CN102532291A - NF-YAI protein and an application of coding gene thereof in cultivating plant with improved content of fatty acid - Google Patents
NF-YAI protein and an application of coding gene thereof in cultivating plant with improved content of fatty acid Download PDFInfo
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- CN102532291A CN102532291A CN2012100055575A CN201210005557A CN102532291A CN 102532291 A CN102532291 A CN 102532291A CN 2012100055575 A CN2012100055575 A CN 2012100055575A CN 201210005557 A CN201210005557 A CN 201210005557A CN 102532291 A CN102532291 A CN 102532291A
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Abstract
The invention discloses NF-YAI protein and an application of a coding gene thereof in cultivating a plant with improved content of fatty acid. A method for cultivating a transgenic plant comprises the step of introducing the coding gene of the NF-YAI protein into a target plant to obtain the transgenic plant. The transgenic plant is characterized in that 1) The content of the fatty acid in the transgenic plant is higher than that of the target plant; and an experiment proves that a transcription factor NF-YAI for adjusting and controlling the metabolism of the fatty acid in the plant and a coding gene thereof are introduced into an arabidopsis thaliana Columbia ecotype to obtain the transgenic plant and discovers that the transcription factor can be used for adjusting and controlling the metabolism of the fatty acid in the plant.
Description
Technical field
The present invention relates to biological technical field, relate in particular to the application in the plant of cultivating the fatty acid content raising of a kind of NF-YA1 albumen and encoding sox thereof.
Background technology
Lipid acid is store energy form main in the organism, and (triacylglycerol TAG) can store in fatty tissue, plant seed or the fruit of animal its main verivate triglyceride in a large number.Wherein, vegetables oil is as edible oil and a kind of important renewable energy material and receive much concern.Triglyceride is the natural high moleculer eompound that lipid acid and glycerine chemical combination form.Triglyceride synthetic process comprises the assembling process of de novo synthesis process, modification in endoplasmic reticulum and the triglyceride of lipid acid in plastid.In plant materials, the de novo synthesis of lipid acid (FAS) carries out in plastid.Lipid acid synthetic precursor is acetyl-CoA (acetyl-CoA).It is at first at acetyl CoA carboxylase (acetyl coenzyme A carboxylase; ACCase) synthetic malonyl-CoA (malonyl-CoA) under the effect; Fatty acid synthetase is that substrate carries out the successive polyreaction with malonyl-CoA then; With the frequency synthesis acyl group carbochain of two carbon of each circulation increase, the sfas of synthetic 16 to 18 carbon at last.Be transported to endoplasmic reticulum after the synthetic free fatty acids is activated in plastid, further modify (desaturation, dehydrogenation prolong etc.), form glyceride and film fat at last.The biosynthesizing of TAG is carried out in endoplasmic reticulum in the plant seed.TAG synthetic precursor is glycerol-3-phosphate and acyl CoA; The synthetic process needs three kinds of acyltransferases and a kind of phosphohydrolase altogether; Be respectively glycerol-3-phosphate acyltransferase (GPAT), lysophosphatidate acyltransferase (LPAT), diacylglycerol acyltransferase (DGAT) and phosphatide phosphohydrolase (PAPase) (Barron etc.; Biochim Biophys Acta, 60:329-337).The polarity of TAG is very low, therefore can make grease constantly accumulation in fatty body.
The transcription factor of the involved in plant fatty acid metabolism regulation and control of having found at present, has (Focks etc., 1998, Plant Physiology, 18:91-101 such as WRI1, LEC1, FUS3 and LEC2; Santos Mendoza etc., 2005, FEBS Letter, 579:4666-4670; Wang etc., 2007, Planta, 226:773-783; Mu etc., 2008, Plant Physiology, 148:1042-1054).Wherein, the WRI1 AP2/EREB transcription factor protein of encoding, it possibly be a key transforming to TAG by sucrose in the plant materials regulatory factor (Cernac etc., 2004, Plant Journal, 40:575-585).LEC1 belongs to one type of transcription factor that can combine cis-acting elements CCAAT box in the promotor, and regulation and control embryo's generation and seed maturity possibly regulated and control (Lotan etc. to the accumulation of embryo's storage thing in embryo's forming process simultaneously; 1998; Cell, 1998,93 (7): 1195-1205; To etc., 2006, The Plant Cell, 18 (7): 1642-1651).Overexpression LEC1 can increase considerably fatty acid content in the transgenic arabidopsis seedling (Mu etc., 2008, Plant Physiology, 148:1042-1054).FUS3 and LEC2 are the transcription factors of a B3 family of having encoded, and they all are important controlling gene (Reidt etc., 2000, Plant Journal, 21:401-408 in the embryo development procedure; Stone etc., 2001, Proc Natl Acad Sci 98:11806-11811).In the seed of fus3 and lec2 two mutants, fat content all significantly reduces (Wang etc., 2007, Planta, 226:773-783; Santos Mendoza etc., 2008, Plant Journal, 54:608-620); Overexpression FUS3 and LEC2 can both increase (Santos Mendoza etc., 2005, FEBS Letter, 579:4666-4670 by the intravital fat content of render transgenic plant; Wang etc., 2007, Planta, 226:773-783).Above-mentioned research shows that the regulation and control of oil and fat accumulation are that the important regulating and controlling factor that relies in embryo's generating process is regulated and control to a great extent in the plant seed ripening process.
In the arabidopsis gene group, the gene of the encoding transcription factor has accounted for greatly, has 1500 at least, account for more than 5% of whole genome (Riechmann etc., 2000, Science, 290:2110-2113).These transcription factors belong to big gene family mostly, and the gene family that has comprises many subtribes again, and some transcription factor family is that plant is peculiar.Result of study to transcription factor shows that a transcription factor possibly implemented to regulate control to a lot of genes of one type of correlated character, thereby effectively changes the correlation properties of plant.The CCAAT box is a cis-acting elements that extensively is present on the gene promoter, is called nuclear factor Y (NF-Y) or CCAAT binding factor (CBF) again with CCAAT sequence bonded transcription factor.NF-Y is a heterotrimer mixture that is made up of three kinds of different subunits, and it discerns the CCAAT sequence on the DNA specifically, and combines with it, thus the expression of regulatory gene.Three kinds of different subunits of NF-Y belong to 3 subtribe: NF-YA (being called HAP2 or CBF-B again) respectively, NF-YB (be called not only HAP3 or CBF-A) and NF-YC (but also being called HAP5 or CBF-C).There is very high homology in the core texture territory of NF-YB and NF-YC with histone H2A and H2B on sequence.NF-YB/NF-YC forms a heterodimer mixture of combining closely, and NF-YA is combined in the surface of this mixture again, forms the heterotrimer mixture.
Summary of the invention
An object of the present invention is to provide a kind of method of cultivating transgenic plant.
Method provided by the invention for the proteic encoding sox of NF-YA1 is imported in the purpose plant, obtains transgenic plant, and said transgenic plant have following 1)-5) in any characteristic:
1) fatty acid content of said transgenic plant the root or the hypocotyl that are higher than said purpose plant, said transgenic plant plant all forms cells,primordial, said transgenic plant plant strain growth is suppressed with said transgenic plant be higher than said purpose plant with lipid acid synthesis related gene expression level;
2) fatty acid content of said transgenic plant is higher than said purpose plant;
3) said transgenic plant plant strain growth is suppressed;
4) root of said transgenic plant plant or hypocotyl all form cells,primordial;
5) said transgenic plant is higher than said purpose plant with lipid acid synthesis related gene expression level;
The proteic aminoacid sequence of said NF-YA1 is the sequence 1 in the sequence table.
In aforesaid method, the nucleotides sequence of the proteic encoding sox of said NF-YA1 is classified sequence 2 or the sequence 3 in the sequence table in the sequence table as;
Said lipid acid is C16:0, C18:0, C18:1, C18:2, C18:3 and/or C20:1.
In aforesaid method, said transgenic plant plant strain growth is suppressed the plant height that is embodied in said transgenic plant or cotyledon size all less than said purpose plant;
Root of said transgenic plant plant or hypocotyl all have root or hypocotyl that cells,primordial is embodied in said transgenic plant plant all greater than said purpose plant;
Said synthesis related because FAB2, FAD2, FAD3 or FAE1 gene with lipid acid.
In aforesaid method, the proteic encoding sox of above-mentioned NF-YA1 imports the purpose plant through recombinant vectors;
Above-mentioned recombinant vectors is following 1) or 2):
1) the proteic encoding sox of NF-YA1 is inserted the carrier that obtains between XhoI and the SpeI site of PER10;
2) the proteic encoding sox of NF-YA1 is inserted the carrier that obtains between XhoI and the SpeI site of pBA002.
In aforesaid method, the above-mentioned purpose plant is dicotyledons or monocotyledons.
In aforesaid method, said dicotyledons is Arabidopis thaliana, rape, peanut, cotton, soybean, Sunflower Receptacle, palm tree, olive, castor-oil plant, yam or tobacco; Said monocotyledons is paddy rice, corn, wheat, barley, oat, rye, jowar or turfgrass.Concrete usefulness is Arabidopis thaliana in an embodiment of the present invention.
Another object of the present invention provides a kind of recombinant vectors.
The invention provides a kind of recombinant vectors, for the proteic encoding sox of said NF-YA1 is inserted the carrier that obtains among the PER10.
Experiment of the present invention proves; The transcription factor NF-YA1 and the encoding sox thereof that the invention provides a regulation and control vegetable fatty acid metabolism import in Arabidopis thaliana (Arabidopsis thaliana) Colombia's ecotype; Obtain transgenic plant, find that this transcription factor can be used for regulating and control plant body fat acid metabolic, the experiment proof; In the NF-YA1 transgenic arabidopsis body; Not only fatty acid content is significantly improved, and participates in lipid acid synthetic enzyme FAE1 and the enzyme FAB2 that participates in fatty acid modifying, and the expression of the corresponding gene of FAD2 and FAD3 is regulated and control by it also all.Transcription factor of the present invention and encoding sox thereof are for the vegetable fatty acid metabolism Molecular Study; And improve the fatty acid content of plant (particularly oil crops) and the improvement of correlated character has important theory and practical significance through biotechnology, have wide application and market outlook at agriculture field.The present invention has found NF-YA1 regulating and controlling effect to oil and fat accumulation in the plant seed growth course in research process.Through being that the genetic manipulation of target spot gene can improve the intravital fatty acid content of transgenic plant effectively with NF-YA1.
Description of drawings
Fig. 1 is the gene structure frame diagram of NF-YA1
Wherein, black box is represented exon, and ' with 3 ' non-coding region, straight line is represented intron in grey box indicating 5.ATG and TAA represent the initiation codon and the termination codon of gene respectively.
Fig. 2 sprouts the phenotype after a week for the pER10-NF-YA1 transgenic plant
Fig. 3 sprouts the Sudan red dyeing after a week for the pER10-NF-YA1 transgenic plant
Fig. 4 is the phenotype of pBA-NF-YA1 transgenic plant sprouting after 10 days
Fig. 5 is the Sudan red dyeing of pBA-NF-YA1 transgenic plant sprouting after 10 days
Fig. 6 detects fatty acid content for GS-MS
Fig. 7 is the expression level of lipid acid synthesis related gene in wild-type and transgenic plant
Embodiment
Employed experimental technique is ordinary method like no specified otherwise among the following embodiment.
Used material, reagent etc. like no specified otherwise, all can obtain from commercial sources among the following embodiment.
One, changes the acquisition of NF-YA1 Arabidopis thaliana
1, the clone of the transcription factor gene NF-YA1 of regulation and control vegetable fatty acid metabolism
The design primer sequence is following:
P1 (upstream primer): 5 '-CCCTCGAGATGCAATCAAAACCGGGAAG-3 '
P2 (downstream primer): 5 '-AACCCGGGTGGTGCACCAGAAGAATTCA-3 '
With plant total RNA extraction reagent box (available from sky root biotech firm; Catalog number (Cat.No.): DP432) and reference reagent box specification sheets extract wild-type Arabidopis thaliana (Arabidopsis thaliana; Columbia is environmental, available from U.S. Arabidopis thaliana Biological resources center, and The Arabidopsis Biological Resource Center; ABRC, PIN are CS28166.Be designated hereinafter simply as the wild-type Arabidopis thaliana) total RNA of 2 all seedling, use SuperScript then
TMII Reverse Transcriptase test kit is (available from full Shi Jin biotech firm; Catalog number (Cat.No.): AH301-02) and synthetic its first chain cDNA of reference reagent box specification sheets reverse transcription; Be template with the synthetic cDNA of institute again, under the guiding of primer P1 and P2, carry out pcr amplification, obtain the PCR product.
After reaction finishes, pcr amplification product is carried out 1% agarose gel electrophoresis detect, reclaim the purpose fragment of the about 819bp of length and it is carried out purifying; With its with cut the pBluescript SK carrier segments (can be available from Merck & Co., Inc., catalog number is ST212205) that obtains through same enzyme and connect, will connect again product with heat shock method transformed into escherichia coli (E.coli) DH5 α competent cell (available from full Shi Jin biotech firm; Catalog number (Cat.No.): CD201-03), screening positive clone is inoculated in it in 5mL LB liquid nutrient medium that contains the 50mg/L penbritin; Under 37 ℃, 200rpm, cultivated 12-16 hour, the upgrading grain obtains containing and reclaims segmental recombinant plasmid; Called after SK-NF-YA1; It is checked order, and sequencing result shows that the gene of the PCR product that this plasmid contains has the nucleotide sequence of the sequence 2 in the sequence table, by 819 based compositions; Its encoding sequence is that its coding has the protein of the amino acid residue sequence of sequence 1 in the sequence table from 5 ' end 1-819 bit base.Wherein, sequence 2 is from 5 ' end 525-585 bit base proteins encoded-protein-interacting structural domain, from 5 ' end 621-681 bit base coding DNA binding domains in the sequence table.
The nucleotide sequence that the corresponding genome sequence of said gene has sequence 3 in the sequence table; By 2695 based compositions; From 5 ' end 574-850 bit base is first exon of this genomic gene; From 5 ' end 851-1203 bit base is first intron of this genomic gene; From 5 ' end 1204-1304 bit base is second exon of this genomic gene, is second intron of this genomic gene from 5 ' end 1305-1455 bit base, is the 3rd exon of this genomic gene from 5 ' end 1456-1527 bit base; From 5 ' end 1528-1838 bit base is the 3rd intron of this genomic gene; From 5 ' end 1839-2003 bit base is the 4th exon of this genomic gene, is the 4th intron of this genomic gene from 5 ' end 2004-2184 bit base, is the 5th exon of this genomic gene from 5 ' end 2185-2389 bit base; From 5 ' end 1-573 bit base is the 5 ' non-coding region (UTR) of this genomic gene, is 3 ' non-coding region of this genomic gene from 5 ' end 2390-2695 bit base.The skeleton construction of this gene is seen Fig. 1, is Nuclear Factor YA1 (being called for short NF-YA1) with this unnamed gene, with its proteins encoded called after Nuclear Factor YA1 (being called for short NF-YA1).
2, the structure that contains the plant inducible expression carrier of NF-YA1
With restriction enzyme Xho I and Spe I the above-mentioned 1 plasmid SK-NF-YA1 that makes up is carried out double digestion; The double digestion product is carried out 1% agarose gel electrophoresis to be detected; Reclaim the NF-YA1 gene fragment of the about 819bp of length and it is carried out purifying; Be connected to then with carrier PER10 and be connected, obtain connecting product through the same enzyme double digestion.
PER10 is one has Theelin,dihydro-inductive plant expression vector; Be documented in Applications of Chemical-Inducible Expression Systems in Functional Genomics and Biotechnology, Nam-Hai Chua, Methods in Molecular Biology; 323 (III); 329-342,2006, the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity.
To connect product with heat shock method transformed into escherichia coli (E.coli) DH5 α competent cell, screening positive clone is inoculated in it in 5mL LB liquid nutrient medium that contains the 50mg/L Totomycin; Under 37 ℃, 200rpm, cultivated 16 hours, the upgrading grain carries out the double digestion evaluation to recombinant plasmid with restriction enzyme Xho I and Spe I; The result cuts through enzyme and has obtained the 819bp dna fragmentation, conforms to expected results, is further PCR with primer P1 and P2 again and identifies; The result has obtained the dna fragmentation of 819bp through pcr amplification; Also consistent with expected results, again this plasmid is sent to order-checking, the result is the carrier that obtains between the Xho I of the 2 insertion PER10 of the sequence in the sequence table and Spe I restriction enzyme site; Show the plant expression vector that contains that has obtained insertion sequence and correct position, called after PER10-NF-YA1.
3, change the acquisition of NF-YA1 Arabidopis thaliana
The plant expression vector PER10-NF-YA1 that step 2 is made up transforms Agrobacterium GV3101 competent cell (available from BIOVECTOR CO. with electrization; LTD; Product article No.: BIOVECTOR-375); It is coated on the LB resistant panel that contains 50mg/L spectinomycin and 50mg/L Rifampin under 28 ℃, 150rpm cultivated 16 hours, the single bacterium colony of the Agrobacterium that picking grows is through the PCR evaluation with primer P1 and P2, and the result has obtained the dna fragmentation of 819bp through pcr amplification; Be illustrated as positive reorganization Agrobacterium, called after GV3101/PER10-NF-YA1.
Again GV3101/PER10-NF-YA1 was inoculated in the 20mLLB liquid nutrient medium that contains 50mg/L spectinomycin and 50mg/L Rifampin under 28 ℃, 150rpm shaking culture 2 days; Then, bacterium liquid was inoculated in the 300mL LB liquid nutrient medium that contains 50mg/L spectinomycin and 50mg/L Rifampin under 28 ℃, 150rpm shaking culture 18 hours by 2% inoculum size bacterium again.After cultivating end, 5000rpm collected thalline in centrifugal 20 minutes, again thalline was dissolved in 250mL and contained infecting in the liquid of 5% sucrose, slowly shook up.At last, bacterium liquid is changeed in the 250mL beaker, be inverted in the beaker 5 minutes, obtain T0 for changeing the NF-YA1 plant with removing the wild-type Arabidopis thaliana of flower with the fruit pod.
Above-mentioned positive T0 is carried out the routine cultivation for changeing the NF-YA1 plant, and results seed, gained seed obtain 20 strain T1 for changeing the NF-YA1 plant after the screening of 50mg/L kantlex.Through selfing, obtain about 500 strain T2 for changeing the NF-YA1 plant.
Two, change the phenotypic evaluation of NF-YA1 Arabidopis thaliana
1, changes the evaluation of PER10-NF-YA1 plant
The T2 that is numbered #1 and #3 that step 1 is obtained broadcasts for seed for the T2 of commentaries on classics NF-YA1 Arabidopis thaliana (PER10-NF-YA1) and is containing 10 μ M oestrogenic hormon (17-β-Estradiol; Sigma; Product article No. E8875) (1/2MS on the MS substratum; 1% sucrose, 0.8% agar) seedling of cultivating 10 days is extracted the RNA of full stand, with the wild-type Arabidopis thaliana as contrast.Reverse transcription obtains cDNA; With P1 (upstream primer): 5 '-CCCTCGAGATGCAATCAAAACCGGGAAG-3 ' and P2 (downstream primer): 5 '-AACCCGGGTGGTGCACCAGAAGAATTCA-3 ' carries out RT-PCR; Detected the NF-YA1 gene at T2 for the expression level in the transgenic plant; With the wild-type Arabidopis thaliana is contrast; With Actin7 is confidential reference items, and the primer of confidential reference items is ACTIN7F:5 '-GGAACTGGAATGGTGAAGGCTG-3 ' and ACTIN7B:5 '-CGATTGGATACTTCAGAGTGAGGA-3 '.The result is shown in Fig. 2 A; Be RT-PCR technology for detection NF-YA1 expression of gene level; From figure, find out; Compare with the wild-type Arabidopis thaliana, the T2 that is numbered #1 and #3 all has rising to some extent for NF-YA1 expression of gene level in the commentaries on classics PER10-NF-YA1 Arabidopis thaliana, explains that the T2 that is numbered #1 and #3 is for changeing the positive expression Arabidopis thaliana excessively of PER10-NF-YA1 Arabidopis thaliana.
2. change the phenotype analytical of PER10-NF-YA1 plant
Be accredited as T2 that male is numbered #1 and #3 and broadcast for the seed that changes NF-YA1 Arabidopis thaliana (PER10-NF-YA1) and containing 10 μ M oestrogenic hormon (17-β-Estradiol above-mentioned; Sigma, product article No. E8875) (1/2MS, 1% sucrose on the MS substratum; 0.8% agar) (wherein; MS salt, agar and sucrose is available from Beijing Xi Meijie Science and Technology Ltd., product article No. M531, A7848 and S391) place the greenhouse at 22 ℃, intensity of illumination 80-120 μ E
-2S
-1Cultivated under the condition 16 hours, with the wild-type Arabidopis thaliana as contrast.
After 10 days; Observe T2 for the growing state that changes NF-YA1 Arabidopis thaliana and wild-type plant; The result can find out shown in Fig. 2 B, and T2 grow on inducing culture obvious growth inhibition phenotype was arranged 10 days the time for changeing the NF-YA1 Arabidopis thaliana; Be embodied in NF-YA1 cross express that plant is short and small, cotyledon diminishes or can not normal development, the zoon of apical meristem is obstructed, growing of root is unusual.Simultaneously, form tangible embryonal connective tissue for the hypocotyl and the root that change the NF-YA1 Arabidopis thaliana, mainly show as root and the hypocotyl color turns green, obviously expand chap at T2.
Three, the acquisition of NF-YA1 constitutive expression transgenic arabidopsis
1, makes up the recombinant expression vector of NF-YA1 genome moulding overexpression
Above-mentioned one SK-NF-YA1 that obtains cut through XhoI and SpeI enzyme obtain enzyme and cut product and pass through the plant inducible expression carrier pBA002 that same enzyme cuts and (be documented in A GFP-mouse talin fusion protein labels plant actin filaments in vivo and visualizes the actin cytoskeleton in growing pollen tubes, Benedikt Kost, Pius Spielhofer; Nam-Hai Chua, The Plant Journal, 16 (3); 393-401,1998, the public can obtain with developmental biology institute from Chinese Academy of Sciences's heredity) be connected; Connect the product transformed into escherichia coli; Obtain the clone, 37 degree were cultivated 16 hours on the LB flat board that contains the grand enzyme element of 50 mg/litre, and screening at last obtains having the clone of anti-grand enzyme element resistance; Extract this clone's plasmid; Send to order-checking, the result is the plasmid that obtains between the XhoI of sequence 2 insertion pBA002 and SpeI restriction enzyme site, this plasmid called after pBA-NF-YA1.
2, change the acquisition of pBA-NF-YA1 Arabidopis thaliana
With the pBA-NF-YA1 of above-mentioned acquisition transform Agrobacterium GV3101 (available from Biovector Co., LTD, product article No.: Biovector-375); Infect method for transformation with flower then and transform wild-type Arabidopis thaliana Col-0 (available from U.S. Arabidopis thaliana Biological resources center; The Arabidopsis Biological Resource Center, ABRC, PIN are CS28166; Hereinafter to be referred as the wild-type Arabidopis thaliana) inflorescence; Filter out 12 strain T1 for changeing the pBA-NF-YA1 Arabidopis thaliana through weedicide Basta (50mg/L), numerous through expanding, obtain about 300 strain T2 for changeing the pBA-NF-YA1 Arabidopis thaliana.
3, change the phenotype analytical of pBA-NF-YA1 Arabidopis thaliana
The T2 that will be numbered #4 and #6 respectively for the planting seed that changes the pBA-NF-YA1 Arabidopis thaliana after at MS substratum (1/2MS; 1% sucrose; 0.8% agar) (wherein, MS salt, agar and sucrose is available from Beijing Xi Meijie Science and Technology Ltd., product article No. M531, A7848 and S391;) 24 hours illumination cultivation of last 22 degree 7 days, the phenotype of growing of making plant.The result shows, with constitutive promoter driving N F-YA1 overexpression, for the not obviously influence of growth of plant in seedling stage, but has tangible embryonal connective tissue to form at the hypocotyl place of seedling, is embodied in hypocotyl and obviously expands chap, color greener (Fig. 4).
Embodiment 2, the fatty acid metabolism detection and the Mechanism Study thereof of changeing the NF-YA1 Arabidopis thaliana
One, the variation of NF-YA1 Arabidopis thaliana body fat acid content is changeed in Sudan red dyeing indication
Sudan red be a kind of can with the dyestuff of the distinctive neutral fat specific combination of seed, the painted depth of vegetable material can the plant indicator body in the content height of neutral fat.To obtain T2 by one of embodiment 1 and be soaked in 1% Sudan red (Fat Red 7B) (Sigma respectively with the wild-type Arabidopis thaliana for changeing NF-YA1 Arabidopis thaliana (PER10-NF-YA1); Product article No. 201161-8), room temperature (25 ℃) dyeing 6 hours is with washed with de-ionized water 3 times; Every all over 30s; Coloration result is as shown in Figure 3, locates for root that changes the NF-YA1 Arabidopis thaliana and hypocotyl at T2, and the painted degree of Sudan red is apparently higher than wild-type plant; Explain to form cells,primordial in the transgenic plant body, and the accumulation volume of the lipid acid of the seed specific in the cells,primordial is apparently higher than wild-type.
To obtain T2 by one of embodiment 1 according to the method described above and carry out Sudan red dyeing for changeing the pBA-NF-YA1 Arabidopis thaliana; The result is as shown in Figure 5; Expand the place at T2 for the hypocotyl that changes the pBA-NF-YA1 Arabidopis thaliana; The coloring degree of Sudan red is explained because the overexpression of NF-YA1 gene causes the neutral fat acid of seed specific to accumulate at the hypocotyl position apparently higher than the hypocotyl of wild-type.
Two, the GC-MS method detect to be changeed NF-YA1 Arabidopis thaliana content situation of various fatty acid components in the body after inducing
Get the T2 that obtains being numbered 1 (#1) and 3 (#3) by one of embodiment 1 respectively and (contain 10 μ M oestrogenic hormon, 17-β-Estradiol, Sigma, product article No. E8875 at inducing culture for changeing NF-YA1 Arabidopis thaliana and wild-type Arabidopis thaliana; The MS substratum on (prescription for 1/2MS, 1% sucrose, 0.8% agar)) in sprout back growth 10 days; Respectively get the whole strain seedling of 0.1 gram plant, in liquid nitrogen, grind to form dry powder, transfer to then in the test tube of with closure; Add 3mL methyl alcohol (containing 2.5% (V/V) vitriol oil),, add the 4.5mL 0.9%NaCl aqueous solution and 1mL normal hexane again 80 ℃ of heating in water bath 90 minutes; Mixing, centrifugal 10 minutes of 4000rpm collects the normal hexane phase.Vacuum is drained, and uses the 100ul acetic acid ethyl dissolution, gets 1ul and goes up appearance; Analyze the content situation of various fatty acid components with the TurboMass GC/MS appearance of PerkinElmer company, used GC post is 30m * 0.25mmBPX-70 post, and the GC heating schedule is: 120 ℃ of initial temperatures; Kept 1 minute, the speed with 10 ℃ of PMs rises to 150 ℃ again, and the speed with 4 ℃ of PMs is warming up to 230 ℃ then; Kept 10 minutes, with trig lyceride (Sigma, the article No.: 201161-8) make interior mark of C17:0.
The time and the molecular weight of mass spectrum appearance can be confirmed component per sample, compare with interior target area according to the area of mass spectra peak figure, can calculate content.The calculation formula of fatty acid content is: mark content/example weight in samples contg=(the sample peak area of pictural surface/interior mark peak area of pictural surface) X.
The result is as shown in Figure 6, and wild-type and the T2 that is numbered 1 (#1), 3 (#3) are respectively 0.56 μ g/mg, 0.84 μ g/mg, 0.95 μ g/mg for the content of the palmitinic acid (C16:0) that changes the NF-YA1 Arabidopis thaliana;
Wild-type and the T2 that is numbered 1 (#1), 3 (#3) are respectively 0.14 μ g/mg, 0.3 μ g/mg, 0.27 μ g/mg for the content of the Triple Pressed Stearic Acid (C18:0) that changes the NF-YA1 Arabidopis thaliana;
Wild-type and the T2 that is numbered 1 (#1), 3 (#3) are respectively 0.12 μ g/mg, 0.3 μ g/mg, 0.27 μ g/mg for the content of the oleic acid (C18:1) that changes the NF-YA1 Arabidopis thaliana;
Wild-type and the T2 that is numbered 1 (#1), 3 (#3) are respectively 0.55 μ g/mg, 0.90 μ g/mg, 0.88 μ g/mg for the content of the linolic acid (C18:2) that changes the NF-YA1 Arabidopis thaliana;
Wild-type and the T2 that is numbered 1 (#1), 3 (#3) are respectively 1.0 μ g/mg, 1.29 μ g/mg, 1.26 μ g/mg for the content of the linolenic acid (C18:3) that changes the NF-YA1 Arabidopis thaliana;
Wild-type and the T2 that is numbered 1 (#1), 3 (#3) are respectively 0 μ g/mg, 0.27 μ g/mg, 0.14 μ g/mg for the content of the eicosenoic acid (C20:1) that changes the NF-YA1 Arabidopis thaliana;
Can find out from above-mentioned, compare with the wild-type Arabidopis thaliana that the T2 that is numbered 1 (#1) and 3 (#3) obviously rises for each component concentration that changes NF-YA1 Arabidopis thaliana lipid acid after inducing, wherein, C16:0 has raise 49.8% and 67.7% respectively; C18:0 has raise 116% and 95.3% respectively; C18:1 has raise 151.5% and 129% respectively; C18:2 has raise 64% and 59.5% respectively; C18:3 has raise 28.9% and 26.3% respectively.Especially it should be noted that distinctive C20:1 in the seed grease in 10 days wild-type seedling be detect less than, but in transgenic plant, this components contents has reached 0.27 μ g/mg and 0.14 μ g/mg respectively.
The above results shows that NF-YA1 is the lipid acid synthetic positive regulation factor, and this gene of overexpression can significantly improve the intravital fatty acid content of plant.
Three, NF-YA1 regulation and control lipid acid synthetic Study on Molecular Mechanism
The #1 T2 that is numbered that is obtained by one of embodiment 1 respectively sprouts the RNA of 10 days extraction full stand of back growth for changeing NF-YA1 Arabidopis thaliana (PER10-NF-YA1) in inducing culture; Reverse transcription obtains cDNA; Increasing with following primer respectively, is contrast with wild-type Arabidopis thaliana (col-0).
FAD2?F1(5‘-ATGGGTGCAGGTGGAAGAAT-3’)
FAD2?B1(5‘-CCAGGAGAAGTAAGGGACGA-3’)
FAD3?F1(5‘-CATAAGCGGCGTGCATTTTG-3’)
FAD3?B1(5‘-ACGAAAGCAGCTAAACATGT-3’)
FAB2?F1(5‘-CGCTGTGCATAAGCATTCTC-3’)
FAB2?B1(5‘-TTGGGGCCGGAGCTGAGAGC-3’)
FAE1?F1(5‘-CGTTAAGCTCCTTTACCGTT-3’)
FAE1?B1(5‘-TCTGTCTCTTCACGTGACGC-3’)
UBQ5?F1(5‘-CTTCAGCAGCCGTTGCCTCA-3’)
UBQ5?B1(5‘-CTGGTAAACGTAGGTGAGTCC-3’)
Carrying out RT-PCR, detected and the expression level of lipid acid synthesis related gene in wild-type and NF-YA1 transfer-gen plant, is confidential reference items with UBQ5.
The result is as shown in Figure 7; From figure, find out, compare, be numbered #1 T2 for changeing in the NF-YA1 Arabidopis thaliana (PER10-NF-YA1) and the lipid acid synthesis related gene with the wild-type Arabidopis thaliana; Comprise FAB2 (At2g43710); FAD2 (At3g12120), the expression level of FAD3 (At2g29980) and FAE1 (At4g34520) all have rising significantly, explain that it is the expression realization through the sour synthesis related gene of induced lipolysis with accumulating that NF-YA1 regulates and control the synthetic of lipid acid.
Claims (7)
1. method of cultivating transgenic plant for the proteic encoding sox of NF-YA1 is imported in the purpose plant, obtains transgenic plant, and said transgenic plant have following 1)-5) in any characteristic:
1) fatty acid content of said transgenic plant the root or the hypocotyl that are higher than said purpose plant, said transgenic plant plant all forms cells,primordial, said transgenic plant plant strain growth is suppressed with said transgenic plant be higher than said purpose plant with lipid acid synthesis related gene expression level;
2) fatty acid content of said transgenic plant is higher than said purpose plant;
3) said transgenic plant plant strain growth is suppressed;
4) root of said transgenic plant plant or hypocotyl all form cells,primordial;
5) said transgenic plant is higher than said purpose plant with lipid acid synthesis related gene expression level;
The proteic aminoacid sequence of said NF-YA1 is the sequence 1 in the sequence table.
2. method according to claim 1 is characterized in that:
The nucleotides sequence of the proteic encoding sox of said NF-YA1 is classified sequence 2 or the sequence 3 in the sequence table in the sequence table as;
Said lipid acid is C16:0, C18:0, C18:1, C18:2, C18:3 and/or C20:1.
3. method according to claim 1 and 2 is characterized in that:
Said transgenic plant plant strain growth is suppressed the plant height that is embodied in said transgenic plant or cotyledon size all less than said purpose plant;
Root of said transgenic plant plant or hypocotyl all have root or hypocotyl that cells,primordial is embodied in said transgenic plant plant all greater than said purpose plant;
Said synthesis related because FAB2, FAD2, FAD3 and FAE1 gene with lipid acid.
4. according to arbitrary described method among the claim 1-3, it is characterized in that:
The proteic encoding sox of said NF-YA1 imports the purpose plant through recombinant vectors;
Said recombinant vectors is following 1) or 2):
1) the proteic encoding sox of said NF-YA1 is inserted the carrier that obtains among the PER10;
2) the proteic encoding sox of said NF-YA1 is inserted the carrier that obtains among the pBA002.
5. according to arbitrary described method among the claim 1-4, it is characterized in that:
Said purpose plant is dicotyledons or monocotyledons.
6. according to arbitrary described method among the claim 1-5, it is characterized in that: said dicotyledons is Arabidopis thaliana, rape, peanut, cotton, soybean, Sunflower Receptacle, palm tree, olive, castor-oil plant, yam or tobacco; Said monocotyledons is paddy rice, corn, wheat, barley, oat, rye, jowar or turfgrass.
7. a recombinant vectors is following 1) or 2):
1) the proteic encoding sox of said NF-YA1 is inserted the carrier that obtains among the PER10;
2) the proteic encoding sox of said NF-YA1 is inserted the carrier that obtains among the pBA002.
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| CN105400792A (en) * | 2015-12-23 | 2016-03-16 | 山东大学 | Application of corn kernel factor gene ZmNF-YA3 to changing plant resistance tolerance |
| CN105732782A (en) * | 2014-12-08 | 2016-07-06 | 中国农业科学院作物科学研究所 | Use of stress-resistant associated protein in regulation and control of plant stress resistance |
| CN107936098A (en) * | 2017-12-21 | 2018-04-20 | 中国科学院遗传与发育生物学研究所 | Chlorella ellipsoidea NF YA genes and its application |
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| CN101597329A (en) * | 2008-06-06 | 2009-12-09 | 中国科学院遗传与发育生物学研究所 | The transcription factor relevant and encoding gene and application with vegetable fatty acid and fat metabolic |
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| CN105732782A (en) * | 2014-12-08 | 2016-07-06 | 中国农业科学院作物科学研究所 | Use of stress-resistant associated protein in regulation and control of plant stress resistance |
| CN105732782B (en) * | 2014-12-08 | 2019-08-20 | 中国农业科学院作物科学研究所 | Application of the resistance relevant protein in regulation stress resistance of plant |
| CN105400792A (en) * | 2015-12-23 | 2016-03-16 | 山东大学 | Application of corn kernel factor gene ZmNF-YA3 to changing plant resistance tolerance |
| CN107936098A (en) * | 2017-12-21 | 2018-04-20 | 中国科学院遗传与发育生物学研究所 | Chlorella ellipsoidea NF YA genes and its application |
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