carrying
Body pGWB412 carries out homologous recombination in the presence of recombinase, builds plant expression vector pGWB412-GmAREB3.
It is described to be organized as seed in the above method；The plant is monocotyledon or dicotyledon.The present invention's
In embodiment, dicotyledon is specially legume or crucifer, and dicotyledon is arabidopsis.
Third object of the present invention is to provide a kind of recombinant vector.
The recombinant vector that the present invention is provided, for encoding proteins GmAREB3 DNA molecular is inserted in expression vector, is obtained
Expressing protein GmAREB3 recombinant vector；
The Protein G mAREB3 is following (1) or (2)：
(1) protein being made up of the amino acid sequence shown in sequence in sequence table 2；
(2) by the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues substitution and/or
Missing and/or addition and the protein as derived from (1) with identical function.
In above-mentioned recombinant vector, the nucleotides sequence of the DNA molecular of the encoding proteins GmAREB3 is classified as the sequence in sequence table
Row 1.
Above-mentioned recombinant vector is to insert said gene in expression vector, obtains expressing the recombinant vector of above-mentioned albumen.
In embodiments of the invention, expression vector is pGWB412, and recombinant vector is by the nucleotides inserted shown in sequence in sequence table 1
Carrier pGWB412, obtains recombinant vector；Inserted by the way of homologous recombination, specific method is shown in embodiment.
The above-mentioned carrier that sets out for being used to build the plant expression vector can be any one double base agrobacterium vector or can
Carrier for plant micropellet bombardment etc., such as pBin438, pCAMBIA1302, pCAMBIA2301, pCAMBIA1301,
PCAMBIA1300, pBI121, pCAMBIA1391-Xa or pCAMBIA1391-Xb (CAMBIA companies).Built using GmAREB3
During plant expression vector, plus any enhanced, composing type, organizing specific type or it can be lured before its transcription initiation nucleotides
Conductivity type promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, general raw plain gene Ubiquitin promoters (pUbi),
They can be used alone or are used in combination with other plant promoters；In addition, being expressed using the gene constructed plant of the present invention
During carrier, enhancer, including translational enhancer or transcriptional enhancer are it is also possible to use, these enhancer regions can be ATG startings
Codon or neighboring region initiation codon etc., but must be identical with the reading frame of coded sequence, to ensure whole sequence just
Really translation.The source of the translation control signal and initiation codon is extensive, can be natural or synthesis
's.Translation initiation region can come from transcription initiation region or structural gene.
For the ease of transgenic plant cells or plant are identified and screened, plant expression vector used can be carried out
Processing, as add the coding that can be expressed in plant can produce color change enzyme or luminophor gene (gus gene,
Luciferase genes etc.), resistant antibiotic marker (gentamicin label, kanamycins label etc.) or anti-
Chemical reagent marker gene (such as anti-herbicide gene).From the security consideration of genetically modified plants, any selectivity can be not added with
Marker gene, directly screens transformed plant with adverse circumstance.
Carrying GmAREB3 of the present invention plant expression vector can be carried by using Ti-plasmids, Ri plasmids, plant virus
The conventional biology methods such as body, directly delivered DNA, microinjection, conductance, agriculture bacillus mediated convert plant cell or tissue, and will
The plant cell or tissue cultivating of conversion are into plant.The plant host being converted both can be monocotyledon, such as paddy rice, small
Wheat, corn etc. or dicotyledon, such as soybean, tobacco, arabidopsis or cotton.
The experiment proves that, the invention provides transcription factor GmAREB3 is transferred in wildtype Arabidopsis thaliana, obtain
Transgenic arabidopsis, the transgenic arabidopsis is compared with wildtype Arabidopsis thaliana, fat content and partial fatty acid in its seed
Content (such as palmitic acid, oleic acid, linoleic acid and/or tribute polyacid) is improved.Illustrate that transcription factor GmAREB3 and its encoding gene can
Contained with regulating and controlling to improve grease and/or aliphatic acid in vegetable seeds in vegetable seeds after grease and/or content of fatty acid, overexpression
Amount.The gene pairs is improved with Crop Improvement grease composition, especially for grease composition in the oilseed plant seeds such as raising soybean,
Cultivating high grease kind has important theory and realistic meaning.
The present invention is described in further details with reference to specific embodiment.
Brief description of the drawings
Fig. 1 is cloning vector and plant expression vector schematic diagram.
Fig. 2 is expression analysis of the GmAREB3 in soybean Different Organs.
Fig. 3 is the Molecular Identification for turning GmAREB3 plant pure lines.
Fig. 4 determines to turn fat content in GmAREB3 plant seeds.
Fig. 5 determines to turn content of fatty acid in GmAREB3 plant seeds.
Embodiment
Experimental method used in following embodiments is conventional method unless otherwise specified.
Material, reagent used etc., unless otherwise specified, are commercially obtained in following embodiments.
The primer is by three rich biotech firm's synthesis in following embodiments.
The black agriculture 44 (HN44) of soybean is recorded in the following literature：Man Weiqun etc., the seed selection of the black agriculture 44 of new soybean varieties and not
Influence with planting patterns to its yield and kind, Exploitation of Agriculture in Heilongjiang science 5 phases in 2004,1-5；The public can be from the Chinese Academy of Sciences
Heredity is obtained with Developmental Biology research；The soybean is obtained from soybean research institute of the Exploitation of Agriculture in Heilongjiang academy of sciences in 2006；By black dragon
The soybean research of river Academy of Agricultural Sciences the soybean varieties through Heilongjiang Province's crop varietal approval committee in 2002, first
It is bred as artificial Du Weiguang researcher, Patent No.：CNA20020216.2, authorization number is：Black careful beans 2002003.
Expression vector pGWB412 is recorded in the following literature：Department of Molecular and
Functional Genomics,Shimane University,Aatsue,Shimane 690-8504,Japan,E.mail:
[email protected] Isuyoshi Nakagawa,et al.,Gatway Vectors for
Plant Transformation,Plant Biotechnology,2009,26,275-284.By Tsuyoshi Nakagawa
Doctor provides, and the public obtains can be from Chinese Academy of Sciences's heredity and Developmental Biology research institute after Tsuyoshi doctors Nakagawa agree to
Obtain.
Agrobacterium GV3101, the public can be obtained from Inst. of Genetics and Development Biology, CAS's Developmental Biology research, be documented in as follows
In document：Lee CW etc., Agrobacterium tumefaciens promotes tumor induction by
modulating pathogen defense in Arabidopsis thaliana,Plant Cell,2009,21(9),
2948-62。
The structure of embodiment 1, soybean transcription factor GmAREB3 cDNA clone and plant expression vector
1st, transcription factor GmAREB3 acquisition
A bZIP transcription factors man is obtained in the analysis of fatty acid accumulation metabolism in carrying out Developing Soybean Seeds
GmAREB3 genes have higher expression in race.Therefore the correlation that the gene regulates and controls with oil synthesis is studied.
The total serum IgE of the black seedling of agriculture 44 of soybean is extracted, RNA is synthesized into cDNA with reverse transcriptase reverse transcription.
According to the information of the GmAREB3 full length cDNA sequences in PlantGDB soybean genomic sequence, primer is designed, is drawn
Thing sequence is as follows：
GmAREB3-up：5 '-ATGGGGTCTCAATGTGGTGGTG (sequence 3)；
GmAREB3-dp：5 '-GAATGAAGCCGATGATGTTCGGC (sequence 4).
CDNA using black agriculture 44, as template, is primer with GmAREB3-up and GmAREB3-dp, enters performing PCR amplification, obtains
About 1Kb PCR primer.By sequencing, the PCR primer is 951bp, and it has the nucleotides shown in sequence 1 in sequence table, the core
Gene shown in thuja acid is GmAREB3, and code area is sequence 1 1-951；The albumen of the gene code is named as GmAREB3,
The albumen is containing 316 amino acid, and its amino acid sequence is the sequence 2 in sequence table.
2nd, the structure of plant expression vector
The Gateway systems that gene cloning is provided using invitrogen the companies ,-T jags of carrier 3 ', for directly connecting
Connect the PCR primer of Taq enzyme amplification.
The PCR primer for the 951bp that above-mentioned 1 is obtained is with the TA principle clones cloned and carrier8/GW/TOPO it is upper (/GW/TA Cloning Kit,Catalog number:K2500-20,Invitogen Corporation,
Carlsbad, CA, USA carrier schematic diagram such as Figure 1A) connection, obtain intermediate carrier/GW/TOPO-GmAREB3。
Due toRecombination site attL1 and attL2 are carried on/GW/TOPO carriers and expression vector pGWB412, because
The intermediate carrier of this connection target gene can carry out LR recombining reactions with expression vector pGWB412 in the presence of recombinase,
Final purpose gene GmAREB3 is successfully building up on over-express vector pGWB412, obtains recombinant vector.
Specific method is as follows：1ul/GW/TOPO-GmAREB3,1ul pGWB412,1ul LR buffer,1ul
LR Enzyme mix, 1ul TE buffer PH8.0,25 DEG C of 6h, plus after 0.5ul Proteinase Ks, 37 DEG C, 10min obtains weight
Group carrier (operation is shown in the specification of company's offer or with reference to above-mentioned document in detail).
Recombinant vector is by sequencing, and the carrier is by the DNA molecular homologous recombination shown in sequence in sequence table 1 to carrier
The carrier obtained in pGWB412, is named as pGWB412-GmAREB3 (part-structure schematic diagram such as Figure 1B).
3rd, expression analysis of the GmAREB3 in soybean Different Organs
The total serum IgE of root, stem, leaf, flower and the seed of the black agriculture 44 of soybean is taken, cDNA is synthesized with reverse transcriptase reverse transcription.Primer
For：5 '-GGAATCTGCTGCCCGTTCTA and 5 '-TGATGTTCGGCGAATCTGGT, carry out RealTime-PCR identifications.Soybean
Tublin genes are internal standard, and the primer is Primer-TF：5 '-AACCTCCTCCTCATCGTACT, and Primer-TR：5’-
GACAGCATCAGCCATGTTCA-3’。
With wildtype Arabidopsis thaliana (col-0), (seed is purchased from Arabidopsis Biological Resource Center
(ABRC) it is) control.
As a result as shown in Fig. 2 in root, stem, leaf and the transcription that GmAREB3 genes are nearly no detectable in spending, and in seed
In its expression quantity it is very high, therefore GmAREB3 is the gene of seed specific expression.
The application of embodiment 2, GmAREB3 in the regulation and control total grease of plant tissue or content of fatty acid
First, the acquisition of recombinational agrobacterium
Recombinant vector pGWB412-GmAREB3 electric shocking method importing Agrobacterium GV3101 are obtained by the 2 of embodiment 1, are obtained
Recombinant bacterium.
The plasmid of recombinant bacterium is extracted, by sequencing, the plasmid is pGWB412-GmAREB3, by the restructuring containing the plasmid
Bacterium is named as GV3101/GmAREB3, as recombinational agrobacterium.
2nd, the acquisition and identification of GmAREB3 arabidopsis are turned
Recombinational agrobacterium GV3101/GmAREB3 is cultivated to logarithmic phase, then Colombia is converted with vacuumizing method
Arabidopsis thaliana ecotype (col-0) (seed is purchased from Arabidopsis Biological Resource Center (ABRC)) flower
In, seed is harvested after cultivation, seed is sowed on the MS screening and culturing mediums containing kanamycins (50mg/L), it is to be screened to obtain
T1Move on on vermiculite and grow during for plant length to 6 leaf, harvest T1For individual plant, each single-strain seed is sowed respectively, is screened with identical MS
Culture medium continues to screen to observe T2The separation situation in generation, such repeat number generation are until obtain the transgenic homozygous strain of inheritance stability
System, obtains 14 T5In generation, turns GmAREB3 arabidopsis pure lines.
Extract 14 T of above-mentioned acquisition5In generation, turns the RNA of GmAREB3 arabidopsis strain pure lines seedling, and reverse transcription obtains cDNA conducts
Template, primer is：5 '-GGAATCTGCTGCCCGTTCTA and 5 '-TGATGTTCGGCGAATCTGGT, carry out Real Time-
PCR is identified.Arabidopsis AtActin2 genes are internal standard, and the primer is Primer-TF：5 '-ATGCCCAGAAGTCTTGTTCC,
And Primer-TR：5’-TGCTCATACGGTCAGCGATA-3’.It is control with wildtype Arabidopsis thaliana (col0).Experiment repeats three
It is secondary, results averaged ± standard deviation.
The result of GmAREB3 expression quantity in wildtype Arabidopsis thaliana (col0) as shown in figure 3, fail to detect GmAREB3's
Expression quantity；T5In generation, turns detect GmAREB3 transcription, OE-2, OE-3, OE-7, OE-17 in GmAREB3 arabidopsis strains
Relative expression quantity with GmAREB3 in OE-18 is about 0.095,0,051,0.117,0,083 and 0.086.The above results prove,
GmAREB3 is transferred in arabidopsis and expressed, it was demonstrated that numbering is OE-2, OE-3, OE-7, OE-17 and OE-18 T5In generation, turns
GmAREB3 arabidopsis is positive transgenic plant.
It is five T of OE-2, OE-3, OE-7, OE-17 and OE-18 to choose numbering5Generation turn GmAREB3 arabidopsis pure lines do into
One step Function detection.
Empty carrier pGWB412 is transferred in wildtype Arabidopsis thaliana using same method, T is obtained0In generation, turns empty carrier and intends south
Mustard, sowing, sowing, until obtaining T5In generation, turns empty carrier arabidopsis.
3rd, the phenotypic analysis of GmAREB3 gene arabidopsis is turned
Determine wildtype Arabidopsis thaliana (col0), T5It is OE-2, OE-3, OE-7, OE-17 that generation, which turns empty carrier arabidopsis, numbering,
With OE-18 T5In generation, turns the total fat content of seed of GmAREB3 arabidopsis.
Specific method is as follows：
The total fat content of seed is determined:Dry seed is pulverized, 100mg is weighed into centrifuge tube, it is parallel to weigh
Four parts.500 μ l n-hexane is added, is fully mixed, 37 DEG C overnight.Slow speed centrifugation 3 minutes, weighed new pipe is sucked by n-hexane
In.Remaining powder continues plus n-hexane repeats to soak, be then centrifuged for and then collect n-hexane into same centrifuge tube.Will
Centrifuge tube is put into vavuum pump, is vacuumized, and n-hexane is volatilized completely.Then the weight of centrifuge tube is weighed again.Before centrifuge tube
The change of weight is the w lipid of extraction afterwards；The calculation formula of total oil quantity (%) is total oil quantity (%)=(extraction
W lipid/seed weight) X100%.
Each strain takes 30 plants of seed, tests in triplicate, results averaged ± standard deviation.
As a result as shown in figure 4,
The total oil quantity of wildtype Arabidopsis thaliana seed is 34.2 ± 0.1% (being the percentage of seed weight), wild type
Arabidopsis and turn the result of empty carrier arabidopsis without significant difference.
T5In generation, turns the total oil quantity of GmAREB3 arabidopsis strain OE-2, OE-3, OE-7, OE-17 and OE-18 seeds and is about
36.0 ± 0.4%；34.5 ± 0.4%；35.7 ± 0.4%；34.8 ± 0.4% and 34.6 ± 0.3%.Wherein OE-2's and OE-7
Seed fat content pole is significantly higher than control, and the total oil quantity of OE-17 and OE-18 seeds is significantly higher than control, and OE-3 seeds are total
Oil quantity is only slightly higher than control.
As a result show, total fat content in 4 transgenic line seeds is apparently higher than control.
Above-mentioned experiment shows that synthesis of the soybean bZIP class transcription factor GmAREB3 to total grease in seed is made in positive regulation and control
With its encoding gene GmAREB3 excess can improve the content of total grease in transfer-gen plant seed.
The detection of content of fatty acid in the seed of each plant:Seed to be measured is thoroughly dried, is pulverized, takes 10mg to add spiral shell
In the 2ml centrifuge tubes of mouth, every part of sample is parallel to weigh four parts.Add the 17 of 10 μ l:0 aliphatic acid (10mg/ml) does internal standard.Plus contain
The methanol solution 1ml of 2.5% concentrated sulfuric acid, is incubated 1 hour in 85 DEG C of water-baths, during which rocks for several times.After natural cooling, supernatant is taken
500 μ l add 600 μ l 0.9%NaCl solution, 300 n-hexanes into new pipe, and concussion mixes a few minutes, and 4000 leave the heart 10
Minute, supernatant is taken into new pipe.Make n-hexane volatilization complete overnight in fume hood, then add 50 μ l ethyl acetate dissolving methyl esters
The aliphatic acid of change.The fatty acid sample of esterification is surveyed (Perkin-Elmer Turbomass) with gas chromatography-mass spectrometry
The relative amount of each component, then the aliphatic acid of each composition with add Heptadecanoic acide 17:0 internal standard (Sigma, 51610)
Compare and show that (method may refer to relative amount：Shen,B.,et al.,The homeobox gene GLABRA2affects
seed oil content in Arabidopsis,Plant Mol.Biol.,60,377-387,2006).Each strain takes 30
The seed of strain, is tested in triplicate, results averaged ± standard deviation.
As a result as shown in figure 5, wherein numbering is OE-2, OE-7, OE-17 and OE-18 T5In generation, turns GmAREB3 arabidopsis kinds
Sinapic acid (22 in son:1) content equal no significant difference compared with wildtype Arabidopsis thaliana；Leukotrienes (18：3) with stearic acid (18:
0) content is lower than compareing, but total oil quantity significantly rises.
Wildtype Arabidopsis thaliana (Col0), the T that numbering is OE-2, OE-7, OE-17 and OE-185In generation, turns GmAREB3 arabidopsis
Palmitic acid (16 in seed:0) percentage for accounting for seed weight respectively may be about 2.8%, 3.2%, 3.3%, 3.1% and 3.2%；
Wildtype Arabidopsis thaliana, the T that numbering is OE-2, OE-7, OE-17 and OE-185In generation, turns in GmAREB3 arabidopsis seeds
Oleic acid (18:1) it is about 4.3%, 5.3%, 5.3%, 5.4% and 5.5%；
Wildtype Arabidopsis thaliana, the T that numbering is OE-2, OE-7, OE-17 and OE-185In generation, turns in GmAREB3 arabidopsis seeds
Linoleic acid (18:2) it is about 10.0%, 12.1%, 11.4%, 11.1% and 11.3%；
Wildtype Arabidopsis thaliana, the T that numbering is OE-2, OE-7, OE-17 and OE-185In generation, turns in GmAREB3 arabidopsis seeds
Tribute polyacid (20:1) it is about 5.1%, 6.6%, 6.2%, 6.1% and 6.3%.
Above-mentioned experiment shows that synthesis of the soybean bZIP class transcription factor GmAREB3 to grease in seed is in positive regulating and controlling effect,
Its encoding gene GmAREB3 overexpression, can improve in transfer-gen plant seed the content of total grease and some aliphatic acid such as
Palmitic acid (16:0), oleic acid (18:1), linoleic acid (18:2) with tribute polyacid (20:1) content.
Claims (10)
1. following 1) -3) any material aliphatic acid in the regulation and control total fat content of plant tissue and/or regulation and control plant tissue in
Application in content：
1) Protein G mAREB3；
2) encoding proteins GmAREB3 DNA molecular；
3) recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium of the DNA molecular containing encoding proteins GmAREB3；
The Protein G mAREB3 is following (1) or (2)：
(1) protein being made up of the amino acid sequence shown in sequence in sequence table 2；
(2) substitution by the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from (1) with identical function.
2. following 1) -3) application of any material in the plant that grease and/or content of fatty acid are improved is cultivated in：
1) Protein G mAREB3；
2) encoding proteins GmAREB3 DNA molecular；
3) recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium of the DNA molecular containing encoding proteins GmAREB3；
The Protein G mAREB3 is following (1) or (2)：
(1) protein being made up of the amino acid sequence shown in sequence in sequence table 2；
(2) substitution by the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from (1) with identical function.
3. application according to claim 1 or 2, it is characterised in that：
The DNA molecular of the encoding proteins GmAREB3 is following 1) to any described DNA molecular in 4)：
1) DNA molecular in sequence table shown in sequence 1；
2) DNA molecular in sequence table shown in sequence 1 1-948；
1) or 2) 3) under strict conditions with the DNA molecular hybridization that is limited and encoding as the amino shown in sequence in sequence table 2
The protein DNA molecule of acid sequence composition；
1) or 2) 4) at least have 70% with the DNA molecular that is limited, at least have 75%, at least have 80%, at least have
85%th, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have
The protein DNA molecule being made up of 99% homology and coding the amino acid sequence shown in sequence in sequence table 2.
4. according to any described application in claim 1-3, it is characterised in that：It is described regulation and control the total fat content of plant tissue be
Improve the total fat content of plant tissue；
Content of fatty acid is content of fatty acid in raising plant tissue in the regulation and control plant tissue；
The aliphatic acid is palmitic acid, oleic acid, linoleic acid and/or tribute polyacid.
5. according to any described application in claim 1-4, it is characterised in that：It is described to be organized as seed.
6. according to any described application in claim 1-5, it is characterised in that：The plant is monocotyledon or dicotyledonous
Plant；
The dicotyledon is specially legume or crucifer.
7. a kind of method for cultivating the genetically modified plants that grease and/or content of fatty acid are improved, for by encoding proteins GmAREB3's
DNA molecular is imported in purpose plant, obtains genetically modified plants；Total fat content and/or fat in the Transgenic plant tissue
Acid content is higher than the purpose plant；
1) Protein G mAREB3；
2) encoding proteins GmAREB3 DNA molecular；
3) recombinant vector, expression cassette, transgenic cell line or the recombinant bacterium of the DNA molecular containing encoding proteins GmAREB3；
The Protein G mAREB3 is following (1) or (2)：
(1) protein being made up of the amino acid sequence shown in sequence in sequence table 2；
(2) substitution by the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from (1) with identical function.
8. method according to claim 7, it is characterised in that：The aliphatic acid be palmitic acid, oleic acid, linoleic acid and/or
Tribute polyacid；
The DNA molecular of the encoding proteins GmAREB3 is imported in the purpose plant by recombinant vector；
The DNA molecular of the encoding proteins GmAREB3 is following 1) to any described DNA molecular in 4)：
1) DNA molecular in sequence table shown in sequence 1；
2) DNA molecular in sequence table shown in sequence 1 1-948；
1) or 2) 3) under strict conditions with the DNA molecular hybridization that is limited and encoding as the amino shown in sequence in sequence table 2
The protein DNA molecule of acid sequence composition；
1) or 2) 4) at least have 70% with the DNA molecular that is limited, at least have 75%, at least have 80%, at least have
85%th, at least have 90%, at least have 95%, at least have 96%, at least have 97%, at least have 98% or at least have
The protein DNA molecule being made up of 99% homology and coding the amino acid sequence shown in sequence in sequence table 2.
9. the method according to claim 7 or 8, it is characterised in that：It is described to be organized as seed；The plant is planted for unifacial leaf
Thing or dicotyledon；
The dicotyledon is specially legume or crucifer.
10. a kind of recombinant vector, for encoding proteins GmAREB3 DNA molecular is inserted in expression vector, obtains expressing protein
GmAREB3 recombinant vector；
The Protein G mAREB3 is following (1) or (2)：
(1) protein being made up of the amino acid sequence shown in sequence in sequence table 2；
(2) substitution by the amino acid sequence shown in sequence in sequence table 2 by one or several amino acid residues and/or missing
And/or addition and the protein as derived from (1) with identical function.
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| CN110592104A (en) * | 2019-10-28 | 2019-12-20 | 晋中学院 | Camelina sativa CsDGAT gene and application thereof in plant salt resistance |
| CN110819633A (en) * | 2018-08-09 | 2020-02-21 | 南京农业大学 | Sequence of carrot ABA response element binding protein gene DcABF3 and application thereof |
| CN112851781A (en) * | 2021-02-01 | 2021-05-28 | 华中农业大学 | Application of citrus bZIP transcription factor in shortening plant childhood |
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| CN110819633A (en) * | 2018-08-09 | 2020-02-21 | 南京农业大学 | Sequence of carrot ABA response element binding protein gene DcABF3 and application thereof |
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| CN112851781A (en) * | 2021-02-01 | 2021-05-28 | 华中农业大学 | Application of citrus bZIP transcription factor in shortening plant childhood |
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