CN1884538A - Synergistic control Gene of AtbHLH29 for plant ferro element absorb and its coded protein and uses - Google Patents

Abstract

The invention discloses a gene for cooperating with AtbHLH29 to regulate plant iron absorption and metabolism, its encoded protein and its application. Its purpose is to provide a gene and its coded protein that cooperates with AtbHLH29 to regulate plant iron absorption and metabolism and its application in regulating plant iron absorption and metabolism. The cDNA of the gene is one of the following nucleotide sequences: 1) the DNA sequence of SEQ ID No.: 1 and SEQ ID No.: 4 in the sequence listing; 2) SEQ ID No.: 3 and SEQ ID No.: 4 in the coding sequence listing: 6 DNA sequence; 3) a nucleotide sequence that has more than 90% homology with the nucleotide sequence defined by Sequence 1 or SEQ ID No. 4 in the sequence listing and plays an important role in regulating plant iron absorption and metabolism ; 4) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID No.: 1 or SEQ ID No.: 4 in the sequence listing under high stringency conditions.

Description

Genes that cooperate with AtbHLH29 to regulate iron uptake in plants, their encoded proteins and their applications

technical field

The present invention relates to plant genes and their coded proteins and applications, in particular to two genes that work with AtbHLH29 to synergistically regulate plant iron absorption and metabolism and their coded proteins and their application in regulating plant iron absorption and metabolism.

Background technique

As a component of many important enzymes and proteins, iron is involved in the most basic biochemical processes in life activities, such as respiration and photosynthesis. Iron deficiency in infants can lead to anemia, which seriously affects the intellectual and physical development of infants. Improving iron content and iron bioavailability in agricultural products through biological means is the most economical, effective and long-lasting way to improve iron nutrition in humans. In agricultural production, iron is also one of the main factors limiting the growth and development of crops. The total content of iron in soil is generally high, but most iron exists in the form of ferric oxide. In neutral and alkaline soils, the solubility of ferric iron is extremely low and cannot be absorbed and utilized by plants. In the long-term evolution process, in order to meet the needs of their growth and development, plants have evolved two efficient ways to activate and absorb iron, which are 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). Poaceae plants belong to strategy II plants, and plants other than grasses belong to strategy I plants. The cloning of the key genes that regulate the absorption and metabolism of iron in plants will open up a new way to increase the iron content in plants by biotechnology.

AtbHLH29 (GenBank number: NM_102582/NP_174138) is a homologous gene of the tomato FER gene previously identified from the Arabidopsis genome by the inventors of the present invention, and is involved in the regulation of iron absorption and metabolism in plants. With the deletion of FER and AtbHLH29 genes, the mutant plants could not initiate the physiological adaptive response to iron deficiency stress and effectively obtain iron from the soil, showing yellowing and lethality (The tomato FER gene encoding a bHLHprotein controls iron-uptake responses in roots by Ling H-Q, Bauer P, Bereczky Z, Keller B and Ganal M, Proc Natl Acad Sci USA 99, 13938-43, 2002; ML, Plant Cell 16, 3400-3412, 2004). Transforming the Arabidopsis thaliana AtbHLH29 gene into the tomato FER gene deletion mutant T3238fer by Agrobacterium-mediated method can restore the function of FER loss, and the transgenic plants can start the whole iron deficiency physiological adaptive response and iron uptake under low iron stress The expression of related genes, so that the iron content in the leaves of transgenic plants returned to normal levels (AtbHLH29 of Arabidopsis thaliana is a functional ortholog of tomato FER involved in controlling iron acquisition in strategy I plants by Yuan YX, ZhangJ, Wang DW and, Ling H-Q Cell Research 15, 613-621, 2005).

Contents of the invention

The purpose of the present invention is to provide two genes that work together with AtbHLH29 to regulate plant iron absorption and metabolism.

The genes provided by the present invention that cooperate with AtbHLH29 to regulate the absorption and metabolism of plant iron elements are named AtbHLH38 and AtbHLH39, and are derived from Arabidopsis thaliana (Arabidopsis thaliana), and its cDNA is one of the following nucleotide sequences:

1) SEQ ID №: 1 and SEQ ID №: 4 in the sequence listing are the coding sequences of AtbHLH38 and AtbHLH39 genes, respectively;

2) DNA sequences of SEQ ID №: 3 and SEQ ID №: 6 in the coding sequence list;

3) A nucleotide sequence that has more than 90% homology with the nucleotide sequence defined by Sequence 1 or SEQ ID No.: 4 in the sequence listing and plays an important role in regulating iron absorption and metabolism in plants;

4) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID №: 1 or SEQ ID №: 4 in the sequence listing under high stringency conditions.

The high stringency condition is to hybridize and wash the membrane at 65° C. in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

SEQ ID №: 1 in the sequence listing consists of 762 bases, and its coding sequence is the 1-762 bases from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID №: 3 in the sequence listing, From the 202-369th base at the 5' end to encode the bHLH conserved domain, the gene with the cDNA sequence of SEQ ID №: 1 is named AtbHLH38; the SEQ ID № in the sequence table: 4 consists of 777 bases, which The coding sequence is bases 1-777 from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID №6 in the sequence listing, bases 217-384 from the 5' end encode the bHLH conserved domain, This gene with SEQ ID No.: 4 cDNA sequence was named AtbHLH39.

Its genome sequence is one of the following nucleotide sequences:

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

2) A nucleotide sequence that has more than 90% homology with the nucleotide sequence defined by Sequence 2 or SEQ ID No. 5 in the sequence listing and plays an important role in regulating iron absorption and metabolism in plants;

3) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID №: 2 or SEQ ID №: 5 in the sequence listing under high stringency conditions.

The high stringency condition is to hybridize and wash the membrane at 65° C. in a solution of 0.1×SSPE (or 0.1×SSC) and 0.1% SDS.

The SEQ ID № in the sequence listing: 2 consists of 985 bases, the 1st-312th base from the 5' end is the first exon of the genome gene, and the 313th-441st base from the 5' end It is the first intron of the genome gene, the 442-891 base from the 5' end is the second exon of the genome gene, and the 1-3 base from the 5' end is the genome gene The start codon ATG of the 5' end base 889-891 is the stop codon TAG of the genome gene, and the 202-498 base base encoding bHLH conserved domain from the 5' end; SEQ in the sequence listing ID №: 5 consists of 949 bases, bases 1-327 from the 5' end are the first exon of the genome gene, bases 328-433 from the 5' end are the bases of the genome gene The first intron, the 434th-883th base from the 5' end is the second exon of the genomic gene, and the 1st-3rd base from the 5' end is the start codon of the genomic gene ATG, bases 881-883 from the 5' end are the stop codon TGA of the genomic gene, bases 217-490 from the 5' end encode the bHLH conserved domain.

The protein (AtbHLH38 and AtbHLH39) of the present invention that cooperates with AtbHLH29 to regulate plant iron absorption and metabolism genes has one of the following amino acid residue sequences:

1) SEQ ID №: 3 and SEQ ID №: 6 in the sequence listing are the amino acid residue sequences of AtbHLH38 and AtbHLH39 proteins, respectively;

2) The amino acid residue sequence of SEQ ID №: 3 or SEQ ID №: 6 in the sequence listing undergoes one to ten substitutions, deletions or additions of amino acid residues and has the function of regulating plant iron absorption and metabolism by cooperating with AtbHLH29 protein.

SEQ ID No. 3 in the sequence listing is the amino acid sequence of AtbHLH38, consisting of 253 amino acid residues, and amino acid residues 68-123 from the amino terminal (N-terminal) are bHLH conserved domains; SEQ ID in the sequence listing №: 6 is the amino acid sequence of AtbHLH39, which consists of 258 amino acid residues, and the 73rd-128th amino acid residues from the amino terminal are the bHLH conserved domain.

The expression vector, transgenic cell line and host bacteria containing the gene of the present invention all belong to the protection scope of the present invention.

The primer pair for amplifying any fragment of AtbHLH38/AtbHLH39 is also within the protection scope of the present invention.

Another object of the present invention is to provide a method for regulating iron absorption and metabolism by plants.

The method for regulating iron absorption and metabolism of plants provided by the present invention is to introduce AtbHLH29 and the gene AtbHLH38/AtbHLH39, which cooperates with AtbHLH29 to regulate iron absorption and metabolism in plants, into plant tissues or cells, and the absorption of iron by plants and metabolism are regulated.

The AtbHLH29 and AtbHLH38/AtbHLH39 can be introduced into explants by containing AtbHLH29, AtbHLH38/AtbHLH39 respectively, or plant expression vectors containing AtbHLH29 and AtbHLH38/AtbHLH39; the starting vector for constructing the plant expression vector can be any double Agrobacterium vectors or vectors that can be used for plant microprojectile bombardment, such 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 derived plant expression vectors.

When using AtbHLH29 and AtbHLH38/AtbHLH39 to construct plant expression vectors, any enhanced, constitutive, tissue-specific or inducible promoter can be added before the transcription start nucleotide, such as cauliflower mosaic virus (CAMV) 35S promoter, ubiquitin gene Ubiquitin promoter (pUbi) and rice actin gene promoter (Actin), etc., they can be used alone or in combination with other plant promoters; in addition, use the gene of the present invention to construct plant When expressing vectors, enhancers can also be used, including translation enhancers or transcription enhancers. These enhancer regions can be ATG start codons or adjacent region start codons, etc., but must be the same as the reading frame of the coding sequence, to Guarantees correct translation of the entire sequence. The sources of the translation control signals and initiation codons are extensive and can be natural or synthetic. The translation initiation region can be from a transcription initiation region or a structural gene.

In order to facilitate the identification and screening of transgenic plant cells or plants, the plant expression vectors used can be processed, such as adding genes (GUS genes, GFP genes, fluorescent luciferase gene, etc.), antibiotic markers with resistance (gentamycin marker, kanamycin marker, etc.) or chemical resistance marker genes (such as herbicide resistance genes), etc. Considering the safety of the transgenic plants, the transformed plants can be screened directly by adversity without adding any selectable marker gene.

Using pBI121 as the starting vector, the constructed plant expression vector containing AtbHLH38 was pBI-38, and the plant expression vector containing AtbHLH39 was pBI-39; using pBinplus as the starting vector, the constructed plant expression vector containing AtbHLH29 was pBinplus-29.

Plant expression vectors carrying AtbHLH29, AtbHLH38, AtbHLH39, or AtbHLH29 and AtbHLH38/AtbHLH39, respectively, can be obtained by conventional biological methods such as Ti plasmids, Ri plasmids, plant virus vectors, direct DNA transformation, microinjection, electrical conductivity, and Agrobacterium-mediated Plant cells or tissues are transformed by scientific methods, and the transformed plant cells or tissues are cultivated into plants.

The method for regulating iron absorption and metabolism in plants is applicable to all plants, not only to monocotyledonous plants such as corn, rice, and wheat, but also to dicotyledonous plants such as Arabidopsis, tobacco, and cotton.

The invention provides genes AtbHLH38 and AtbHLH39 and their coded proteins, which work together with AtbHLH29 to coordinately regulate plant iron absorption and metabolism. Transgenic experiments showed that AtbHLH38 and AtbHLH39 acted together with AtbHLH29 at the protein level to significantly improve the plant's ability to absorb Fe. This gene has important theoretical and practical significance for improving the low iron tolerance and iron utilization efficiency of plants, and provides an economical and rapid method for the biofortification of iron content in agricultural products and the bioremediation of heavy metal-contaminated soils. , An effective way. The invention has broad application and market prospects in the fields of agriculture and bioremediation.

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

Description of drawings

Figure 1 shows the results of quantitative PCR detection of the expression of AtbHLH38 and AtbHLH39 under iron-deficiency conditions

Figure 2 is the results of GUS histochemical staining and GUS protein enzymatic activity assay for detecting the transcriptional regulation of AtbHLH38, AtbHLH39 and AtbHLH29 on ferric reductase gene AtFRO2 and ferrous ion transporter gene AtIRT1 in yeast cells

Figure 3 shows the growth of single and double overexpression plants of AtbHLH38, AtbHLH39 and AtbHLH29 after low iron stress treatment

Figure 4 shows the determination results of iron content in shoot leaves of AtbHLH38 and AtbHLH39 single overexpression plants and AtbHLH29 double overexpression plants after normal growth and low iron stress treatment

Figure 5 shows the RT-PCR detection results of single and double overexpression of AtbHLH38/AtbHLH39 and AtbHLH29 to increase the transcription levels of ferric reductase gene AtFRO2 and ferrous ion transporter gene AtIRT1

Figure 6A is a partial physical map of pBI-38 and pBI-39

Figure 6B is a partial physical map of pBinplus-29

Detailed ways

The methods used in the following examples are conventional methods unless otherwise specified, and the primer synthesis and sequencing work used were completed by Beijing Aoke Biotechnology Co., Ltd.

Example 1 Cloning of genes AtbHLH38 and AtbHLH39 that cooperate with AtbHLH29 to regulate iron absorption and metabolism in plants

Two pairs of primers (P38 and P39) were designed according to the Arabidopsis genomic DNA sequence and the predicted coding sequence of genes (named AtbHLH38 and AtbHLH39) that cooperate with AtbHLH29 to regulate plant iron uptake and metabolism. The primer sequences are as follows:

Primer pair P38:

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

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

Primer pair P39:

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

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

Arabidopsis thaliana seedlings were treated with iron deficiency for 4 days on iron-free MS medium, then total RNA was extracted from the iron-deficiency-treated seedlings, and its cDNA was synthesized by reverse transcription, and the cDNA was used as a template, respectively, in Under the guidance of primers P38 and P39, PCR amplification was carried out. The PCR reaction conditions were as follows: first 95°C for 2 minutes; then 95°C for 1 minute, 50°C for 1 minute, 72°C for 1 minute, a total of 35 cycles; finally 72°C for 5 minutes . After the reaction, the PCR product was detected by 1.0% agarose gel electrophoresis. As a result, under the guidance of the primer pair P38 and P39, bands of 770 bp and 785 bp were respectively amplified, and the two target fragments were recovered and purified by DNA kit (Phamacia company) to purify it, then the purified target fragments were cloned into the vector pGEM T-easy (Promega company), and the recombinant vector containing the primer pair P38 amplified fragment was named as pGEM T-easy/AtbHLH38 , the recombinant vector containing the amplified fragment of the primer pair P39 was named pGEM T-easy/AtbHLH39, and then pGEMT-easy/AtbHLH38 and pGEM T-easy/AtbHLH39 were transformed into E. coli DH5a competent cells by the CaCl ₂ method, and the positive results were screened Single clone, plasmid extraction, sequencing, sequencing results show that the primer pair P38 amplified fragment has the nucleotide sequence of SEQ ID №: 1 in the sequence listing, and SEQ ID №: 1 in the sequence listing consists of 762 bases, and its encoding The sequence is bases 1-762 from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID No. 3 in the sequence listing, and bases 202-369 from the 5' end encode the bHLH conserved domain. The gene with the DNA sequence of SEQ ID №: 1 is named AtbHLHt38; the primer pair P39 amplified fragment has the nucleotide sequence of SEQ ID №: 4 in the sequence listing, and the SEQ ID № in the sequence listing: 4 consists of 777 bases , its coding sequence is the 1st-777th base from the 5' end, encoding a protein with the amino acid residue sequence of SEQ ID No. 6 in the sequence listing, and the bHLH conservative structure from the 217th-384th base at the 5' end Domain, the gene with the DNA sequence of SEQ ID No.: 4 was named AtbHLH39.

Obtained the genomic DNA sequence of AtbHLH38 and AtbHLH39 according to the cDNA sequence of the amplified AtbHLH38 and AtbHLH39 and the genomic DNA sequence of Arabidopsis, the genomic DNA sequence of AtbHLH38 has the nucleotide sequence of SEQ ID No. 2 in the sequence listing, sequence listing The SEQ ID №: 2 consists of 985 bases, the 1st-312th base from the 5' end is the first exon of the genome gene, and the 313th-441st base from the 5' end is the The first intron of the genome gene, bases 442-891 from the 5' end are the second exon of the genome gene, bases 1-3 from the 5' end are the beginning of the genome gene The start codon ATG, bases 889-891 from the 5' end are the stop codon TAG of the genomic gene, bases 202-498 from the 5' end encode the bHLH conserved domain; the genomic DNA sequence of AtbHLH39 has the sequence The nucleotide sequence of SEQ ID №: 5 in the list, the SEQ ID №: 5 in the sequence list is composed of 949 bases, and the 1-327 bases from the 5′ end are the first exome of the genome gene The 328-433rd base from the 5' end is the first intron of the genome gene, and the 434-883rd base from the 5' end is the second exon of the genome gene, from the 5' end Bases 1-3 at the 'end are the start codon ATG of the genome gene, bases 881-883 at the 5' end are the stop codon TGA of the genome gene, bases 217-490 at the 5' end The bases encode the conserved domain of bHLH.

Example 2, quantitative PCR detection of the expression of AtbHLH38 and AtbHLH39 under iron-deficiency conditions

Arabidopsis seeds were placed on MS medium (sigma, USA) to germinate (1 week), and then the seedlings were placed in MS culture without iron (Fe), manganese (Mn), and zinc (Zn) elements. The stress treatment lacking different metal nutrients was carried out on the base for 4 days, and the plants without stress treatment were used as the control, and then the total RNA of the roots and shoots of the stress-treated seedlings and the control were respectively extracted and used as templates. : 5'-GACGGTACCACAGACTTATGAAGT-3' and primer 2: 5'-TAAGCTCTTTGAAACCGTTTCAGGA-3'guided quantitative PCR detection/expression of AtbHLH38, primer 3: 5'-GACTTATGGAGCTGTTACAGCGGT-3' and primer 4: 5'-CTTCAAGCTTCGAGAAACCGTCGCA The expression of AtbHLH39 was detected by quantitative PCR under the guidance of -3', and the ferric reductase gene AtFRO2 was detected by quantitative PCR under the guidance of primer 5: 5'-GATCGAAAAAAGCAATAACGGTGGTT-3' and primer 6: 5'-GATGTGGCAACCACTTGGTTCGATA-3' ( GenBank No. NM_100040/NP_171664), under the guidance of primer 7: 5'-GAGTTTCCGTTCACAGGCTTTATC-3' and primer 8: 5'-TTCCACGCCAACAATCCCGT-3', the zinc transporter gene ZIP5 (GenBank No.: NP_973762) was detected by quantitative PCR Expression situation, detection result is as shown in Figure 1 (A, B, C, D figure in Fig. 1 is the expression situation of AtbHLH38, AtbHLH39, AtFRO2, ZIP5 in root and shoot under control and different stress treatment situations respectively), shows The expression of AtbHLH38 and AtbHLH39 in both roots and shoots was induced by iron deficiency, and the expression was up-regulated under iron deficiency, but not affected by other metal elements (such as manganese and zinc).

Example 3. Detecting the transcriptional regulation of AtbHLH38/AtbHLH39 and AtbHLH29 on ferric reductase gene AtFRO2 and ferrous ion transporter gene AtIRT1

1. Yeast two-hybrid experiment to detect the interaction between AtbHLH38/AtbHLH39 and AtbHLH29

Yeast two-hybrid assay was used to detect the interaction between AtbHLH38/AtbHLH39 and AtbHLH29. Using Strategene's yeast two-hybrid detection kit and operating according to the kit instructions, AtbHLH38/AtbHLH39 and AtbHLH29 were cloned into two yeast expression vectors pBD-GAL4 Cam and pAD-GAL4-2.1 respectively, and then double-transformed by heat shock Yeast strain YRG-2. According to the principle of yeast two-hybrid, if the two genes can interact, the transformed yeast strain can grow normally on the medium without Histidine, and X-GAL enzyme activity can be detected, otherwise the strain cannot grow normally and cannot be detected X-GAL enzyme activity. The X-GAL activity expressed by the reporter gene lacZ and the Histidine expressed by HIS3 were detected in the positive clones, and the positive strains could grow normally on the medium without Histidine, indicating that AtbHLH38 and AtbHLH39 can interact with AtbHLH29 at the protein level.

2. Detection of the transcriptional regulation of the interaction between AtbHLH38/AtbHLH39 and AtbHLH29 on the ferric reductase gene AtFRO2 and the ferrous ion transporter gene AtIRT1 in yeast cells

Since the AtbHLH38/AtbHLH39-encoded proteins both contain the bHLH conserved domain, and the gene is induced by low iron, it is speculated that AtbHLH38/AtbHLH39 may be a transcription factor involved in the regulation of iron uptake by plants. In addition, the yeast two-hybrid results showed that AtbHLH38 and AtbHLH39 can interact with AtbHLH29 at the protein level. Therefore, it is speculated that AtbHLH38 and AtbHLH39 can interact with AtbHLH29 to form heterodimers and regulate the expression of downstream functional genes, such as ferric reductase gene AtFRO2 and ferrous ion Transporter gene AtIRT1. The following methods were used for verification: 1) Using pBD-GAL4 Cam and pAD-GAL4-2.1 (Stategene, USA) as starting vectors, construct recombinant yeast containing the coding sequences of AtbHLH38, AtbHLH39, AtbHLH29, AtbHLH38+AtbHLH29, AtbHLH39+AtbHLH29 respectively Expression vector, the position of the above-mentioned gene in the yeast expression vector is between the EcoRI and SalI restriction sites of the multi-cloning site, and then the above-mentioned recombinant yeast expression vector is transformed into yeast cell YRG-2 (Strategene, USA) to obtain There are recombinant yeast cells of the above-mentioned genes; 2) the promoter sequence of the ferric reductase gene AtFRO2 (AtFRO2 initiation codon ATG to the upstream 947bp, the promoter is named as P _FRO2 ) and the ferrous ion transporter gene AtIRT1 The promoter sequence (from -18bp to -1598bp upstream of the start codon of AtIRT1, named _PIRT1 ) was fused with the DNA sequence of the reporter gene GUS (GenBank number: AAL92040), and the promoter sequence was located upstream of GUS, and then the two The fusion genes (P _FRO2 ::GUS and _PIRT1 ::GUS) were respectively cloned into the PmacI restriction sites of the yeast expression vectors pBD-GAL4 Cam and pBD-GAL4-AtbHLH29 Cam (Stategene, USA) to obtain the above-mentioned different fusion genes respectively. Gene recombinant yeast expression vectors, and then transfer two recombinant yeast expression vectors carrying different fusion genes into step 1) to obtain different gene combinations (AtbHLH38, AtbHLH39, AtbHLH29, AtbHLH38+AtbHLH29, AtbHLH39+AtbHLH29) 3) performing GUS histochemical staining on the recombinant yeast cells obtained in step 2) and measuring the enzyme activity of the GUS protein. The results are shown in Figure 2 (Figures A and B are the results of GUS histochemical staining of yeast cells: 1.pAD-WT+pBD-WT-P _FRO2 ::GUS; 2.pAD-WT+pBD-A tbHLH29-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- _PIRT1 ::GUS; 10. pAD-WT+pBD-AtbHLH29- _PIRT1 ::GUS; 11. pAD-AtbHLH38+pBD-WT- _PIRT1 ::GUS; 12. pAD-AtbHLH38+pBD-AtbHLH29- _PIRT1 ::GUS; 13. pAD-AtbHLH39+pBD-WT- _PIRT1 ::GUS; 14. pAD-AtbHLH39+pBD-AtbHLH29- _PIRT1 ::GUS; 15. pGAL4+pBD-AtbHLH29- _PIRT1 ::GUS; 16. pAD-AtbHLH40+pBD -WT-P _IRT1 ::GUS. Figures C and D show the enzyme activity assay results of GUS protein, * indicates that there is a significant difference with other strains), AtbHLH29, AtbHLH38 and AtbHLH39 can not be activated in yeast cells _PFRO2 ::GUS and The transcription of _PIRT1 ::GUS, when AtbHLH29 and AtbHLH38 or AtbHLH39 are co-expressed in yeast cells, can promote the expression of _PFRO2 ::GUS and _PIRT1 ::GUS.

Embodiment 4, the acquisition of transgenic Arabidopsis and the functional verification experiment of the gene

Taking Arabidopsis thaliana as an example, the gene of the present invention is subjected to a functional verification experiment, and the specific experimental method is as follows:

1. Acquisition of AtbHLH38 and AtbHLH39 Single-gene Arabidopsis

AtbHLH38 and AtbHLH39 were separated from the recombinant plasmids pGEM T-easy/AtbHLH38 and pGEM T-easy/AtbHLH39 constructed in Example 1 with restriction endonucleases Xba I and Sma I, and then the two fragments were cloned into the recombinant plasmids containing 35S respectively. The overexpression vectors of AtbHLH38 and AtbHLH39 were obtained between the Xba I and Sma I restriction sites of the enhanced promoter vector pBI121, which were named pBI121-38 and pBI121-39, respectively. Part of the physical map is shown in Figure 6A. Then, the recombinant vectors pBI121-38 and pBI121-39 were introduced into Agrobacterium GV3101 by heat shock method, and Arabidopsis thaliana was transformed by flower invasion method to obtain transgenic plants with AtbHLH38 and AtbHLH39 respectively, which were named AT38 and AT39 respectively.

2. Acquisition of AtbHLH38/AtbHLH39 and AtbHLH29 double-gene Arabidopsis

The cDNA sequence of AtbHLH29 was amplified by PCR under the guidance of primer P9 (5'-cacccatggaaggaagagtcaac-3') and primer P10 (5'-ttagtcgacctagtaaatgacttgatg-3') using the cDNA obtained by reverse transcription of Arabidopsis total RNA , and then clone the fragment into the pGEM T-easy vector multi-cloning site between the Xba I and Sac I restriction sites to obtain an intermediate vector, named pGEMT-easy/AtbHLH29, which was sequenced, and the sequencing results showed that the clone The cDNA sequence of AtbHLH29 was correct, AtbHLH29 was isolated from the recombinant vector pGEM T-easy/AtbHLH29 with restriction endonucleases Xba I and SacI, and then cloned into the Agrobacterium transformation vector pBinplus containing 35S enhanced promoter The AtbHLH29 transformation vector was obtained between the Nco I and Sal I restriction sites, named pBinplus-29, and part of its physical map is shown in Figure 6B. Then the vector was introduced into the Agrobacterium strain GV3101, and the AT38 and AT39 transgenic plants obtained in step 1 were respectively transformed to obtain double-gene overexpression plants carrying AtbHLH29+AtbHLH38 and AtbHLH29+AtbHLH39, which were named AT38/29 and AT39/29 respectively. .

3. Low iron tolerance test of single and double overexpression plants of AtbHLH38/AtbHLH39 and AtbHLH29

The single overexpression plants AT38 and AT39 of AtbHLH38 and AtbHLH39 obtained in step 1 and step 2 and the double overexpression plants AT38/29 and AT39/29 of AtbHLH29 were transplanted on the MS medium containing 10 μM Fe-EDTA under low iron Conditions were treated for 10 days, and the plants grown on MS medium were used as controls. The growth of each plant under low iron conditions was shown in Figure 3 (WT is a wild-type control). As a result, the double overexpression plants AT38/29 and At39/29 grew normally under low iron stress, while wild-type and single overexpression plants showed iron-deficiency yellow flower symptoms. Simultaneously, the iron content of the above-ground leaves of the single overexpression plants of AtbHLH38 and AtbHLH39 and the double overexpression plants of AtbHLH29 was measured, and the measurement results are shown in Figure 4 (A graph represents each plant grown on normal MS medium The Fe content of , the B graph represents the Fe content of each plant grown on the iron-deficiency medium, ☆ represents the significant difference α=0.05 level, and ★ represents the significant difference α=0.01 level), indicating that AtbHLH38+AtbHLH29 and AtbHLH39+AtbHLH29 The iron content of leaves of double overexpression plants was significantly higher than that of wild type and AtbHLH38, AtbHLH29, AtbHLH39 single overexpression plants, while the iron content of single overexpression plants was not significantly different from wild type.

4. Verification experiment of single and double overexpression of AtbHLH38/AtbHLH39 and AtbHLH29 to increase transcription levels of ferric iron reductase gene AtFRO2 and ferrous ion transporter gene AtIRT1

The seeds of the single overexpression plant AT38 and AT39 of AtbHLH38 and AtbHLH39 obtained in step one and step two and the double overexpression plant AT38/29 and AT39/29 of AtbHLH29 were placed on MS medium to germinate, and then 1 week Seedlings of different sizes were transferred to MS solid medium containing 100 μM iron concentration and iron-free medium to grow for 4 days, and then the total RNA of the roots and leaves of the above-mentioned transgenic plants were extracted respectively (without any gene transfection and under the same conditions treated Arabidopsis plants as controls), and using different extracted total RNAs as templates, RT-PCR was used to analyze AtbHLH38/AtbHLH39 and AtbHLH29 and their downstream functional genes (ferric iron reductase gene AtFRO2 and ferrous The expression abundance of the ion transporter gene AtIRT1), under the guidance of primer P11: 5'-GACGGTACCACAGACTTATGAAGT-3' and primer P12: 5'-TAAGCTCTTTGAAACCGTTTCAGGA-3', AtbHLH38 was amplified by PCR; under the guidance of primer P13: 5'-GACTTATGGAGCTGTTACAGCGGT Under the guidance of -3' and primer P14: 5'-CTTCAAGCTTCGAGAAACCGTCGCA-3', AtbHLH39 was amplified by PCR; Amplify AtbHLH29; Under the guidance of primer P17; 5'-GATCGAAAAAAGCAATAACGGTGGTT-3' and primer P18: 5'-GATGTGGCAACCACTTGGTTCGATA-3', the ferric reductase gene AtFRO2 (GenBank number NM_100040/NP_171664) was amplified by PCR; Under the guidance of P19: 5'-GAATGTGGAAGCGAGTCAGCGA-3' and primer P20: 5'-GATCCCGGAGGCGAAACACTTA-3', PCR amplified the ferrous ion transporter gene AtIRT1 (GenBank No. NM_118089/NP_567590); under the guidance of primer P21: 5'-GCTGTTCGTGGTGTTGAGATGC Under the guidance of -3' and primer P22: 5'-AGGCTCTGAGGTGAGGAAGTCT-3', EF1B-a was amplified by PCR (GenBank number: NM_121956). After the reaction was finished, the above-mentioned PCR amplification products were detected by 1% agarose gel electrophoresis, and the detection results were as shown in Figure 5 ("+" means: under the iron concentration of 100 μM, "-" means: under the iron-free situation Bottom), WT is the Arabidopsis control plant without any gene transfection and treated under the same conditions, AT38-18, AT39-17, AT29-3, AT38/29-2, AT39/29-12 are different AtbHLH38 The single overexpression line with AtbHLH39 and the double overexpression line with AtbHLH29 showed that the transcription levels of the ferric reductase gene AtFRO2 and the ferrous ion transporter gene AtIRT1 in the double overexpression plants were significantly higher than those of the control, especially under iron-sufficient conditions.

Sequence Listing

<160>6

<210>1

<211>762

<212>DNA

<213> 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 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 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 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 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 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 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 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 Ser Arg Ser Gln Gly Glu Arg Leu Phe Tyr 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 (10)

1. The cDNA of a gene cooperating with AtbHLH29 to regulate iron absorption and metabolism in plants is one of the following nucleotide sequences: 1) SEQ ID №: 1 and SEQ ID №: 4 in the sequence listing are the coding sequences of AtbHLH38 and AtbHLH39 genes, respectively; 2) DNA sequences of SEQ ID №: 3 and SEQ ID №: 6 in the coding sequence list; 3) A nucleotide sequence that has more than 90% homology with the nucleotide sequence defined by Sequence 1 or SEQ ID No.: 4 in the sequence listing and plays an important role in regulating iron absorption and metabolism in plants; 4) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID №: 1 or SEQ ID №: 4 in the sequence listing under high stringency conditions. 2. The gene according to claim 1, characterized in that its cDNA has the DNA sequence of SEQ ID №: 1 or SEQ ID №: 4 in the sequence listing. 3. The genome sequence of the gene that cooperates with AtbHLH29 to regulate iron absorption and metabolism in plants is one of the following nucleotide sequences: 1) SEQ ID №: 2 and SEQ ID №: 5 in the sequence listing are the genomic DNA sequences of AtbHLH38 and AtbHLH39, respectively; 2) A nucleotide sequence that has more than 90% homology with the nucleotide sequence defined by Sequence 2 or SEQ ID No. 5 in the sequence listing and plays an important role in regulating iron absorption and metabolism in plants; 3) A nucleotide sequence that can hybridize to the DNA sequence defined by SEQ ID №: 2 or SEQ ID №: 5 in the sequence listing under high stringency conditions. 4. The protein encoded by the gene of claim 1, having one of the following amino acid residue sequences: 1) SEQ ID №: 3 and SEQ ID №: 6 in the sequence listing are the amino acid residue sequences of AtbHLH38 and AtbHLH39 proteins, respectively; 2) The amino acid residue sequence of SEQ ID №: 3 or SEQ ID №: 6 in the sequence listing undergoes one to ten substitutions, deletions or additions of amino acid residues and has the function of regulating plant iron absorption and metabolism by cooperating with AtbHLH29 protein. 5. The protein according to claim 4, characterized in that: the protein has the amino acid residue sequence of SEQ ID №: 3 or SEQ ID №: 6 in the sequence listing. 6. The expression vector, transgenic cell line and host bacterium containing the gene of claim 1 or 2 or 3. 7. A method for regulating the absorption and metabolism of iron elements by plants, which is to introduce AtbHLH29 and the gene described in claim 1 that cooperates with AtbHLH29 to regulate the absorption and metabolism of iron elements in plants into plant tissues or cells, and the absorption of iron elements by plants and metabolism are regulated. 8. The method according to claim 7, characterized in that: the AtbHLH29 and the genes that cooperate with AtbHLH29 to regulate the absorption and metabolism of iron elements in plants are respectively contained in AtbHLH29 and the genes that cooperate with AtbHLH29 to regulate the absorption and metabolism of iron elements in plants , or the plant expression vector containing AtbHLH29 and AtbHLH29 synergistically regulating the plant’s iron absorption and metabolism is introduced into the explant; the starting vector for constructing the plant expression vector is pBinplus, pBI121, pBin19, pCAMBIA2301 or pCAMBIA1300. 9. The method according to claim 8, characterized in that: the plant expression vector containing the gene that cooperates with AtbHLH29 to regulate iron absorption and metabolism in plants is pBI-38 or pBI-39, and the plant expression vector containing AtbHLH29 is pBinplus-29. 10. The method according to claim 7, 8 or 9, characterized in that said plants include monocots and dicots.

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