CN103351429A

CN103351429A - Transcription factor for regulating plant oils and fatty acids, coding gene and application thereof

Info

Publication number: CN103351429A
Application number: CN2013101412979A
Authority: CN
Inventors: 汪阳东; 陈益存
Original assignee: Research Institute of Subtropical Forestry of Chinese Academy of Forestry
Current assignee: Research Institute of Subtropical Forestry of Chinese Academy of Forestry
Priority date: 2013-04-23
Filing date: 2013-04-23
Publication date: 2013-10-16
Anticipated expiration: 2033-04-23
Also published as: CN103351429B

Abstract

The present invention discloses a transcription factor for regulating plant oils and fatty acids, an coding gene and an application thereof. The transcription factor is named VfLEC1, wherein the amino acid sequence of the transcription factor is represented by SEQIDNO:1. The transcription factor and the coding gene thereof provide regulation effects for components and contents of plant oils and fatty acids.

Description

The transcription factor of regulating plant grease and lipid acid and encoding gene thereof and application

Technical field

The invention belongs to plant genetic engineering field, specifically, the present invention relates to transcription factor and encoding gene and the application of a regulating plant grease and lipid acid.

Background technology

Vegetable oil lipoprotein receives much concern as main edible oil and biomass energy.In the most plants, Vegetable oil lipoprotein is stored in the plant seed with the form of triacylglycerol (Triacylglycerol, TAG), is rich in unsaturated fatty acids.

The biosynthesizing of Vegetable oil lipoprotein is the physiological and biochemical procedure of a complexity, mainly comprises generation, glycerol 3-phosphate and 3 processes of free fatty acids dehydrating condensation of lipid acid and aliphatic alcohol.Lipid acid is synthetic mainly to carry out in plastid.In the growth course of seed, the sucrose that photosynthesis produces is the main carbon source of synthetic fatty acid, generates hexose by glycolytic pathway, and further generates pyruvic acid.Under pyruvate carboxylase catalysis, generate the synthetic precursor acetyl-CoA (acetyl-CoA) of lipid acid.Subsequently; acetyl-CoA is at acetyl-CoA carboxylase (acetyl-CoA carboxylase; ACCase) under the catalysis; form malonyl-list acyl coenzyme A; the latter carries out the successive polymerization reaction under the effect of fatty acid synthetase; two carbon of each increase, acyl group carbochain enter the lipid acid route of synthesis with acyl carrier protein (acyl carrier protein, ACP) combination again.Fatty acid modifying subsequently and triacylglycerol ester synthesis etc. mainly carry out in endoplasmic reticulum.On the net through the effect of glycerol 3-phosphate acyltransferase (GPAT), lysophosphatidic acyltransferase (LPAAT), diacylglycerol transaldolase (DGAT) and phosphatidic acid phosphohydrolase (PAPase), attached lipid acid synthesizes triglyceride to acyl-CoA on glycerol backbone respectively built-in.Scientists is by having carried out useful exploration to the regulation and control of above-mentioned key gene with raising fat content and improvement fatty acid component and having made some progress.Roesaler etc. utilize seed specific promoters to cross expression ACCase gene in rape, make the oleaginousness of its seed improve 3-5%(Roesler et al., 1997, Plant physiology. 113:75-81).Simultaneously, by the research discovery, the acid of plant body fat and grease are synthetic to be the network regulation of a complexity, can't effectively reach the purpose of change fat content and optimization fatty acid component to the regulation and control of indivedual or individual gene.Therefore, by the regulation and control to transcription factor or protein kinase, (Girke et al., Plant Physiol. 2000,124 (4): 1570-81.) can to reach the purpose of regulation and control a plurality of genes and network.

By the research to model plant and crop, find transcription factor to the important regulating and controlling effect of lipid acid, wherein LEC(Leafy Cotyledon 1) be to participate in one of most important transcription factor of fatty acid metabolism.The homologue of LEC coding CCAAT-box binding factor HAP3 (Heme-activated protein 3) subunit.Overexpression LEC1 can make the main fatty acid component concentration increase substantially that (Tan et al., Plant Physiol. 2011,156 (3): 1577-88. Mu et al., Plant Physiol. 2008,148 (2): 1042-54).And the bibliographical information that the key factor of regulation and control grease and lipid acid is relevant in the traditional oil tree is less.Yet, the molecule of the special grease of some chemical industry oilseed plants and lipid acid is resolved, can more enrich the genetic resources of Vegetable oil lipoprotein.The applicant is also finding to account for 2.5% transcription factor (Chen transcribing of special industry purposes traditional oil tree tung oil tree in the group, et al., Industrial Crops and Products 2010,32:684 – 686), and to its regulating and controlling effect in fat content and composition are synthetic carried out positive exploration.

Summary of the invention

The transcription factor that the purpose of this invention is to provide a regulating plant grease and lipid acid.

The transcription factor of regulating plant grease provided by the present invention and lipid acid, called after VfLEC1, derive from tung oil tree ( Vernicia fordii), be following 1) or 2) albumen:

1) protein that is formed by the aminoacid sequence shown in the sequence in the sequence table 1;

2) in sequence table the aminoacid sequence of sequence 1 through replacement and/or disappearance and/or add one or several amino acid and relevant with vegetable fatty acid metabolism by 1) protein of deriving.

Wherein, the sequence in the sequence table 1 is comprised of 242 amino-acid residues.Wherein, be conservative region from aminoterminal 71-136 amino acids.

Replace and/or lack and/or add one or several amino-acid residue, can be the amino-acid residue in the non-structural domain, its change can not exert an influence to the function of albumen.

Above-mentioned is 2) but middle VfLEC1 synthetic also can synthesize first its encoding gene, carries out biological expression again and obtains.Above-mentioned 2) encoding gene of the VfLEC1 in can be by the codon with sequence in the sequence table 2 certain one or several amino-acid residue in the dna sequence dna shown in 5 ' the end 33-796 bit base, and/or carry out the missense mutation of one or several base pair, and/or connect the label coding sequence at its 5 ' end and/or 3 ' end and obtain.

Above-mentioned cDNA gene with the Fatty Acids in Seeds synthesis associated protein (called after VfLEC) also belongs to protection scope of the present invention.

Specifically can be following 1 with the synthetic relevant cDNA gene of Fatty Acids in Seeds)-4) in any one described gene:

1) its encoding sequence be in the sequence table sequence 2 from 5 ' terminal 33-796 position deoxyribonucleotide;

2) its nucleotide sequence is the sequence 2 in the sequence table;

3) under stringent condition with 1) or 2) gene recombination and the gene of encoding said proteins;

4) with 1) or 2) the gene of gene with 90% above homology and encoding said proteins.

Sequence 2 in the sequence table is by 1334 based compositions, and its open reading frame (Open Reading Frame, ORF) is from 5 ' terminal 33-796 bit base, the aminoacid sequence VfLEC1 albumen of sequence 1 in the code sequence tabulation; Sequence 2 is from 5 ' terminal 71-136 bit base coding NF-YB structural domain.

The gene of the synthetic transcription factor of described coding regulating plant lipid acid both can VfLEC1 the cDNA sequence, also can fear the genomic gene sequence of VfLEC1, with the dna sequence dna that VfLEC1 has 90% above homology and coding same protein, be that cDNA with VfLEC1 separates and/or modifies and/or design with known method and obtains.

The gene VfLEC1 of the synthetic transcription factor of described coding regulating plant lipid acid or its homologous sequence can just direction or antisense orientation importing plant tissue, cell or organs.

The primer of above-mentioned VfLEC1 full length gene or its arbitrary fragment of increasing also belongs to protection scope of the present invention.

Contain above-mentioned recombinant vectors, transgenic cell line and recombinant bacterium with the synthetic protein coding gene that closes first of Fatty Acids in Seeds and also belong to protection scope of the present invention.

Description of drawings

Fig. 1 is the homogenic evolutionary relationship tree of VfLEC1 and known other species.

Fig. 2 is restructuring pB110-VfLEC1 carrier.PB110-VfLEC1 contains plant seed expression specificity promotor β-half glycinin (Beta-Conglycinin) promotor, is positioned to insert before the gene VfLEC1 gene order.

Fig. 3 is that the fat content composition changes in the VfLEC1 transgenic arabidopsis seed.

Fig. 4 is fatty acid component content in the VfLEC1 transgenic arabidopsis seed.

Embodiment

The employed molecular biology of following examples of the present invention and biochemical method are known technology.Write at Current Protocols in Molecular Biology(Ausubel, John Wiley and Sons company publishes), write the Molecular Cloning:A Labortory Manual that Cold Spring Harbor Laboratory Press (2001) publishes with J. Sambrook etc., the documents such as 3rd ED. all have detailed explanation.

Embodiment 1, the gene VfLEC1 synthetic relevant with vegetable fatty acid and grease separate

(1) extraction of total RNA and the purifying of mRNA

When extracting total RNA with the Trizol method, increased chloroform extracting number of times, the total RNA that obtains utilizes 1% agarose gel electrophoresis can detect 28S RNA, 18S RNA and 5S RNA three bands, and the brightness ratio of 28S RNA and 18S RNA is about 2:1,5S RNA band a little less than, not degraded of total RNA is described, more complete, meet the requirement that makes up the high quality cDNA library fully.Detect OD260/280 ratio between 1.9-2.0 with spectrophotometer.With paramagnetic particle method separation and purification mRNA the time, the mRNA quality that obtains after magnetic bead after total RNA is concentrated is better.

(2) the synthetic and fractional separation of ds cDNA

Because the ZAP-cDNA Gigapack III Gold Cloning Kit operation instruction according to Stratagene company, when fractional separation, need to fill post, time is long, and produce easily bubble, so reelect the CHROMA SPIN-400 Column with fractional separation part among the Creator SMART cDNA Library Construction Kit of Clontech company, Hoarding segment length crossing post liquid and be mixed in the same pipe more than 500 bp.

(3) detection of cDNA library quality

ZAP-cDNA Gigapack III Gold Cloning Kit according to Stratagene company detects, and the titre in the elementary library of tung oil tree kind benevolence cDNA is 1 * 10 ⁶Pfu/mL, recombination fraction are 99.7%, and the titre in library is 1.2 * 10 after the amplification ⁹Pfu/mL, 20 positive colonies of random choose carry out PCR and detect, and the agarose gel electrophoresis detected result shows the Insert Fragment size between 0.5～2.5 kb, and average about 1 kb carries out plasmid DNA after the cyclisation EcoRI and XhoThe I double digestion, detected result is consistent with PCR.The titre in library, recombination fraction and integrity all meet the high quality library standard that makes up.

(4) tung oil tree kind benevolence cDNA serves in initial library the marine life chip companies and checks order acquisition transcription factor VfLEC sequence.The original series that records utilizes crossmatch software to remove 5 ' end carrier sequence, library joint sequence and poly(A) sequence; Splice with phrap; By local BLAST obtain 515 with the sequence (singlets) of other sequences without homologous region, it is 228 contigs that remaining 1849 sequence is gathered according to homology.Transcription factor VfLEC sequence obtains by the contig splicing.

(5) separation of VfLEC gene

The sequences Design Auele Specific Primer of general layout VfLEC increases from the tung oil tree cDNA library, behind the acquisition specific fragment, carries out the TA cloning and sequencing.

Embodiment 2, turn the acquisition of VfLEC gene Arabidopis thaliana

For verifying better the function of VfLEC, select wild-type Arabidopis thaliana (Col-0), mutant Arabidopis thaliana.The mutant Arabidopis thaliana is available from Arabidopsis Biological Resource Center (ABRC; Ohio State University), be respectively the mutant of the different sites of Arabidopis thaliana LEC1 gene (GenBank accession number AT1G21970), article number is SALK_000450.

Make up recombinant plant expression vector pB110-VfLEC, Electroporation Agrobacterium GV3101 receives the antibiotic-screening positive transformant by card.The agrobacterium-mediated transformation arabidopsis thaliana transformation is by red fluorescence screening transgenic seed.

Embodiment 3, turn VfLEC gene Arabidopis thaliana seed grease and fatyy acids

Collect the seed of wild-type Arabidopis thaliana and transgenic arabidopsis, 60 ℃ of oven dry are spent the night, and pulverizer grinds again oven dry, and the Soxhlet extraction process extracts grease, and poor heavy method is calculated oleaginousness, and the GC-MS method detects fatty acid component and changes.

Show such as Fig. 3,4 results, the interior fat content of Arabidopis thaliana seed that turns the VfLEC gene promotes 5.6%-7.1%, and fatty acid content significantly promotes, and fat content, oleic acid and linoleic acid content are higher than wild-type in the VfLEC transposon mutant body Arabidopis thaliana, this explanation, the VfLEC gene can compensate Arabidopis thaliana LEC1Function, and be better than to a certain extent Arabidopis thaliana LEC1Gene.

Sequence table

Sequence ID 1

＜110〉Subtropical Zone Forestry Inst., Chinese Academy of Forest Science

＜120〉the synthetic transcription factor encoding gene of tung oil tree regulation and control grease VfLEC

<130> patent

<160> 3

<170> PatentIn version 3.2

<210> 1

<211> Length : 242

SequenceName : vfLEC

<212> Type : DNA

<221> CDS

＜213〉tung oil tree (Valernicia fordii)

<400> 1

Met Glu Arg Gly Gly Arg Phe His Arg Tyr Arg Arg His Ala Lys

1 5 10 15

Gln Gln Ala Ser Ile Ser Ser Ala Ala Ser Ser Gly Val Asn Leu

20 25 30

Leu Arg Ala Asn Asn Pro Pro Asn Phe Asn Leu Pro Thr Asn Leu

35 40 45

Asn Pro Ser Gln Ala Asn Pro Thr Met Val Pro Asn Pro Gln Gln

50 55 60

Pro Pro Gln Cys Thr Val Arg Glu Gln Asp Gln Tyr Met Pro Ile

65 70 75

Ala Asn Val Ile Arg Ile Met Arg Arg Ile Leu Pro Ala His Ala

80 85 90

Lys Ile Ser Asp Asp Ala Lys Glu Thr Ile Gln Glu Cys Val Ser

95 100 105

Glu Tyr Ile Ser Phe Ile Thr Gly Glu Ala Asn Asp Arg Cys Gln

110 115 120

His Glu Gln Arg Lys Thr Ile Thr Ala Glu Asp Val Leu Trp Ser

125 130 135

Met Gly Lys Leu Gly Phe Asp Asp Tyr Val Glu Pro Leu Thr Leu

140 145 150

Phe Leu Asn Arg Tyr Arg Glu Ala Glu Asn Glu Arg Gly Ser Val

155 160 165

Arg Asp Pro Leu Leu Lys Arg Ser Asn Val Gly Val Val Asp Tyr

170 175 180

Gly Asn Ile Gly Met Pro Pro Phe Met Pro Ser Phe Ala Val Gly

185 190 195

Pro Pro Pro Pro Gln Gly Val Phe Asp Pro Ala Met Phe Gly Ala

200 205 210

Tyr Tyr Arg Asp Val Ser Asp Ala Gly Ala Gly Met Gly Gly Pro

215 220 225

Ser Ser Ser Asn Asn Pro Tyr Ile Asn Phe Asp Pro Phe Thr Gln

230 235 240

Phe Lys

SEG ID NO：2

<210> 2

<211> 1334

<212> DNA

＜213〉tung oil tree (Vernicia fordii)

<400> 2

cattttattt ttattattat tttagtttaa ttatggaacg tggaggcagg ttccatcgct 60

atcgaaggca tgcaaaacag caagcttcaa taagctctgc tgctagctct ggcgtgaatt 120

tactgcgagc aaataaccca ccaaacttca atctccccac caatctgaac ccaagtcagg 180

ccaacccaac aatggtgcca aacccccagc agccacctca atgcactgtg cgtgaacaag 240

atcagtacat gccaatagcc aatgtgattc gcatcatgcg ccgtattctc ccagctcatg 300

caaaaatctc tgatgatgcc aaagaaacta tccaagaatg tgtctctgaa tacataagct 360

tcatcactgg agaggcaaat gatcgttgcc agcatgagca gcgcaagact atcactgctg 420

aggatgtgct ttggtctatg ggaaagctgg gattcgatga ctatgtcgag cctctaactc 480

tttttctgaa tcgctatcgc gaggcagaaa atgaacgtgg ctctgttcgt gatccactcc 540

tgaagcgtag caatgttggt gttgttgatt atggaaatat tgggatgcct ccttttatgc 600

caagttttgc tgtgggacct cccccaccac agggtgtttt tgatcctgca atgtttggtg 660

cctattatag ggatgtttct gatgctggtg ctggtatggg tggcccctct tcttcgaaca 720

atccttatat caacttcgat ccatttactc agttcaagtg atgacagttt gatagttcga 780

caagaaagga ggaacatata tgactattaa ctataaaaga aataaaggga taatatcata 840

aacttttttt tttttaaatt aaagaaatgt agtttaagga gtagatggca ctagtgacta 900

gctagtgaga taccatttgc tcctcctcct cctcctttta tatgttgttt acttctatct 960

gatgtaacaa catacctgct atattttgaa tttcaagcat atatatggaa agaaatttca 1020

attaaaaaaa aaaaaaaaaa actcgagggg gggcccggta cccaattcgc cctatagtga 1080

gtcgtattac aattcactgg ccgtcgtttt acaacgtcgt gactgggaaa accctggcgt 1140

tacccaactt aatcgccttg cagcacatcc ccctttcgcc agctggcgta atagcgaaga 1200

ggcccgcacc gatcgccctt cccaacagtt gcgcagcctg aatggcgaat ggcaaattgt 1260

aagcgttaat attttgttaa aattcgcgtt aaatttttgt taaatcagct cattttttaa 1320

ccaataggcc gaaa 1380

Claims

1. the transcription factor of regulating plant grease and lipid acid, its aminoacid sequence is shown in SEQ ID NO:1.

2. the cDNA gene order of the described transcription factor of claim 1 of encoding.

3. cDNA gene order according to claim 2 is characterized in that, the base sequence of this cDNA sequence is shown in SEQ ID NO:2.

4. the expression vector that contains the 2 or 3 described genes of having the right.

5. the transgenic cell line that contains the 2 or 3 described genes of having the right.

6. the Host Strains that contains the 2 or 3 described genes of having the right.

7. the method for a regulating plant grease and fatty acid component and content is characterized in that, is that vegetation fat acid constituents and content are regulated with right 2 or 3 described gene transfered plant tissues, cell or organ.

8. method according to claim 7 is characterized in that, described gene imports plant tissue, cell or organ by the plant expression vector that contains this gene; The carrier that sets out that is used for making up described plant expression vector is plant expression vector.

9. according to claim 7 or 8 described methods, it is characterized in that described plant is dicotyledons.