CN102757488B

CN102757488B - Transcription factor for improving plant seed fatty acid content, coding gene and application thereof

Info

Publication number: CN102757488B
Application number: CN201210205390.7A
Authority: CN
Inventors: 华金平; 刘正杰; 张园; 赵鹏; 赵清翠; 李玉华; 王玉美
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2012-06-18
Filing date: 2012-06-18
Publication date: 2014-04-23
Anticipated expiration: 2032-06-18
Also published as: CN102757488A

Abstract

The invention discloses a transcription factor associated for improving plant seed fatty acid content, a coding gene and application thereof. The transcription factor provided by the invention is (a) or (b): (a) any protein segment with length of 155th to 315th amino acids which is obtained by extending the amino acid sequences of 161th to 315th sites with carboxyl terminals containing at least sequence 2 in a sequence table from 161th site in the sequence 2 towards an amino acid terminal of the sequence 2 according to the direction of the amino acid sequence of the sequence 2, wherein the protein segment has transcription activity; and (b) protein which is obtained by substituting, deleting and/or adding one or more amino acid residues for the amino acid sequence defined by (a) and has transcription activity or is associated for improving the plant seed fatty acid content and is derived from the amino acid sequence defined by (a). According to the transcription factor GhDof1 provided by the invention, the fatty acid content of plant seeds can be obviously increased; and the transcription factor is good in application prospect.

Description

Improve transcription factor and encoding gene and the application of plant seed fatty acid content

Technical field

The present invention relates to a kind of transcription factor and encoding gene and the application relevant to improving plant seed fatty acid content, particularly a kind of transcription factor and encoding gene and application relevant to improving plant seed fatty acid content that derives from cotton.

Background technology

Cotton is the important cash crop of China and oil crops.China plants cotton 6,000,000 hectares throughout the year, approximately produces 7,600,000 tons of gined cottons, 1,300 ten thousand tons of cottonseeds.As the main byproduct of Cotton Production, cottonseed be rich in grease and albumen (Xu Hongxia, Yang Wei China, Wang Yanqin, Zhou great Yun, rectifies violent, Feng Xinai.China's oil is analyzed with cotton seed quality condition.Cotton, 2009,36 (7): 2-3), the cotton benevolence lipid content 30%～40% after peeling off, can produce grease.In recent years, day by day urgent along with world's oil resource and Energy resources, the production of cottonseed wet goods oil crops more and more comes into one's own, and the cultivated area that expands oil crops has become the important channel that solves China's oil resource anxiety.In line with the principle of " not striving ground with food crop ", for increasing the fat content of cotton seeds in unit cultivated area, by the synthetic transcription factor of clone's regulating plant lipid acid, application genetic transformation means import in cotton acceptor plant, thereby improve the fatty acid content of plant, and then the fat content in increase Cotton Seed, improve the grease yield of oil crops.This has Important Economic benefit and social benefit to increasing China's grease supply.

By utilizing the relevant genetic expressions of approach such as lipid acid synthesizes in transcription factor regulation and control seed, thereby the research of raising fatty acid content and fat content more and more comes into one's own.The homologue of LEC1 coding CCAAT binding factor HAP3 subunit, it is transcription regulaton factor important in Embryos Development of Plant process, (the Mu et al..LEAFY COTYLEDON1 is a key regulator of fatty acid biosynthesis in Arabidopsis.2008 such as Mu, 148 (2): 1042-1054) research shows, the increase that causes lipid acid synthetic gene expression amount integral level is expressed in crossing of Arabidopis thaliana LEC1 gene, at LEC1, cross in the transfer-gen plant of expression, the enzyme gene that exceedes 58% known participation plastid lipid acid route of synthesis just shows.WRINKLED1(WRI1) transcription factor direct regulation and control glycolysis-and fatty acid metabolism process, can improve the overall expression level of lipid acid synthesis related gene, improve lipid acid and fat content (Baud et al..WRINKLED 1specifies the regulatory action of LEAFY COTYLEDON2towards fatty acid metabolism during seed maturation in Arabidopsis, Plant Journal, 2007,50 (5): 825-838).In Arabidopis thaliana, excessively expressing Bnwri1 makes the oleaginousness of seed increase 10%-40%, further analysis finds that the cell volume of transgenic arabidopsis rataria becomes large but not quantity becomes many (Liu et al..Increasing seed mass and oil content in transgenic Arabidopsis by the overexpression of wri1-like gene from Brassica napus.Plant Physiology and Biochemistry, 2010,48:9-15).But, also there is no at present the research about the transcription factor of regulation and control cotton fatty acid content.

At home about regulating in the patented technology report of fatty acid content or composition, (protein of peanut phosphoric acid enol type pyruvate carboxylase gene and coding thereof and the cloning process such as Yu Shanlin, application number: 200810002795.4) disclose in Ecological Property of Peanut Seeds peanut stearyl-carrier proteins desaturase gene and coded protein thereof in fatty acid desaturation reaction process, the expression of this gene can improve the content of unsaturated fatty acids in peanut, and can also increase the winter resistance of peanut.The concrete technology that has not yet to see cotton transcription factor regulation and control fatty acid content or composition is delivered.

Summary of the invention

The object of this invention is to provide a kind of albumen and encoding gene and the application relevant to improving plant seed fatty acid content, this albumen has functional transcription factor.

Protein provided by the present invention, derives from cotton (Gossypiumhirsutum L.), and name is called GhDof1, is following (a) or (b):

(a) carboxyl terminal at least contains the 161-315 amino acids sequence of sequence 2 in ordered list, and from the 161st of sequence 2, according to the aminoterminal of the aminoacid sequence direction sequence 2 of sequence 2, extend, obtaining length is 155 to 315 amino acid whose any one protein fragments; Described protein fragments has transcriptional activity.

(b) replacement and/or disappearance and/or the interpolation of one or several amino-acid residue of aminoacid sequence process (a) being limited, and there is transcriptional activity or the aminoacid sequence that by (a) the limited derivative protein relevant to improving plant seed fatty acid content.

In of the present invention, described protein is specially as follows (c) or (d):

(c) protein being formed by the aminoacid sequence shown in sequence in sequence table 2;

(d) protein being formed by the 161-315 amino acids sequence of sequence in sequence table 2.

In sequence table, sequence 2 is comprised of 315 amino acid.

For the ease of described protein purification, can be in by sequence table N-terminal or the C-terminal of the protein that forms of the amino acid residue sequence of 161-315 position of sequence 2 or sequence 2 connect label as shown in the table.

Table: the sequence of label

Label	Residue	Sequence
			Poly-Arg	5-6(is generally 5)	RRRRR
Poly-His	2-10(is generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

The nucleic acid molecule of code for said proteins also belongs to protection scope of the present invention.

Described nucleic acid molecule can be DNA, as cDNA, genomic dna or recombinant DNA; Described nucleic acid molecule can be also RNA, as mRNA, hnRNA or tRNA etc.

In the present invention, described nucleic acid molecule is the gene of code for said proteins; Described gene is following 1) or 2) or 3):

1) 3 ' end at least contains the nucleotide sequence of the 578-1045 position of sequence 3 in ordered list, and from the 578th of sequence 3, according to the nucleotide sequence of sequence 3, to 5 of sequence 3 ' end, extend, and/or from the 578th of sequence 3, according to the nucleotide sequence of sequence 3, to 3 of sequence 3 ' end, extend, obtain length and be any one DNA fragmentation of 468 to 1249bp;

2) 3 ' end is at least containing the nucleotide sequence of the 481-948 position of sequence 1 in ordered list, and from the 481st of sequence 1, according to the nucleotide sequence of sequence 1, to the prolongation of 5 of sequence 1 ' end, obtains length and be any one DNA fragmentation of 468 to 948bp;

3) with 1) or 2) nucleotide sequence that limits has 75% or 75% above homology, and the DNA fragmentation relevant to raising plant seed fatty acid content.

More specifically, described gene is following 4)-8) in any:

4) encoding sequence is the DNA molecular shown in sequence 1 in sequence table;

5) DNA molecular shown in sequence 1 in sequence table;

6) DNA molecular shown in the 481-948 position of sequence 1 in sequence table;

7) DNA molecular shown in sequence 3 in sequence table;

8) DNA molecular shown in the 95-1153 position of sequence 3 in sequence table.

Sequence 3 is comprised of 1294 Nucleotide, and wherein 98-1045 position is encoding sequence, i.e. sequence 1 in sequence table, is comprised of 948 Nucleotide, the protein shown in sequence 2 in code sequence list; Wherein, the 161-315 amino acids of the corresponding sequence 2 of 481-948 position Nucleotide of sequence 1.

The recombinant vectors, expression cassette, transgenic cell line or the recombinant bacterium that contain described nucleic acid molecule also belong to protection scope of the present invention.

Described recombinant vectors can be recombinant expression vector, also can be recombinant cloning vector.

Described recombinant expression vector can build with existing plant expression vector or Yeast expression carrier.Described plant expression vector comprises double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment, as pCAMBIA3301, pCAMBIA1300, pBI121, pBin19, pCAMBIA2301, pCAMBIA1301-UbiN or other derivative plant expression vector.Described Yeast expression carrier is as pGBT9 etc.Described plant expression vector or described Yeast expression carrier also can comprise 3 ' end untranslated region of foreign gene, comprise the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.While using described nucleic acid molecule (gene) to build recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promotor (pUbi), stress induced promoter Rd29A etc., they can be used alone or are combined with other promotor; In addition, while using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser regions can be ATG initiator codon or neighboring region initiator codon etc., but must be identical with the reading frame of encoding sequence, to guarantee the correct translation of whole sequence.The source of described translation control signal and initiator codon is widely, can be natural, also can synthesize.Translation initiation region can be from transcription initiation region or structure gene.For the ease of transgenic plant cells or plant are identified and are screened, can process recombinant expression vector used, the coding that can express in plant as added can produce the enzyme of colour-change or the gene of luminophor, have the antibiotic marker thing of resistance or anti-chemical reagent marker gene etc.Also can not add any selected marker, directly with adverse circumstance screening transformed plant.

In an embodiment of the present invention, the promotor that starts described genetic transcription in described recombinant expression vector is specially (1) or (2) as follows:

(1) P _aDH1promotor;

(2) 35S promoter, as CaMV 35S promoter.

More specifically, described recombinant expression vector can be (1) or (2) as follows:

(1) at pGBT9 carrier (P _aDH1promotor) multiple clone site place insert the recombinant plasmid that described gene obtains; Described multiple clone site is specially BamH I and Pst I.

(2) at the multiple clone site place of pBI121 carrier (CaMV 35S promoter), insert the recombinant plasmid that described gene obtains.Described multiple clone site is specially Xba I and Sac I.

Described expression cassette is by the promotor that can start described genetic expression, described gene, and transcription termination sequence composition.

In one embodiment of the invention, described recombinant bacterium is specially the recombination yeast that carries described gene; Described yeast specifically can be yeast strain Y187.

Described protein, or described nucleic acid molecule, or described recombinant expression vector, expression cassette or recombinant bacterium are at following a1) or a2) in application also belong to protection scope of the present invention:

A1) regulating plant seed fat acid content;

A2) the high plant variety of seed selection seed fat acid content.

In one embodiment of the invention, described regulating plant seed fat acid content is specially and improves plant seed fatty acid content.

The method of the plant variety that described seed selection seed fat acid content is high, specifically can comprise the step that the higher plant of described protein expression amount is hybridized as parent.

Another object of the present invention is to provide a kind of method of transgenic plant of cultivating the raising of seed fat acid content.

The method can comprise the step in the gene importing object plant of code for said proteins; Described transgenic plant are compared with described object plant, and seed fat acid content improves.

The expression amount of described protein in described transgenic plant is higher than described object plant; The gene of code for said proteins is that described gene is following 1) or 2) or 3):

More specifically, described gene is following 4)-8) in any:

5) DNA molecular shown in sequence 1 in sequence table;

6) DNA molecular shown in the 481-948 position of sequence 1 in sequence table;

7) DNA molecular shown in sequence 3 in sequence table;

8) DNA molecular shown in the 95-1153 position of sequence 3 in sequence table.

Described gene specifically can import in described object plant by above-mentioned arbitrary described recombinant expression vector (plant expression vector), obtains described transgenic plant.Specifically can be by using the conventional biological method such as Ti-plasmids, Ri plasmid, plant viral vector, directly delivered DNA, microinjection, electricity be led, agriculture bacillus mediated, particle gun by described recombinant expression vector transformed plant cells or tissue, and the plant tissue of conversion is cultivated into plant.The agriculture bacillus mediated biological method that waits is transformed in vegetable cell or tissue.

Described plant can be dicotyledons, also can be monocotyledons, and described dicotyledonous plant is as Arabidopis thaliana.

In one embodiment of the invention, described plant is specially Col type Arabidopis thaliana.

Described protein also belongs to protection scope of the present invention in the application as in transcription factor.

The transcription factor GhDof1 that the present invention obtains from cotton can significantly improve the fatty acid content of plant seed, and this transcription factor possesses good application prospect.

Accompanying drawing explanation

Fig. 1 is the pcr amplification result of GhDof1 gene (containing ORF).Wherein, swimming lane 1 is D2000DNA Ldder molecular weight standard; Swimming lane 1 and swimming lane 2 are the pcr amplification result of GhDof1 gene (containing ORF).

Fig. 2 is the qualitative detection result of GhDof1 albumen betagalactosidase activity.Wherein, A is three primary yeast carrier sketches, and a-e is respectively pGBT9, pGBT9-GhDof1, pGBT9-GhDof1-N, pGBT9-Dof1-C, pGBT9-WER; B is yeast filter paper colour developing situation, and conversion carrier a-e is the same, and " left side " represents yeast colony growing state on SD/-Trp defective type substratum, and " right side " represents yeast colony colour developing situation on filter paper, and 1-3 represents 3 single bacterium colonies.

Fig. 3 is the detection by quantitative result of GhDof1 albumen betagalactosidase activity.Wherein, the Dof in container name represents GhDof1.

Fig. 4 is the half-quantitative detection result that GhDof1 gene is expressed in different tissues.Wherein, Ova: ovary, A: flower pesticide, Se: calyx, P: petal, Ovu: ovule, R: root; S: stem; L: leaf.

Fig. 5 is recombinant expression vector pBI-Dof1 double digestion qualification result.Wherein, swimming lane M is 1Kb DNA Ladder molecular weight standard; Swimming lane 1 is the double digestion product of recombinant expression vector pBI-Dof1.

Fig. 6 is that pBI121-Dof1 transforms Agrobacterium bacterium colony PCR the result.Wherein, swimming lane M is D2000DNALadder molecular weight standard; Swimming lane 1-3 is the PCR detected result of three positive monoclonal bacterium colonies.

Fig. 7 is the PCR qualification result that turns GhDof1 gene Arabidopis thaliana plant.Wherein, swimming lane M is D2000DNALadder molecular weight standard; The positive contrast of swimming lane 1 (recombinant expression vector pBI-Dof1); The negative contrast of swimming lane 2 (not genetically modified wild-type Arabidopis thaliana plant); Swimming lane 3-8 is the PCR detected result of 8 positive transfer-gen plants.

Fig. 8 is T ₃for the pure and mild growth phenotype observations that turns GhDof1 gene strain.Wherein, A and B are two repetitions, and 1 in A and B is not genetically modified wild-type (Col type) Arabidopis thaliana contrast, and 2 are and turn GhDof1 gene Arabidopis thaliana plant.

Fig. 9 is that Arabidopis thaliana turns GhDof1 gene strain T ₃for Fatty Acids in Seeds content analysis result.Wherein, Col represents not genetically modified wild-type Arabidopis thaliana plant contrast; D1-2, D1-9, D2-6, D2-8 and D3-2 represent respectively 5 T ₃in generation, turns GhDof1 gene Arabidopis thaliana strain.* be illustrated in 0.05 level and organize significant difference with Col, * * is illustrated in 0.01 level and organizes significant difference with Col.

Embodiment

The experimental technique using in following embodiment if no special instructions, is ordinary method.

In following embodiment, material used, reagent etc., if no special instructions, all can obtain from commercial channels.

The acquisition of the encoding gene GhDof1 of embodiment 1, cotton transcription factor GhDof1

1, total RNA extracts: select upland cotton (Gossypium hirsutum L.) kind Coker201(military elegant bright, Liu Chuanliang, Zhang Chaojun, Li Fuguang.The progress that cotton somatic embryos fetal hair is raw.BULLETIN OF BOTANY Vol., 2008,25 (4): 469-475) be experiment material, take away the rataria material of spending latter 20 days, quick-frozen, in liquid nitrogen, is applied CTAB method (reference: Liu Yang, He Xinyao, Ma Hongbo, Wu Yongli, Yang Youming.By CTAB-PVP method, extract the research of the each total tissue RNA of cotton.China Agricultural University's journal, 2006,11 (1): 53-56) extraction RNA.

2, the synthetic cDNA of RNA reverse transcription that utilizes the RT-PCR reaction system of Promaga that step 1 is extracted.

3, RT-PCR amplification:

According to the EST information of cotton in ncbi database, be spliced into contig, design following primer 1 and primer 2:

The 95-113 position of primer 1:5'-GTAATGCAAGACATCCACT-3'(sequence 3)

Primer 2: the reverse complementary sequence of the 1134-1153 position of 5'-CAGTTAAATCCTGCCTTAAG-3'(sequence 3)

The cDNA of the Upland Cotton Coker201 obtaining take step 2 is masterplate, carries out pcr amplification with primer 1 and primer 2.Reaction system: 2.5 μ l 10 × PCR damping fluids are (containing MgCl ₂), each 0.5 μ l primer 1 and primer 2 (10 μ M), 4 μ l 2.5mM dNTPs, 1 μ l cDNA sample, 0.25 μ l LA enzyme (TaKaRa), 16.25 μ l distilled waters.Response procedures: 94 ℃ of 3min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 2min, 35 circulations; 72 ℃ of 10min.

Through above-mentioned PCR reaction, amplify the object band (Fig. 1) that size is about 1059bp.After PCR product is reclaimed, be connected with pMD18-T carrier (TaKaRa), connect product and transform bacillus coli DH 5 alpha competent cell, adopt blue hickie method screening positive clone, sample presentation order-checking.Result shows, in this PCR product, containing the DNA fragmentation (wherein the 98-1045 position nucleotides sequence of sequence 3 is classified sequence 1 in sequence table as) shown in the nucleotide sequence of the 95-1153 position of sequence 3 in ordered list, by this DNA fragmentation place unnamed gene, be GhDof1 gene.Whole sequence 1 is the coding region sequence of GhDof1 gene, the protein shown in sequence 2 in code sequence list, called after GhDof1 albumen.

4, RACE amplification obtains the 3' terminal sequence of GhDof1 gene cDNA: according to Invitrogen Gene Racer Kit explanation, carry out 3'RACE.With the 567-590 position of the GhDof1 gene 3'RACE special primer Wri3GSP1:5'-GCTAGAACAAGGATCAGACTGCGG-3'(sequence 1 designing, the 664-687 position of sequence 3) with the 836-857 position of Wri3NGSP1:5'-GGCAAGGGAACGAAGATCAAGG-3'(sequence 1, the 933-954 position of sequence 3) and primer corresponding to test kit carry out following twice PCR:

PCR reaction (primer sequence GeneRacer 3 ' primer:3 '-GCAATGCATCGCATAGCAACTGTCG-5 ' that Wri3GSP1 and test kit provide) for the first time: 94 ℃ of 3min; 94 ℃ of 30s, 70 ℃ ~ 60 ℃ (Touch Down, the each circulation of front 10 cycle annealing temperature declines 1 ℃, rear 25 cycle annealing temperature are 60 ℃) 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.Nest-type PRC reaction (primer sequence GeneRacer 3 ' nested primer:3 '-GTGACAGTACGGCAATGCATCGC-5 ' that Wri3NGSP1 and test kit provide) for the second time: 94 ℃ of 3min; 94 ℃ of 30s, 60 ℃ of 30s, 72 ℃ of 1min, 35 circulations; 72 ℃ of 10min.By above-mentioned twice PCR amplification, obtain the 3' terminal sequence of cDNA, and sample presentation order-checking, sequencing result shows that gained 3' terminal sequence is the 933-1294 position of sequence 3 in sequence table.

5, GhDof1 full length gene obtains and analyzes: the 3' terminal sequence splicing of the GhDof1 gene cDNA obtaining through coding region sequence (sequence 1) and the step 4 of the GhDof1 gene that Cap3 splicing software (http://pbil.univ-lyon1.fr/cap3.php) obtains step 3 online, to splice the upper est database of sequence and NCBI compares again, through electronic splicing, obtain the total length of this gene, this full length gene sequence is 1294bp, comprise the 948bp core coding region (sequence 1) of 98-1045 position, 97bp 5 ' UTR and 249bp 3 ' UTR.Sequence 3 is the GhDof1 albumen shown in sequence 2 in code sequence list also.With ProtParam tool(http: //web.expasy.org/protparam/) analyze the physico-chemical property of this albumen, the molecular weight of finding this albumen is 35.339KD, the theoretical iso-electric point of albumen is 8.33.Application SMART software (http://smart.embl-heidelberg.de/) is analyzed the conservative functional domain of GhDof1 aminoacid sequence, find the motif that the 157-208 amino acids of GhDof1 protein N terminal contains a C2C2-type class zinc fingers, by DOF protein family peculiar.Application is predicted its Subcellular Localization mode through online software prediction (www.bioinfo.tsinghua.edu.cn/SubLoc/), finds that GhDof1 protein localization is in nucleus.

The transcriptional activation function analysis of embodiment 2, GhDof1 transcription factor

The present embodiment is with pGBT9(red rock in the Meng, Du Xiongming, Zhang Chunyi etc. the clone of cotton transcription factor gene GhMS3 and the evaluation of promoter function thereof. Scientia Agricultura Sinica, 2010,43(17)) be initial carrier, build following three kinds of expression vectors (A in Fig. 2): the recombinant expression vector pGBT-GhDof1 of the CDS total length (sequence 1 is called for short GDof1-CDS) that (1) contains GDof1 gene; (2) the recombinant expression vector pGBT-GhDof1-N of the N end (the 1-501 position Nucleotide of sequence 1 is called for short GDof1-N) that contains GDof1 gene; (3) the recombinant expression vector pGBT-GhDof1-C of the C end (the 481-948 position Nucleotide of sequence 1 is called for short GhDof1-C) that contains GhDof1 gene.Thereby the transcriptional activation function to GhDof1 transcription factor is analyzed.

One, the structure of recombinant expression vector

The coding region sequence (sequence 1) of the GhDof1 gene obtaining take embodiment 1 is template, obtains GDof1-CDS, the GDof1-N and the GDof1-C fragment that contain corresponding restriction enzyme site with following primer amplification; With PCR product corresponding to corresponding restriction enzymes double zyme cutting, the product after enzyme is cut connects with the skeleton fragment of the pGBT9 carrier through same double digestion.To connecting product, transform, shake bacterium, picking plasmid, sample presentation order-checking.Through order-checking, show that the recombinant plasmid that contains GDof1-CDS, GDof1-N and GDof1-C fragment is respectively recombinant expression vector pGBT-GhDof1, pGBT-GhDof1-N and pGBT-GhDof1-C.

The primer sequence of amplification GhDof1-CDS is as follows:

CDS-up:CG gGATCCaTGCAAGACATCCACTCG(underscore part is the recognition sequence of BamH I, and sequence is thereafter the 1-18 position of sequence 1)

CDS-down:AA cTGCAGtTACGGGAGATAGAGGCCATGG(underscore part is the recognition sequence of Pst I, and sequence is thereafter the 927-948 position reverse complementary sequence of sequence 1)

The primer sequence of amplification GDof1-N is as follows:

N-up:CG gGATCCaTGCAAGACATCCACTCG(underscore part is BamH I, and sequence is thereafter the 1-18 position of sequence 1)

N-down:AA cTGCAGgTTAGAATGAGAGGTTACCG(underscore part is Pst I, and sequence is thereafter the reverse complementary sequence of the 482-501 position of sequence 1)

The primer sequence of amplification GDof1-C is as follows:

C-up:CG gGATCCgCGGTAACCTCTCATTCTAA(underscore part is BamH I, and sequence is thereafter the 481-500 position of sequence 1)

C-down:AA cTGCAGtTAAGTGGGATAAAAAAGGGTC(underscore part is the recognition sequence of Pst I)

Two, the transcriptional activation function of the each recombinant expression vector of Whatman filter paper color developing detection step 1

Three recombinant expression vectors that adopt heat shock method that step 1 is built are transformed into respectively yeast strain Y187(red rock in the Meng, Du Xiongming, Zhang Chunyi etc. the clone of cotton transcription factor gene GhMS3 and the evaluation of promoter function thereof. Scientia Agricultura Sinica, 2010, competent cell 43(17)), yeast strain after transforming is divided into two groups, respectively at SD/-Trp substratum (general Jino, Beijing (FunGenome) Science and Technology Ltd., catalog number: YGM003A-2) and SD/-Trp/-His substratum (general Jino, Beijing (FunGenome) Science and Technology Ltd., catalog number: YGM003A-17) 28 ℃ cultivate 2-3 days.Because Y187 is the two deficient strain of Trp and His, and pGBT9 carrier contains Trp encoding gene, so transform the yeast of above carrier, can on SD/-Trp substratum, grow, but can not be on SD/-Trp/-His substratum normal growth.The yeast list bacterium colony that 3 of each pickings are grown on SD/-Trp substratum, by Clontech system operation flow process on Whatman filter paper, carry out betagalactosidase activity qualitative detection (reference: Meng Hongyan. the preliminary study of cotton myb transcription factor GhMS3 biological function. Beijing, the Chinese Academy of Agricultural Sciences, master thesis, pp:16).Then, take ONPG(onitrophenyl-β-D-galactopyranoside) be substrate, carry out betagalactosidase activity detection by quantitative (reference: Meng Hongyan. the preliminary study of cotton myb transcription factor GhMS3 biological function. Beijing, the Chinese Academy of Agricultural Sciences, master thesis, pp:16-17).PGBT9 carrier is set simultaneously as negative control; Arrange pGBT9-WER carrier (Meng Hongyan, Du Xiongming, Zhang Chunyi etc. the clone of cotton transcription factor gene GhMS3 and the evaluation of promoter function thereof. Scientia Agricultura Sinica, 2010,43(17)) as positive control.

With Whatman filter paper, carry out the result of qualitative detection of betagalactosidase activity as shown in B in Fig. 2, pGBT-GhDof1-N is the same with the pGBT9 carrier as negative control, can not develop the color; And pGBT-GhDof1-C, pGBT-GhDof1 are the same with the pGBT9-WER carrier as positive control, can make filter paper colour developing.

Take ONPG(onitrophenyl-β-D-galactopyranoside) as substrate carry out betagalactosidase activity detection by quantitative result as shown in Figure 3, pGBT-GhDof1-N almost can't detect betagalactosidase activity, and the betagalactosidase activity of pGBT-GhDof1-C is far above pGBT-GhDof1.The result shows that transcriptional activation region is positioned at the C end of GhDof1 albumen.

The expression analysis of embodiment 3, GhDof1 gene

Extract the ovary of upland cotton (Gossypium hirsutum L.) kind Coker201, flower pesticide, calyx, petal, ovule, root, the RNA of stem and leaf tissue, through the synthetic cDNA of the RT-PCR of Promaga reaction system reverse transcription, for the 507-526 position of GhDof1 gene design primer 5'-TGATGTGAGTGAACCGAAGT-3'(sequence 1) and 5'-TTTACGGGAGATAGAGGC-3'(2-18 position be the reverse complementary sequence of the 932-948 position of sequence 1), take Ghactin as reference gene, design primer 5'-ATCCTCCGTCTTGACCTTG-3' and 5'-TGTCCGTCAGGCAACTCAT-3', adopt the expression of semi-quantitative RT-PCR analysis GhDof1 gene at different tissues position.

As shown in Figure 4, GhDof1 gene shows certain tissue expression specificity to result, is in particular in: this gene all has expression in the reproductive organ tissues such as ovary, flower pesticide, calyx, and very weak or do not express at the expression amount of petal and ovule; In vegetative organ tissue, GhDof1 gene expression amount in stem and blade is higher, but does not express in root.

Embodiment 4, turn acquisition and the functional verification thereof of GhDof1 gene Arabidopis thaliana

MS screening culture medium: adding final concentration in basic MS nutrient solution is the sucrose of 10g/L, the agar powder that final concentration is 7.6g/L, the kantlex that final concentration is 50mg/L.Wherein, the solvent of MS nutrient solution is that water, solute are as shown in table 1.

The solute of table 1MS basic culture solution

Macroelement	Concentration (gL in substratum ^-1）
		NH ₄NO ₃	1.65
KNO ₃	1.9
		KH ₂PO ₄	0.17
MgSO ₄.7H ₂O	0.37
		CaCl ₂	0.44
Trace element	Concentration (mgL in substratum ^-1）
		FeSO ₄·7H ₂O	27.8
Na ₂EDTA	37.3
		MnSO ₄·4H ₂O	22.3
ZnSO ₄·4H ₂O	8.6
		H ₃BO ₃	6.2
KI	0.83
		Na ₂MoO ₄·2H ₂O	0.25
CuSO ₄·5H ₂O	0.025
		CoCl ₂·6H ₂O	0.025
Organic composition	Concentration (mgL in substratum ^-1）
		Glycine	2.0
Vitamin	0.1
		Pyridoxine hydrochloride	0.5
Nicotinic acid VB5	0.5
		Inositol	100

One, the structure of recombinant expression vector pBI-Dof1

The complete sequence (sequence 3) of the GhDof1 gene obtaining take embodiment 1 is template, obtains the GhDof1 gene fragment that contains Xba I and Sac I restriction enzyme site with primer GDof1-Xba and GhDof1-Sac amplification; With restriction enzyme Xba I and PCR product corresponding to Sac I double digestion, the GhDof1 gene fragment after enzyme is cut and the pBI121 carrier (Chinese plasmid vector strain cell pnca gene preservation center, the product article No. that pass through same double digestion; Biovector008-32) skeleton fragment connects.To connecting product, transform, shake bacterium, picking plasmid, Xba I and SacI double digestion enzyme are cut evaluation.By cut evaluation through enzyme, show to contain the recombinant plasmid sample presentation order-checking that size is about the object band (Fig. 5) of 1077bp.Recombinant plasmid called after recombinant expression vector pBI-Dof1 containing the GhDof1 gene shown in the 95-1153 position Nucleotide of sequence 3 in ordered list will be shown through order-checking.

GhDof1-Xba:5'-GC tCTAGAthe recognition sequence that GCGTAATGCAAGACATCCACT-3'(underscore place is XbaI, 11-29 position is the 95-113 position of sequence 3)

GhDof1-Sac:5'-C gAGCTCgCAGTTAAATCCTGCCTTAAG-3'(underscore place is the recognition sequence of Sac I, and 9-28 position is the 1134-1153 position of sequence 3)

Two, turn the acquisition of GhDof1 gene Arabidopis thaliana

1, recombinant expression vector pBI121-Dof1 arabidopsis thaliana transformation

The recombinant expression vector pBI121-Dof1 that above-mentioned steps one is obtained is converted into Agrobacterium EHA105(China's plasmid vector strain cell pnca gene preservation center, product article No. Biovector008-5 through heat shock method).Picking mono-clonal carries out bacterium colony PCR and identifies positive colony, and the primer is GhDof1-Xba and the GhDof1-Sac described in step 1, and positive colony can increase and obtain the object band (Fig. 6) of size for 1077bp.

By the Agrobacterium called after EHA105/pBI121-Dof1 that shows to contain recombinant expression vector pBI121-Dof1 through above-mentioned evaluation; The Agrobacterium called after EHA105/pBI121 of pBI121 empty carrier will be proceeded to.

Adopt " flower-dipping method " (Gao Jianqiang, Liang Hua, Zhao Jun. Genetic Transformation in Higher Plants Agrobacterium flower-dipping method progress. Chinese agronomy circular, 2010,26 (16): 22-25) (Col type Arabidopis thaliana is bought from the registration of Tair website Agrobacterium EHA105/pBI121-Dof1 and EHA105/pBI121 to be converted into respectively to Col type Arabidopis thaliana, tair accession number: SpeciesVariant:90) in, to ripe results seed (T ₁generation).

2, turn the evaluation of GhDof1 gene Arabidopis thaliana plant

The seed that turns GhDof1 gene Arabidopis thaliana plant that above-mentioned steps 1 is gathered in the crops and proceed to the seed (T of Arabidopis thaliana plant of EHA105/pBI121 ₁generation) through MS screening culture medium (Kan that interpolation final concentration is 50mg/L) screening, obtain resistant plant (T ₁generation), after transplanting and planting, get leaf sample and extract DNA as template, with primer 5'-CACCAACCCTGATCAAT-3 ' and 5'-CATCGCAAGACCGGCAAC-3 ', carry out pcr amplification kantlex marker gene hpt II, identify positive plant.Meanwhile, not genetically modified wild-type Arabidopis thaliana is set as negative control, recombinant expression vector pBI-Dof1 is set as positive control.Experiment in triplicate.

Result as shown in Figure 7, turns the expression that goal gene hpt II all can be detected GhDof1 gene resistant plant blade from major part, for detecting the positive GhDof1 gene resistant plant that turns, illustrates that GhDof1 gene is successfully converted into Arabidopis thaliana.Also positive to proceeding to the detected result of Arabidopis thaliana plant of EHA105/pBI121.

Three, turn the evaluation of GhDof1 gene Arabidopis thaliana plant phenotype and the detection of seed oil-containing situation

1, turn the evaluation of GhDof1 gene Arabidopis thaliana plant phenotype

To be accredited as positive T through above-mentioned steps two ₁the Arabidopis thaliana plant that generation turns GhDof1 gene Arabidopis thaliana plant or proceeds to EHA105/pBI121 is after selfing, to T ₂for plant, carry out kalamycin resistance Screening and Identification, anti-sense, is carried out through selfing these materials as follow-up study material than the strain that meets 3:1, obtains T ₃for isozygotying transfer-gen plant and seed.To T ₃the growth phenotype (as leaf color, root length etc.) that turns GhDof1 gene strain or proceed to the Arabidopis thaliana plant of EHA105/pBI121 for isozygotying is observed.With not genetically modified wild-type (Col type), Arabidopis thaliana plant seed in contrast simultaneously.Experiment in triplicate, is chosen individual plant at every turn and is carried out.

As shown in Figure 8, compared with the control, part turns GhDof1 gene Arabidopis thaliana plant leaf and occurs yellow in various degree result, but root is long, is not affected.The result shows that the expression of GhDof1 gene is subject to some impacts to the growth in acceptor material seedling stage.The Arabidopis thaliana plant detected result that proceeds to EHA105/pBI121 is consistent with not genetically modified wild-type (Col type) Arabidopis thaliana plant.

2, turn the detection of GhDof1 gene Arabidopis thaliana plant seed oil-containing situation

The different strain T that obtain with gas chromatography determination above-mentioned steps 1 ₃in generation, turns the fatty acid content of GhDof1 gene Arabidopis thaliana plant seed.Concrete operation method is as follows:

Get and turn GhDof1 gene Arabidopis thaliana plant T ₃for 1000, seed, dry 60 hours, use mortar grind into powder for 45 ℃.Each sample takes 0.01～0.02g powder puts into the cell culture tube of 15ml, add 0.4ml anhydrous methanol with transfer pipet: the mixed solution of chloracetyl (volume ratio is 10: 1), 0.5ml concentration is about the interior mark (Nonadecanoic acid methylester of 1mg/ml, purchased from the lark waffle company limited that learns a skill, article No.: SFA-013N), vibration mixes, 80 ℃ of water-baths 2 hours.Water-bath finishes rear taking-up, cool to room temperature, adding 0.5ml concentration is 7%(7g/100ml) K ₂cO ₃in the aqueous solution and lipid acid.Draw supernatant in sample bottle, put 4 ℃ of refrigerators and treat machine in gas-chromatography.GC conditions is as follows: crosslinked quartz capillary column, and PEG-20M is stationary phase, nitrogen is carrier gas, constant current flow velocity 1.2ml/min, 200 ℃ of column temperatures, 350 ℃ of detected temperatures, fid detector.The Arabidopis thaliana plant that proceeds to empty carrier pBI121 is set simultaneously, and not genetically modified wild-type Arabidopis thaliana plant in contrast.Test in triplicate results averaged.

Detected result is as shown in Fig. 9 and table 2, and compared with not genetically modified wild-type Arabidopis thaliana plant (Col), the fatty acid content that part turns GhDof1 gene Arabidopis thaliana strain significantly improves.The detected result of Arabidopis thaliana plant that proceeds to empty carrier pBI121 is consistent with not genetically modified wild-type Arabidopis thaliana plant (Col), without significant difference.

Table 2 turns GhDof1 gene Arabidopis thaliana plant T ₃for Fatty Acids in Seeds content detection result

	Col	D1-2	D1-9	D2-6	D2-8	D3-2
							Repeat 1	0.215	0.259	0.226	0.273	0.245	0.279
Repeat 2	0.251	0.288	0.261	0.276	0.279	0.274
							Repeat 3	0.245	0.272	0.286	0.254	0.222	0.311

Mean+SD

0.237±0.019

0.273±

0.258±0.020

0.268±0.012

0.249±0.019

0.288±0.015

Note: Col represents not genetically modified wild-type Arabidopis thaliana plant contrast; D1-2, D1-9, D2-6, D2-8 and D3-2 represent respectively 5 T ₃in generation, turns GhDof1 gene Arabidopis thaliana strain.

Claims

1. protein, for following (c) or (d):

2. the nucleic acid molecule of protein described in coding claim 1.

3. nucleic acid molecule according to claim 2, is characterized in that: described gene is following 4)-8) in any:

5) DNA molecular shown in sequence 1 in sequence table;

6) DNA molecular shown in the 481-948 position of sequence 1 in sequence table;

7) DNA molecular shown in sequence 3 in sequence table;

8) DNA molecular shown in the 95-1153 position of sequence 3 in sequence table.

4. contain recombinant vectors, expression cassette or the recombinant bacterium of nucleic acid molecule described in claim 2 or 3.

5. protein claimed in claim 1, or the nucleic acid molecule described in claim 2 or 3, or recombinant expression vector claimed in claim 4, expression cassette or recombinant bacterium are at following a1) or a2) in application:

A1) regulating plant seed fat acid content;

A2) the high plant variety of seed selection seed fat acid content;

Described plant is cotton or Arabidopis thaliana.

6. cultivate a method for the transgenic plant of seed fat acid content raising, comprise the gene of protein described in coding claim 1 is imported to the step in object plant; Described transgenic plant are compared with described object plant, and seed fat acid content improves;

Described plant is cotton or Arabidopis thaliana.

Described in claim 1 protein in the application as in transcription factor.