CN100455666C

CN100455666C - Synergistic control Gene of AtbHLH29 for plant ferro element absorb and its coded protein and uses

Info

Publication number: CN100455666C
Application number: CNB2006100121942A
Authority: CN
Inventors: 袁有喜; 凌宏清
Original assignee: Institute of Genetics and Developmental Biology of CAS
Current assignee: Institute of Genetics and Developmental Biology of CAS
Priority date: 2006-06-09
Filing date: 2006-06-09
Publication date: 2009-01-28
Anticipated expiration: 2026-06-09
Also published as: CN1884538A

Abstract

The present invention discloses a gene which is coordinated with AtbHLH29 to adjust and control the ferrum element absorption and the metabolism of a plant, a coding protein thereof and the application thereof. The aim of the present invention is to provide a gene coordinated with AtbHLH29 to adjust and control the ferrum element absorption and the metabolism of a plant, a coding protein thereof, and the application thereof on the aspects of the adjustment and the control of the ferrum element absorption and the metabolism of a plant. The cDNA of the gene is one of the following nucleotide sequences: 1. the DNA sequences of SEQ ID No. 1 and SEQ ID No. 4 in a sequence table, 2. the DNA sequences of SEQ ID No. 3 and SEQ ID No. 6 in a coding sequence table, 3. a nucleotide sequence which has homology of more than 90% with a first sequence or the nucleotide sequence limited by the SEQ ID No. 4 in the sequence table and performs an important function on the aspects of the adjustment and the control of the ferrum element absorption and the metabolism of a plant, and 4. a nucleotide sequence which can cross with a DNA sequence limited by the SEQ ID No. 1 or the SEQ ID No. 4 in the sequence table under a highly strict condition.

Description

Gene and proteins encoded and application with the absorption of AtbHLH29 coordinated regulation plant ferro element

Technical field

The present invention relates to plant gene and proteins encoded thereof and application, particularly relate to two and absorb with AtbHLH29 acting in conjunction coordinated regulation plant ferro element and metabolic gene and proteins encoded thereof are being regulated and control plant to the application in ferro element absorption and the metabolism with it.

Background technology

Ferro element participates in the most basic biological process in the vital movement as a lot of important enzymes and proteinic moiety, as breathing and photosynthesis etc.Baby's iron deficiency can cause anemia, and the intelligence and the physical efficiency that have a strong impact on the baby are grown.By the iron level in the biological method raising agricultural-food and the biological effectiveness of iron, be most economical, the effective and persistent method of improving human iron nutrition.In agriculture production, iron also is one of main factor of restriction crop growthing development.The total content of iron is generally very high in the soil, but most iron all exists with the form of trivalent ferric oxide, and in neutral and alkaline soil, ferric solubility is extremely low, can not be by plant absorbing and utilization.In the long-term evolution process, plant is in order to satisfy the needs that it grows, evolve out two activation and absorb the efficient approach of iron, be called strategy I and strategy II (Ion uptake mechanisms of individual cells and roots:in Mineral nutrition of higher plants, ed by Marschner (second edition), Academic Press, New York, 1995,6-78).Grass belongs to strategy II plant, and the plant except that Gramineae all belongs to strategy I plant.Clone the regulation and control plant and will open up a new approach for the iron level that improves in the plant with biotechnological means ferro element absorption and metabolic key gene.

AtbHLH29 (GenBank number: NM_102582/NP_174138) be the homologous gene of the tomato FER gene that before from the arabidopsis gene group, identified of the present inventor, participate in the regulation and control plant ferro element and absorb and metabolism.The disappearance of FER and AtbHLH29 gene, the mutant plant can not start the physiological compatibility reaction that whole iron deficiency coerces and obtain ferro element effectively from soil, show yellow, deadly (The tomato FER gene encoding a bHLHprotein controls iron-uptake responses in roots by Ling H-Q, Bauer P, BereczkyZ, Keller B and Ganal M, Proc Natl Acad Sci USA 99,13938-43,2002; The essentialbasic helix-loop-helix protein FIT1 is required for the iron deficiencyresponse by Colangelo PE and Guerinot ML, Plant Cell 16,3400-3412,2004).Arabidopis thaliana AtbHLH29 gene transformation is arrived the deletion mutant T3238fer of tomato FER gene by agrobacterium mediation method, can recover the disappearance function of FER, transfer-gen plant starts whole iron deficiency physiological compatibility reaction and absorbs relevant expression of gene with iron when low iron is coerced, thereby the iron level in the rotaring gene plant blade has returned to normal level (AtbHLH29 of Arabidopsis thaliana is a functional ortholog of tomato FERinvolved in controlling iron acquisition in strategy I plants by Yuan YX, ZhangJ, Wang DW and Ling H-Q, Cell Research 15,613-621,2005).

Summary of the invention

The purpose of this invention is to provide two and absorption of AtbHLH29 acting in conjunction coordinated regulation plant ferro element and metabolic gene.

Provided by the present invention and AtbHLH29 coordinated regulation plant ferro element absorbs and metabolic gene, and name is called AtbHLH38 and AtbHLH39, derives from Arabidopsis Arabidopis thaliana (Arabidopsis thaliana), and its cDNA is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 1 and SEQ ID №: 4 are respectively the encoding sequence of AtbHLH38 and AtbHLH39 gene;

2) SEQ ID № in the code sequence tabulation: 3 and SEQ ID №: 6 dna sequence dna;

3) with sequence table in sequence 1 or SEQ ID №: the nucleotide sequence that 4 nucleotide sequences that limit have 90% above homology and play an important role in absorption of regulation and control plant ferro element and metabolism;

4) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of 1 or SEQ ID №: the 4 dna sequence dnas hybridization that limit.

The rigorous condition of described height be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridization and wash film under 65 ℃ of conditions.

SEQ ID № in the sequence table: 1 by 762 based compositions, its encoding sequence is from 5 ' end 1-762 bit base, coding has SEQ ID № in the sequence table: the protein of 3 amino acid residue sequence, from 5 ' end 202-369 bit base coding bHLH conserved domain, will have SEQ ID №: this unnamed gene of 1 cDNA sequence is AtbHLH38; SEQ ID № in the sequence table: 4 by 777 based compositions, its encoding sequence is from 5 ' end 1-777 bit base, coding has SEQ ID № in the sequence table: the protein of 6 amino acid residue sequence, from 5 ' end 217-384 bit base coding bHLH conserved domain, will have SEQ ID №: this unnamed gene of 4 cDNA sequences is AtbHLH39.

Its genome sequence is one of following nucleotide sequence:

1) SEQ ID № in the sequence table: 2 and SEQ ID №: 5 are respectively the genome sequence of AtbHLH38 and AtbHLH39;

2) with sequence table in sequence 2 or SEQ ID №: the nucleotide sequence that 5 nucleotide sequences that limit have 90% above homology and play an important role in absorption of regulation and control plant ferro element and metabolism;

3) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of 2 or SEQ ID №: the 5 dna sequence dnas hybridization that limit.

SEQ ID № in the sequence table: 2 by 985 based compositions, from 5 ' end 1-312 bit base is first exon of this genomic gene, from 5 ' end 313-441 bit base is first intron of this genomic gene, from 5 ' end 442-891 bit base is second exon of this genomic gene, from 5 ' end 1-3 bit base is the initiator codon ATG of this genomic gene, from 5 ' end 889-891 bit base is the terminator codon TAG of this genomic gene, from 5 ' end 202-498 bit base coding bHLH conserved domain; SEQ ID № in the sequence table: 5 by 949 based compositions, from 5 ' end 1-327 bit base is first exon of this genomic gene, from 5 ' end 328-433 bit base is first intron of this genomic gene, from 5 ' end 434-883 bit base is second exon of this genomic gene, from 5 ' end 1-3 bit base is the initiator codon ATG of this genomic gene, from 5 ' end 881-883 bit base is the terminator codon TGA of this genomic gene, from 5 ' end 217-490 bit base coding bHLH conserved domain.

The present invention and AtbHLH29 coordinated regulation plant ferro element absorb and metabolic gene encoded protein (AtbHLH38 and AtbHLH39), have one of following amino acid residue sequences:

1) the SEQ ID № in the sequence table: 3 and SEQ ID №: 6 are respectively AtbHLH38 and the proteic amino acid residue sequence of AtbHLH39;

2) with SEQ ID № in the sequence table: 3 or SEQ ID №: 6 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have and absorb with AtbHLH29 acting in conjunction regulation and control plant ferro element and the protein of metabolic function.

SEQ ID № in the sequence table: 3 is the aminoacid sequence of AtbHLH38, forms by 253 amino-acid residues, and be the bHLH conserved domain from aminoterminal (N end) 68-123 amino acids residue; SEQ ID № in the sequence table: 6 is the aminoacid sequence of AtbHLH39, forms by 258 amino-acid residues, and be the bHLH conserved domain from aminoterminal 73-128 amino acids residue.

Contain expression carrier of the present invention, transgenic cell line and host bacterium and all belong to protection scope of the present invention.

Arbitrary segmental primer is to also within protection scope of the present invention among the amplification AtbHLH38/AtbHLH39.

Another object of the present invention provides a kind of regulation and control plant ferro element is absorbed and metabolic method.

Regulation and control plant provided by the present invention absorbs and metabolic method ferro element, be with AtbHLH29 and ferro element is absorbed for described and AtbHLH29 coordinated regulation plant and metabolic gene A tbHLH38/AtbHLH39 imports plant tissue or cell, plant obtains to the absorption and the metabolism of ferro element.

Described AtbHLH29 and AtbHLH38/AtbHLH39 can be by containing AtbHLH29, AtbHLH38/AtbHLH39 respectively, or the plant expression vector that contains AtbHLH29 and AtbHLH38/AtbHLH39 imports explant; The carrier that sets out that is used to make up described plant expression vector can be any one double base agrobacterium vector or can be used for carrier of plant micropellet bombardment etc., as pBinplus (VAN ENGELEN, F.A., et al., 1995 pBINPLUS:an improved plant transformation vector based on pBIN19.Transg.Res.4:288-290), pBI121, pBin19, pCAMBIA2301, pCAMBIA1300 or other plant expression vector of deriving.

When using AtbHLH29 and AtbHLH38/AtbHLH39 to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type, composing type, organizing specific type or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, general living plain gene Ubiquitin promotor (pUbi) and rice actin gene promoter (Actin) etc., they can use separately or be used in combination with other plant promoter; In addition, when using gene constructed plant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhanser zones 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 be synthetic.Translation initiation region can be from transcription initiation zone or structure gene.

For the ease of transgenic plant cells or plant being identified and screening, can process used plant expression vector, can produce the enzyme of colour-change or the gene of luminophor (gus gene, GFP gene, luciferase genes etc.) as adding the coding that in plant, to express, have the antibiotic marker thing (gentamicin marker, kantlex marker etc.) of resistance or anti-chemical reagent marker gene (as anti-herbicide gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.

Be the carrier that sets out with pBI121, the plant expression vector that contains AtbHLH38 of structure is pBI-38, and the plant expression vector that contains AtbHLH39 is pBI-39; Be the carrier that sets out with pBinplus, the plant expression vector that contains AtbHLH29 of structure is pBinplus-29.

Carry AtbHLH29, AtbHLH38, AtbHLH39 respectively, or carry AtbHLH29 and AtbHLH38/AtbHLH39 plant expression vector can Ti-plasmids, Ri plasmid, plant viral vector, directly DNA conversion, microinjection, electricity be led, conventional biological method transformed plant cells or tissue such as agriculture bacillus mediated by using, and the plant transformed cell or tissue is cultivated into plant.

Ferro element is absorbed above-mentioned regulation and control plant and metabolic method all is suitable for all plants, both has been applicable to monocotyledonss such as corn, paddy rice, wheat, also is applicable to dicotyledonss such as Arabidopis thaliana, tobacco, cotton.

The invention provides one and the absorption of AtbHLH29 acting in conjunction coordinated regulation plant ferro element and metabolic gene A tbHLH38 and AtbHLH39 and proteins encoded thereof.Transgenic experiments shows that AtbHLH38 and AtbHLH39 and AtbHLH29 acting in conjunction on protein level can significantly improve the receptivity of plant to the Fe element.Anti-low iron ability and ferro element utilising efficiency that this gene pairs improves plant have important theory and practical significance, and provide an economy, approach fast and effectively for the biological restoration (Phytoremediation) of biological reinforced (Biofortification) of iron level in the agricultural-food and heavy-metal contaminated soil.The present invention has wide application and market outlook in agricultural and biological restoration field.

The present invention will be further described below in conjunction with specific embodiment.

Description of drawings

Fig. 1 is the quantitative PCR detection result of AtbHLH38 and AtbHLH39 expression under the iron deficiency condition

Fig. 2 detects AtbHLH38 and AtbHLH39 and AtbHLH29 to ferric iron reductase gene AtFRO2 and the GUS histochemical stain of ferrous ion transporter gene AtIRT1 transcriptional control effect and the enzyme assay result of gus protein in yeast cell

Fig. 3 is AtbHLH38, the growing state of the single, double overexpression plant of AtbHLH39 and AtbHLH29 after low iron is coerced processing

Fig. 4 for normal growth and after low iron is coerced processing AtbHLH38 and AtbHLH39 single overexpression plant and with the measurement result of two overexpression plant overground part blade iron levels of AtbHLH29

Fig. 5 is the RT-PCR detected result that single, double overexpression AtbHLH38/AtbHLH39 and AtbHLH29 improve ferric iron reductase gene AtFRO2 and ferrous ion transporter gene AtIRT1 transcriptional level

Fig. 6 A is the part physical map of pBI-38 and pBI-39

Fig. 6 B is the part physical map of pBinplus-29

Embodiment

Method therefor is ordinary method if no special instructions among the following embodiment, and the primer synthesizes and examining order is finished by Beijing gram difficult to understand biotechnology limited liability company.

Embodiment 1, absorb with AtbHLH29 coordinated regulation plant ferro element and the clone of metabolic gene A tbHLH38 and AtbHLH39

The encoding sequence with absorption of AtbHLH29 coordinated regulation plant ferro element and metabolic gene (called after AtbHLH38 and AtbHLH39 respectively) according to arabidopsis thaliana genomic dna sequence and prediction designs two pairs of primers (P38 and P39), and primer sequence is as follows:

Primer is to P38:

P38F (upstream primer): 5 '-TCTAGAATGTGTGCATTAGTCCCTTCA-3 '

P38R (downstream primer): 5 '-CCCGGGTTAAACGAGTTTTCACATTT-3 ';

Primer is to P39:

P39F (upstream primer): 5 '-TCTAGAATGTGTGCATTAGTACCTCCA-3 '

P39R (downstream primer): 5 '-CCCGGGATATATGAGTTTCCACATTC-3 '

The Arabidopis thaliana seedling placed on the MS substratum that does not contain ferro element iron deficiency to handle 4 days, extract the total RNA that handles seedling through iron deficiency then, with synthetic its cDNA of reverse transcription method, and be template with this cDNA, carry out pcr amplification at primer under to the guiding of P38 and P39 respectively, the PCR reaction conditions is: earlier 95 ℃ 2 minutes; Then 95 ℃ 1 minute, 50 ℃ 1 minute, 72 ℃ 1 minute, totally 35 circulations; Last 72 ℃ 5 minutes.After reaction finishes, the PCR product is carried out 1.0% agarose gel electrophoresis to be detected, the result has amplified the band of 770bp and 785bp under to the guiding of P38 and P39 respectively at primer, reclaim two purpose segments, and it is carried out purifying with DNA purification kit (Phamacia company), purpose segment with purifying is cloned into respectively among the carrier pGEM T-easy (Promega company) then, to contain primer to the P38 pulsating recombinant vectors called after pGEM T-easy/AtbHLH38 that increases, to contain primer to the P39 pulsating recombinant vectors called after pGEM T-easy/AtbHLH39 that increases, then pGEMT-easy/AtbHLH38 and pGEM T-easy/AtbHLH39 be used CaCl respectively ₂Method transformed into escherichia coli DH5a competent cell, the screening positive monoclonal, the upgrading grain, order-checking, sequencing result shows that primer has SEQ ID № in the sequence table to P38 amplification segment: 1 nucleotide sequence, SEQ ID № in the sequence table: 1 by 762 based compositions, its encoding sequence is from 5 ' end 1-762 bit base, coding has SEQ ID № in the sequence table: the protein of 3 amino acid residue sequence, from 5 ' end 202-369 bit base coding bHLH conserved domain, will have SEQ ID №: the unnamed gene of 1 dna sequence dna is AtbHLH38; Primer has SEQ ID № in the sequence table to P39 amplification segment: 4 nucleotide sequence, SEQ ID № in the sequence table: 4 by 777 based compositions, its encoding sequence is from 5 ' end 1-777 bit base, coding has SEQ ID № in the sequence table: the protein of 6 amino acid residue sequence, from 5 ' end 217-384 bit base coding bHLH conserved domain, will have SEQ ID №: the unnamed gene of 4 dna sequence dnas is AtbHLH39.

Obtained the genomic dna sequence of AtbHLH38 and AtbHLH39 according to the genomic dna sequence of the AtbHLH38 of amplification and the cDNA sequence of AtbHLH39 and Arabidopis thaliana, the genomic dna sequence of AtbHLH38 has SEQ ID № in the sequence table: 2 nucleotide sequence, SEQ ID № in the sequence table: 2 by 985 based compositions, from 5 ' end 1-312 bit base is first exon of this genomic gene, from 5 ' end 313-441 bit base is first intron of this genomic gene, from 5 ' end 442-891 bit base is second exon of this genomic gene, from 5 ' end 1-3 bit base is the initiator codon ATG of this genomic gene, from 5 ' end 889-891 bit base is the terminator codon TAG of this genomic gene, from 5 ' end 202-498 bit base coding bHLH conserved domain; The genomic dna sequence of AtbHLH39 has SEQ ID № in the sequence table: 5 nucleotide sequence, SEQ ID № in the sequence table: 5 by 949 based compositions, from 5 ' end 1-327 bit base is first exon of this genomic gene, from 5 ' end 328-433 bit base is first intron of this genomic gene, from 5 ' end 434-883 bit base is second exon of this genomic gene, from 5 ' end 1-3 bit base is the initiator codon ATG of this genomic gene, from 5 ' end 881-883 bit base is the terminator codon TGA of this genomic gene, from 5 ' end 217-490 bit base coding bHLH conserved domain.

Embodiment 2, quantitative PCR detection AtbHLH38 and the AtbHLH39 expression under the iron deficiency condition

The Arabidopis thaliana seed is placed MS substratum (sigma, USA) make its sprouting (1 week) on, then seedling is placed not iron content (Fe) respectively, manganese (Mn), lacking coercing of different metal nutritive element on the MS substratum of zinc (Zn) element handled 4 days, being contrast without the plant of coercing processing, extract respectively then through coercing the root of handling seedling and contrast and overground part total RNA and as template, the expression of quantitative PCR detection AtbHLH38 under the guiding of primer 1:5 '-GACGGTACCACAGACTTATGAAGT-3 ' and primer 2: 5 '-TAAGCTCTTTGAAACCGTTTCAGGA-3 ', the expression of quantitative PCR detection AtbHLH39 under the guiding of primer 3:5 '-GACTTATGGAGCTGTTACAGCGGT-3 ' and primer 4:5 '-CTTCAAGCTTCGAGAAACCGTCGCA-3 ', the expression of quantitative PCR detection ferric iron reductase gene AtFRO2 (GenBank NM_100040/NP_171664) under the guiding of primer 5:5 '-GATCGAAAAAAGCAATAACGGTGGTT-3 ' and primer 6:5 '-GATGTGGCAACCACTTGGTTCGATA-3 ', quantitative PCR detection zinc transporter gene ZIP5 is (GenBank number: expression NP_973762) under the guiding of primer 7:5 '-GAGTTTCCGTTCACAGGCTTTATC-3 ' and primer 8:5 '-TTCCACGCCAACAATCCCGT-3 ', detected result is the (A among Fig. 1 as shown in Figure 1, B, C, D figure is respectively contrast and coerces AtbHLH38 under the disposition with different, AtbHLH39, AtFRO2, ZIP5 is at the expression of root and overground part), show that AtbHLH38 and AtbHLH39 are induced by iron deficiency all in the expression of root and overground part, up-regulated expression under the iron deficiency situation, and be not subjected to the influence of other metallic element (as manganese and zinc).

Embodiment 3, detection AtbHLH38/AtbHLH39 and AtbHLH29 are to the transcriptional control effect of ferric iron reductase gene AtFRO2 and ferrous ion transporter gene AtIRT1

One, detects the interactional yeast two-hybrid experiment of AtbHLH38/AtbHLH39 and AtbHLH29

Whether interact with yeast two-hybrid experiment detection AtbHLH38/AtbHLH39 and AtbHLH29.Operate with the yeast two-hybrid detection kit of Strategene company and by reference reagent box specification sheets, respectively AtbHLH38/AtbHLH39 and AtbHLH29 are cloned into two Yeast expression carrier pBD-GAL4 Cam and pAD-GAL4-2.1, again by the two transformed yeast bacterial strain YRG-2 of thermal shock.According to the yeast two-hybrid principle, if two genes can take place to do mutually, change yeast strain can be on the substratum that does not contain Histidine normal growth, and can detect the X-GAL enzymic activity, otherwise bacterial strain can not normal growth, also detects the enzymic activity less than X-GAL.Detected the Histidine that X-GAL activity that reporter gene lacZ expresses and HIS3 express in positive colony, or else positive strain can contain normal growth on the substratum of Histidine. show that AtbHLH38 and AtbHLH39 can interact with AtbHLH29 on protein level.

Two, in yeast cell, detect the mutual transcriptional control effect of doing ferric iron reductase gene AtFRO2 and ferrous ion transporter gene AtIRT1 of AtbHLH38/AtbHLH39 and AtbHLH29

In view of the AtbHLH38/AtbHLH39 proteins encoded all contains the bHLH conserved domain, and this gene is subjected to low iron abduction delivering, infer that AtbHLH38/AtbHLH39 may be a transcription factor, the regulation and control that involved in plant absorbs ferro element, in addition, the yeast two-hybrid result shows that AtbHLH38 and AtbHLH39 can interact with AtbHLH29 on protein level, therefore, infer that AtbHLH38 and AtbHLH39 can form heterodimer with AtbHLH29 respectively mutually, the expression of regulation and control downstream functional gene is as ferric iron reductase gene AtFRO2 and ferrous ion transporter gene AtIRT1.Verify with following method: 1) with pBD-GAL4 Cam and pAD-GAL4-2.1 (Stategene, USA) be the carrier that sets out, make up and contain AtbHLH38 respectively, AtbHLH39, AtbHLH29, AtbHLH38+AtbHLH29, the recombinant yeast expression vector of AtbHLH39+AtbHLH29 encoding sequence, the position of said gene in Yeast expression carrier is between the EcoRI and SalI restriction enzyme site of multiple clone site, then with above-mentioned recombinant yeast expression vector transformed yeast cell YRG-2 (Strategene, USA), carried the recombinant yeast cell of said gene respectively; 2) (AtFRO2 initiator codon ATG is to the 947bp of upstream, this promotor called after P with the promoter sequence of ferric iron reductase gene AtFRO2 _FRO2) and the promoter sequence of ferrous ion transporter gene AtIRT1 (AtIRT1 upstream from start codon-18bp is to-1598bp, called after P _IRT1) (GenBank number: AAL92040) merge, promoter sequence is positioned at the upstream of GUS, then with two kinds of fusion gene (P with the dna sequence dna of reporter gene GUS respectively _FRO2:: GUS and P _IRT1:: GUS) be cloned into Yeast expression carrier pBD-GAL4 Cam and pBD-GAL4-AtbHLH29 Cam (Stategene respectively, USA) between the PmacI restriction enzyme site, the recombinant yeast expression vector that is contained above-mentioned fusion genes respectively, and then two kinds of recombinant yeast expression vectors that carry fusion genes are transferred to respectively in the recombinant yeast cell that carries different genes combination (AtbHLH38, AtbHLH39, AtbHLH29, AtbHLH38+AtbHLH29, AtbHLH39+AtbHLH29) that step 1) obtains; 3) to step 2) recombinant yeast cell that obtains carries out the GUS histochemical stain and measures the enzymic activity of gus protein.(A, B figure are the GUS histochemical stain result of yeast cell: 1.pAD-WT+pBD-WT-P to the result as shown in Figure 2 _FRO2:: GUS; 2.pAD-WT+pBD-AtbHLH29-P _FRO2:: GUS; 3.pAD-AtbHLH38+pBD-WT-P _FRO2:: GUS; 4.pAD-AtbHLH38+pBD-AtbHLH29-P _FRO2:: GUS; 5.pAD-AtbHLH39+pBD-WT-P _FRO2:: GUS; 6.pAD-AtbHLH39+pBD-AtbHLH29-P _FRO2:: GUS; 7.pGAL4+pBD-AtbHLH29-P _FRO2:: GUS; 8.pAD-AtbHLH40+pBD-WT-P _FRO2:: GUS; 9.pAD-WT+pBD-WT-P _IRT1:: GUS; 10.pAD-WT+pBD-AtbHLH29-P _IRT1:: GUS; 11.pAD-AtbHLH38+pBD-WT-P _IRT1:: GUS; 12.pAD-AtbHLH38+pBD-AtbHLH29-P _IRT1:: GUS; 13.pAD-AtbHLH39+pBD-WT-P _IRT1:: GUS; 14.pAD-AtbHLH39+pBD-AtbHLH29-P _IRT1:: GUS; 15.pGAL4+pBD-AtbHLH29-P _IRT1:: GUS; 16.pAD-AtbHLH40+pBD-WT-P _IRT1:: GUS.C, D figure are the enzyme assay result of gus protein, and * represents to have significant difference with other bacterial strain), AtbHLH29, AtbHLH38 and AtbHLH39 single expression can not start P in yeast cell _FRO2:: GUS and P _IRT1:: GUS transcribes, when AtbHLH29 and AtbHLH38 or AtbHLH39 can start P during co expression in yeast cell _FRO2:: GUS and P _IRT1:: the expression of GUS.

The acquisition of embodiment 4, transgenic arabidopsis and the functional verification of gene experiment

With the Arabidopis thaliana is example, and gene of the present invention is carried out the functional verification experiment, and concrete experimental technique is as follows:

One, changes the acquisition of AtbHLH38 and AtbHLH39 single-gene Arabidopis thaliana

Separate recombinant plasmid pGEM T-easy/AtbHLH38 that AtbHLH38 and AtbHLH39 is made up from embodiment 1 respectively with restriction enzyme Xba I and Sma I and the pGEM T-easy/AtbHLH39, again two segments are cloned into the overexpression carrier that obtains AtbHLH38 and AtbHLH39 between the Xba I of the carrier pBI121 that contains 35S enhancement type promotor and the Sma I restriction enzyme site respectively, difference called after pBI121-38 and pBI121-39, the part physical map as shown in Figure 6A.Then recombinant vectors pBI121-38 and pBI121-39 are imported respectively among the Agrobacterium GV3101 with the heat shock method,, obtain to change respectively the transfer-gen plant that AtbHLH38 and AtbHLH39 are arranged, respectively called after AT38 and AT39 again with invading colored method arabidopsis thaliana transformation.

Two, change the acquisition of AtbHLH38/AtbHLH39 and the dual-gene Arabidopis thaliana of AtbHLH29

What obtain cDNA through reverse transcription with the total RNA of Arabidopis thaliana is template, under the guiding of primer P9 (5 '-cacccatggaaggaagagtcaac-3 ') and primer P10 (5 '-ttagtcgacctagtaaatgacttgatg-3 '), the cDNA sequence of pcr amplification AtbHLH29, then this segment is cloned between the Xba I and Sac I restriction enzyme site of pGEM T-easy carrier multiple clone site, obtain intermediate carrier, called after pGEMT-easy/AtbHLH29, it is checked order, sequencing result shows that clone's the cDNA sequence of AtbHLH29 is correct, with restriction enzyme Xba I and Sac I AtbHLH29 is separated from recombinant vectors pGEM T-easy/AtbHLH29, again it is cloned between the Nco I of the Agrobacterium-mediated Transformation carrier pBinplus that contains 35S enhancement type promotor and the Sal I restriction enzyme site and obtains the AtbHLH29 conversion carrier, called after pBinplus-29, its part physical map is shown in Fig. 6 B.Then this carrier is imported among the agrobacterium strains GV3101, AT38 and AT39 transfer-gen plant that difference step of converting one obtains, obtain carrying the dual-gene overexpression plant of AtbHLH29+AtbHLH38 and AtbHLH29+AtbHLH39, respectively called after AT38/29 and AT39/29.

Three, the anti-low iron tests of the single, double overexpression plant of AtbHLH38/AtbHLH39 and AtbHLH29

The AtbHLH38 that step 1 and step 2 are obtained and single overexpression plant AT38 of AtbHLH39 and AT39 and with two overexpression plant AT38/29 and the AT39/29 of AtbHLH29, be transplanted on the MS substratum that contains 10 μ M Fe-EDTA and under low iron bar spare, handled 10 days, with the plant that grows on the MS substratum is contrast, the growing state of each plant (WT is the wild-type contrast) as shown in Figure 3 under low iron bar spare, two overexpression plant AT38/29 of result and At39/29 are when low iron is coerced, growth is normal, and wild-type and single overexpression plant show iron deficiency chrysanthemum symptom.Simultaneously, measure to single overexpression plant of AtbHLH38 and AtbHLH39 and with the iron level of two overexpression plant overground part blades of AtbHLH29, (A figure is illustrated in the Fe content of each plant that grows on the normal MS substratum to measurement result as shown in Figure 4, B figure is illustrated in the Fe content of each plant that grows on the iron deficiency substratum, ☆ represents significance of difference α=0.05 level, ★ represents significance of difference α=0.01 level), the iron level that shows the blade of the two overexpression plant of AtbHLH38+AtbHLH29 and AtbHLH39+AtbHLH29 is significantly higher than wild-type and AtbHLH38, AtbHLH29, the single overexpression plant of AtbHLH39, and iron level of single overexpression plant and wild-type do not have significant difference.

Four, single, double overexpression AtbHLH38/AtbHLH39 and AtbHLH29 improve the confirmatory experiment of ferric iron reductase gene AtFRO2 and ferrous ion transporter gene AtIRT1 transcriptional level

The AtbHLH38 that step 1 and step 2 are obtained and single overexpression plant AT38 of AtbHLH39 and AT39 and place with the seed of the two overexpression plant AT38/29 of AtbHLH29 and AT39/29 and to make its germination on the MS substratum, then the seedling of 1 week size was transferred on the MS solid medium that contains 100 μ M concentration of iron and the iron-free substratum growth respectively 4 days, extract total RNA (is contrast with the Arabidopis thaliana plant that does not change any gene and handle under the same conditions) of above-mentioned transfer-gen plant root and overground part leaf more respectively, and be template with the different total RNA that is extracted, expression abundance with methods analyst AtbHLH38/AtbHLH39 and AtbHLH29 and the downstream functional gene (ferric iron reductase gene AtFRO2 and ferrous ion transporter gene AtIRT1) thereof of RT-PCR, under the guiding of primer P11:5 '-GACGGTACCACAGACTTATGAAGT-3 ' and primer P12:5 '-TAAGCTCTTTGAAACCGTTTCAGGA-3 ', pcr amplification AtbHLH38; Under the guiding of primer P13:5 '-GACTTATGGAGCTGTTACAGCGGT-3 ' and primer P14:5 '-CTTCAAGCTTCGAGAAACCGTCGCA-3 ', pcr amplification AtbHLH39; Under the guiding of primer P15:5 '-CAGTCACAAGCGAAGAAACTCA-3 ' and primer P16:5 '-CTTGTAAAGAGATGGAGCAACACC-3 ', pcr amplification AtbHLH29; At primer P17; Under the guiding of 5 '-GATCGAAAAAAGCAATAACGGTGGTT-3 ' and primer P18:5 '-GATGTGGCAACCACTTGGTTCGATA-3 ', pcr amplification ferric iron reductase gene AtFRO2 (GenBank NM_100040/NP_171664); Under the guiding of primer P19:5 '-GAATGTGGAAGCGAGTCAGCGA-3 ' and primer P20:5 '-GATCCCGGAGGCGAAACACTTA-3 ', pcr amplification ferrous ion transporter gene AtIRT1 (GenBank NM_118089/NP_567590); Under the guiding of primer P21:5 '-GCTGTTCGTGGTGTTGAGATGC-3 ' and primer P22:5 '-AGGCTCTGAGGTGAGGAAGTCT-3 ', pcr amplification EF1B-a (GenBank number: NM_121956).After reaction finishes, above-mentioned pcr amplification product is carried out 1% agarose gel electrophoresis to be detected, detected result is ("+" expression: containing under the 100 μ M concentration of iron as shown in Figure 5, "-" expression: under iron content situation not), the Arabidopis thaliana adjoining tree of WT for not changeing any gene and handling under the same conditions, AT38-18, AT39-17, AT29-3, AT38/29-2, AT39/29-12 is respectively single overexpression strain system of different AtbHLH38 and AtbHLH39 and with two overexpression strains of AtbHLH29 is, the transcriptional level comparison illumination that shows ferric iron reductase gene AtFRO2 in two overexpression plant and ferrous ion transporter gene AtIRT1 shows and improves, particularly under the competent condition of iron.

Sequence table

<160>6

<210>1

<211>762

<212>DNA

＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis thaliana)

<400>1

atgtgtgcat?tagtcccttc?atttttcaca?aacttcggtt?ggccgtcaac?gaatcaatac 60

gaaagctatt?acggtgccgg?agataaccta?aataacggca?catttcttga?attgacggta 120

ccacagactt?atgaagtgac?tcatcatcag?aatagcttgg?gagtatctgt?ttcgtcagaa 180

ggaaatgaga?tagacaacaa?tccggttgtg?gtcaagaagc?ttaatcacaa?tgctagtgaa 240

cgtgaccgac?gcaagaagat?caacactttg?ttctcatctc?tccgttcatg?tcttccagct 300

tctgatcaat?cgaagaagct?aagtattcct?gaaacggttt?caaagagctt?aaagtacata 360

ccagagctgc?aacagcaagt?gaagaggcta?atacaaaaga?aggaagaaat?tttggtacga 420

gtatcgggtc?aaagagactt?tgagctttac?gataagcagc?aaccaaaggc?ggtcgcgagt 480

tatctctcaa?cggtttctgc?cactaggctt?ggtgacaacg?aagtgatggt?ccaagtctca 540

tcgtccaaga?ttcataactt?ttcgatatca?aatgtgttgg?gtgggataga?agaagatggg 600

tttgttcttg?tggatgtttc?atcatcaaga?tctcaaggag?agaggctctt?ctacactttg 660

catcttcaag?tggagaatat?ggatgattac?aagattaatt?gcgaagaatt?aagtgaaagg 720

atgttgtact?tgtacgagaa?atgtgaaaac?tcgtttaact?ag 762

<210>2

<211>985

<212>DNA

＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis thaliana)

<400>2

atgtgtgcat?tagtcccttc?atttttcaca?aacttcggtt?ggccgtcaac?gaatcaatac 60

gaaagctatt?acggtgccgg?agataaccta?aataacggca?catttcttga?attgacggta 120

ccacagactt?atgaagtgac?tcatcatcag?aatagcttgg?gagtatctgt?ttcgtcagaa 180

ggaaatgaga?tagacaacaa?tccggttgtg?gtcaagaagc?ttaatcacaa?tgctagtgaa 240

cgtgaccgac?gcaagaagat?caacactttg?ttctcatctc?tccgttcatg?tcttccagct 300

tctgatcaat?cggtaagatc?gctagtagct?actctgatat?gtcatctaca?tctgtttcta 360

ataagctcgt?ctatacagct?atacaattct?aaagaagaac?accgagttaa?cccttatttc 420

tttttcttta?ctttcttgca?gaagaagcta?agtattcctg?aaacggtttc?aaagagctta 480

aagtacatac?cagagctgca?acagcaagtg?aagaggctaa?tacaaaagaa?ggaagaaatt 540

ttggtacgag?tatcgggtca?aagagacttt?gagctttacg?ataagcagca?accaaaggcg 600

gtcgcgagtt?atctctcaac?ggtttctgcc?actaggcttg?gtgacaacga?agtgatggtc 660

caagtctcat?cgtccaagat?tcataacttt?tcgatatcaa?atgtgttggg?tgggatagaa 720

gaagatgggt?ttgttcttgt?ggatgtttca?tcatcaagat?ctcaaggaga?gaggctcttc 780

tacactttgc?atcttcaagt?ggagaatatg?gatgattaca?agattaattg?cgaagaatta 840

agtgaaagga?tgttgtactt?gtacgagaaa?tgtgaaaact?cgtttaacta?ggtgactaat 900

tcatataatg?gtgtgtttat?ccactagttc?tcatttcttt?ttagctgtgt?ccttttctca 960

tatgaatcta?acactgatct?ggacc 985

<210>3

<211>253

<212>PRT

＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis thaliana)

<400>3

Met?Cys?Ala?Leu?Val?Pro?Ser?Phe?Phe?Thr?Asn?Phe?Gly?Trp?Pro?Ser

1 5 10 15

Thr?Asn?Gln?Tyr?Glu?Ser?Tyr?Tyr?Gly?Ala?Gly?Asp?Asn?Leu?Asn?Asn

20 25 30

Gly?Thr?Phe?Leu?Glu?Leu?Thr?Val?Pro?Gln?Thr?Tyr?Glu?Val?Thr?His

35 40 45

His?Gln?Asn?Ser?Leu?Gly?Val?Ser?Val?Ser?Ser?Glu?Gly?Asn?Glu?Ile

50 55 60

Asp?Asn?Asn?Pro?Val?Val?Val?Lys?Lys?Leu?Asn?His?Asn?Ala?Ser?Glu

65 70 75 80

Arg?Asp?Arg?Arg?Lys?Lys?Ile?Asn?Thr?Leu?Phe?Ser?Ser?Leu?Arg?Ser

85 90 95

Cys?Leu?Pro?Ala?Ser?Asp?Gln?Ser?Lys?Lys?Leu?Ser?Ile?Pro?Glu?Thr

100 105 110

Val?Ser?Lys?Ser?Leu?Lys?Tyr?Ile?Pro?Glu?Leu?Gln?Gln?Gln?Val?Lys

115 120 125

Arg?Leu?Ile?Gln?Lys?Lys?Glu?Glu?Ile?Leu?Val?Arg?Val?Ser?Gly?Gln

130 135 140

Arg?Asp?Phe?Glu?Leu?Tyr?Asp?Lys?Gln?Gln?Pro?Lys?Ala?Val?Ala?Ser

145 150 155 160

Tyr?Leu?Ser?Thr?Val?Ser?Ala?Thr?Arg?Leu?Gly?Asp?Asn?Glu?Val?Met

165 170 175

Val?Gln?Val?Ser?Ser?Ser?Lys?Ile?His?Asn?Phe?Ser?Ile?Ser?Asn?Val

180 185 190

Leu?Gly?Gly?Ile?Glu?Glu?Asp?Gly?Phe?Val?Leu?Val?Asp?Val?Ser?Ser

195 200 205

Ser?Arg?Ser?Gln?Gly?Glu?Arg?Leu?Phe?Tyr?Thr?Leu?His?Leu?Gln?Val

210 215 220

Glu?Asn?Met?Asp?Asp?Tyr?Lys?Ile?Asn?Cys?Glu?Glu?Leu?Ser?Glu?Arg

225 230 235 240

Met?Leu?Tyr?Leu?Tyr?Glu?Lys?Cys?Glu?Asn?Ser?Phe?Asn

245 250

<210>4

<211>777

<212>DNA

＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis thaliana)

<400>4

atgtgtgcat?tagtacctcc?attgtttcca?aactttgggt?ggccatcaac?gggagagtac 60

gacagctact?acctcgccgg?agatatcctc?aacaacggcg?ggtttcttga?ttttccggta 120

ccggaggaga?cttatggagc?tgttacagcg?gtgactcaac?atcagaatag?ctttggtgtt 180

tctgtttcgt?cggagggaaa?tgaaatagac?aacaatccgg?tggtcgtcaa?gaagcttaat 240

cacaatgcta?gtgagcgtga?ccgtcgcagg?aaaattaact?ctttgttctc?atctctccgt 300

tcatgtcttc?ctgcctctgg?ccaatcgaag?aagctaagca?ttcctgcgac?ggtttctcga 360

agcttgaagt?acataccaga?gctgcaagag?caagtgaaga?agctaataaa?aaagaaggaa 420

gagctcttgg?tgcaaatttc?aggtcaaaga?aacactgaat?gttacgttaa?gcagccacca 480

aaggccgtcg?cgaattatat?ctcgaccgtt?tctgcgacta?ggcttggtga?caacgaagtg 540

atggtccaaa?tctcatcgtc?caagattcat?aacttttcga?tatctaatgt?tttaagtggg 600

ttagaagaag?ataggtttgt?tcttgtggac?atgtcatctt?caaggtctca?aggagaaagg 660

cttttctaca?ctttgcattt?acaagtggag?aagattgaaa?attacaagct?gaattgcgaa 720

gagttaagtc?agaggatgtt?gtacttgtat?gaggaatgtg?gaaactcata?tatatga 777

<210>5

<211>949

<212>DNA

＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis thaliana)

<400>5

atgtgtgcat?tagtacctcc?attgtttcca?aactttgggt?ggccatcaac?gggagagtac 60

gacagctact?acctcgccgg?agatatcctc?aacaacggcg?ggtttcttga?ttttccggta 120

ccggaggaga?cttatggagc?tgttacagcg?gtgactcaac?atcagaatag?ctttggtgtt 180

tctgtttcgt?cggagggaaa?tgaaatagac?aacaatccgg?tggtcgtcaa?gaagcttaat 240

cacaatgcta?gtgagcgtga?ccgtcgcagg?aaaattaact?ctttgttctc?atctctccgt 300

tcatgtcttc?ctgcctctgg?ccaatcggta?agaagctact?ccgactcatt?gattagaccc 360

gttatagttt?atgcatctat?acaatttaga?agaagaatac?tgcgttaaac?cttttttctt 420

tcctttcttg?cagaagaagc?taagcattcc?tgcgacggtt?tctcgaagct?tgaagtacat 480

accagagctg?caagagcaag?tgaagaagct?aataaaaaag?aaggaagagc?tcttggtgca 540

aatttcaggt?caaagaaaca?ctgaatgtta?cgttaagcag?ccaccaaagg?ccgtcgcgaa 600

ttatatctcg?accgtttctg?cgactaggct?tggtgacaac?gaagtgatgg?tccaaatctc 660

atcgtccaag?attcataact?tttcgatatc?taatgtttta?agtgggttag?aagaagatag 720

gtttgttctt?gtggacatgt?catcttcaag?gtctcaagga?gaaaggcttt?tctacacttt 780

gcatttacaa?gtggagaaga?ttgaaaatta?caagctgaat?tgcgaagagt?taagtcagag 840

gatgttgtac?ttgtatgagg?aatgtggaaa?ctcatatata?tgagaatttg?gtcttgtttc 900

tttatagtta?tgttatgtcg?tcctttttct?cttcgaaatc?taacattct 949

<210>6

<211>258

<212>PRT

＜213〉Arabidopsis Arabidopis thaliana (Arabidopsis thaliana)

<400>6

Met?Cys?Ala?Leu?Val?Pro?Pro?Leu?Phe?Pro?Asn?Phe?Gly?Trp?Pro?Ser

1 5 10 15

Thr?Gly?Glu?Tyr?Asp?Ser?Tyr?Tyr?Leu?Ala?Gly?Asp?Ile?Leu?Asn?Asn

20 25 30

Gly?Gly?Phe?Leu?Asp?Phe?Pro?Val?Pro?Glu?Glu?Thr?Tyr?Gly?Ala?Val

35 40 45

Thr?Ala?Val?Thr?Gln?His?Gln?Asn?Ser?Phe?Gly?Val?Ser?Val?Ser?Ser

50 55 60

Glu?Gly?Asn?Glu?Ile?Asp?Asn?Asn?Pro?Val?Val?Val?Lys?Lys?Leu?Asn

65 70 75 80

His?Asn?Ala?Ser?Glu?Arg?Asp?Arg?Arg?Arg?Lys?Ile?Asn?Ser?Leu?Phe

85 90 95

Ser?Ser?Leu?Arg?Ser?Cys?Leu?Pro?Ala?Ser?Gly?Gln?Ser?Lys?Lys?Leu

100 105 110

Ser?Ile?Pro?Ala?Thr?Val?Ser?Arg?Ser?Leu?Lys?Tyr?Ile?Pro?Glu?Leu

115 120 125

Gln?Glu?Gln?Val?Lys?Lys?Leu?Ile?Lys?Lys?Lys?Glu?Glu?Leu?Leu?Val

130 135 140

Gln?Ile?Ser?Gly?Gln?Arg?Asn?Thr?Glu?Cys?Tyr?Val?Lys?Gln?Pro?Pro

145 150 155 160

Lys?Ala?Val?Ala?Asn?Tyr?Ile?Ser?Thr?Val?Ser?Ala?Thr?Arg?Leu?Gly

165 170 175

Asp?Asn?Glu?Val?Met?Val?Gln?Ile?Ser?Ser?Ser?Lys?Ile?His?Asn?Phe

180 185 190

Ser?Ile?Ser?Asn?Val?Leu?Ser?Gly?Leu?Glu?Glu?Asp?Arg?Phe?Val?Leu

195 200 205

Val?Asp?Met?Ser?Ser?Ser?Arg?Ser?Gln?Gly?Glu?Arg?Leu?PheTyr?Thr

210 215 220

Leu?His?Leu?Gln?Val?Glu?Lys?Ile?Glu?Asn?Tyr?Lys?Leu?Asn?Cys?Glu

225 230 235 240

Glu?Leu?Ser?Gln?Arg?Met?Leu?Tyr?Leu?Tyr?Glu?Glu?Cys?Gly?Asn?Ser

245 250 255

Tyr?Ile

Claims

1, absorbing with AtbHLH29 coordinated regulation plant ferro element and the cDNA of metabolic gene, is one of following nucleotide sequence:

1) dna sequence dna of SEQ ID NO:3 in the code sequence tabulation;

2) dna sequence dna of SEQ ID NO:6 in the code sequence tabulation.

2, gene according to claim 1 is characterized in that: the nucleotide sequence of its cDNA is shown in SEQ ID NO:1 or SEQ ID NO:4.

3, absorbing with AtbHLH29 coordinated regulation plant ferro element and the genomic gene of metabolic gene, is one of following nucleotide sequence:

1) genomic gene of AtbHLH38, its nucleotide sequence is shown in SEQ ID NO:2;

2) genomic gene of AtbHLH39, its nucleotide sequence is shown in SEQ ID NO:5.

4, the albumen of the described genes encoding of claim 1 is one of following amino acid residue sequences:

1) AtbHLH38 albumen, its amino acid residue sequence is shown in SEQ ID NO:3;

2) AtbHLH39 albumen, its amino acid residue sequence is shown in SEQ ID NO:6.

5, contain claim 1 or 2 or 3 described expression carrier.

7, the transgenic cell line that contains claim 1 or 2 or 3 described genes.

8, the host bacterium that contains claim 1 or 2 or 3 described genes.

9, a kind of regulation and control plant absorbs and metabolic method ferro element, is with AtbHLH29 with AtbHLH29 coordinated regulation plant ferro element to be absorbed and the cDNA of metabolic gene imports plant tissue or cell, and plant obtains to the absorption and the metabolism of ferro element; Described and AtbHLH29 coordinated regulation plant ferro element absorb and the cDNA of metabolic gene, are one of following nucleotide sequence:

1) dna sequence dna of SEQ ID NO:3 in the code sequence tabulation;

2) dna sequence dna of SEQ ID NO:6 in the code sequence tabulation.

10, method according to claim 9, it is characterized in that: described AtbHLH29 and with AtbHLH29 coordinated regulation plant ferro element is absorbed and metabolic gene by containing AtbHLH29 respectively, ferro element absorbed and metabolic gene with AtbHLH29 coordinated regulation plant, or contain AtbHLH29 and with AtbHLH29 coordinated regulation plant ferro element absorbed and the plant expression vector importing explant of metabolic gene; The carrier that sets out that is used to make up described plant expression vector is pBinplus, pBI121, pBin19, pCAMBIA2301 or pCAMBIA1300.

11, method according to claim 10, it is characterized in that: described containing absorbs ferro element with AtbHLH29 coordinated regulation plant and metabolic gene plant expression vector is as shown in Figure 6A pBI-38 or pBI-39, and the plant expression vector that contains AtbHLH29 is the pBinplus-29 shown in Fig. 6 B.

12, according to claim 9 or 10 or 11 described methods, it is characterized in that: described plant is monocotyledons or dicotyledons.