CN111944029A

CN111944029A - Low boron tolerance gene bdr1 in arabidopsis thaliana and encoding protein and application thereof

Info

Publication number: CN111944029A
Application number: CN202010848039.4A
Authority: CN
Inventors: 汪社亮; 徐芳森; 淮泽勋; 赵华
Original assignee: Huazhong Agricultural University
Current assignee: Huazhong Agricultural University
Priority date: 2020-08-21
Filing date: 2020-08-21
Publication date: 2020-11-17
Anticipated expiration: 2040-08-21
Also published as: CN111944029B

Abstract

The invention provides a low boron tolerance gene bdr1 in arabidopsis thaliana and a coding protein and application thereof, belonging to the technical field of molecular biology. The invention discloses a low boron tolerance gene bdr1 in Arabidopsis thaliana, which is obtained by mutating the 1165 th base from T to C on the basis of At4g 19180. The gene is proved to have no influence on the absorption and transportation efficiency of boron but can greatly improve the utilization efficiency of boron in cell walls through genetics and gene function analysis. Therefore, the low boron tolerance gene bdr1 and the protein coded by the gene provide a practical means for the creation of low boron tolerance germplasm resources of crops and provide a new reference for the research and application of the utilization efficiency of other essential nutrient elements of plants.

Description

Low boron tolerance gene bdr1 in arabidopsis thaliana and encoding protein and application thereof

Technical Field

The invention belongs to the technical field of molecular biology, and particularly relates to a low boron tolerance gene bdr1 in arabidopsis thaliana and a coding protein and application thereof.

Background

Boron (B) is a micronutrient element essential for plant growth and development. Plants absorb boron mainly through the root system. As boron is very soluble in water, the southern rainy region in China belongs to the region with insufficient effective boron in soil. Boron deficiency is an important factor limiting the yield and quality improvement of crops. The current situation of insufficient boron in soil is relieved by applying boron fertilizer in agricultural production, but the application of boron fertilizer directly increases the agricultural production cost. With the continuous research on the absorption, regulation and utilization mechanism of boron nutrition in plants, plant scientists expect that it is possible to improve the low boron tolerance of plants by genetic engineering means. Meanwhile, the method is an important measure which has low cost and high benefit and meets the agricultural requirements of China.

The japanese scientist raised the boroxin-tobacco boron inward transporter NIP 5; 1 and the boronic acid transporter protein BOR1 (Miwa et al, 2006, Kato et al, 2009). These reported genetic engineering methods for improving low boron tolerance in plants can improve the tolerance of plants under low boron conditions, but essentially at the cost of consuming more soil available boron. In agricultural production, soil available boron is further depleted with increasing cultivation time. Therefore, the improvement of the utilization efficiency of boron by means of bioengineering on the premise of not increasing the absorption of boron in soil becomes a research hotspot of scientific research on plant nutrition.

Disclosure of Invention

In view of the above, the present invention aims to provide a novel arabidopsis low boron tolerance gene bdr1, and a protein encoded by the gene bdr1, and applications of the gene.

The invention provides a low boron tolerance gene bdr1 in Arabidopsis, which is obtained by mutating the 1165 th base from T to C on the basis of At4g 19180.

Preferably, the nucleotide sequence is shown as SEQ ID No. 1.

The invention provides a coding protein of a low boron tolerance gene bdr1 in arabidopsis thaliana, and on the basis of a protein APY7, the 389 th amino acid is mutated from cysteine to arginine.

Preferably, the amino acid sequence is shown in SEQ ID No. 2.

The invention provides a primer pair for cloning a low boron resistance gene bdr1 in arabidopsis thaliana, which comprises an upstream primer shown as SEQ ID No.3 and a downstream primer shown as SEQ ID No. 4.

The invention provides application of the low boron tolerance gene bdr1, the coding protein or the primer pair in constructing low boron tolerance transgenic plants.

The invention provides application of the low boron tolerance gene bdr1, the encoded protein or the primer pair in planting or breeding of low boron tolerance plants.

Preferably, the low boron means that the concentration of boron element in the environment is not higher than 1 μ M.

The low boron tolerance gene bdr1 in Arabidopsis provided by the invention is obtained by mutating the 1165 th base from T to C on the basis of At4g 19180. Under the condition of low boron, compared with the wild type (Col-0) bdr1 mutant, the boron concentration in the overground part and the roots is not increased, but the RG-II double body in the cell wall is obviously increased, which proves that the gene does not influence the absorption and transportation efficiency of boron but can greatly improve the utilization efficiency of boron in the cell wall. Therefore, the invention provides a practical means for the creation of low boron tolerance germplasm resources of crops and provides a new reference for the research and application of the utilization efficiency of other essential nutrient elements of plants.

Drawings

FIG. 1 shows the growth phenotype of Arabidopsis thaliana wild type Col-0 and bdr1 mutants in culture medium conditions of 30. mu. M B and 0.1. mu. M B;

FIG. 2 shows the growth phenotype of Arabidopsis wild type Col-0 and bdr1 mutants on solid media of 30 μ M B and 0.1 μ M B;

FIG. 3 is an isolation of the F2 phenotype of the Arabidopsis thaliana wild type Col-0 and bdr1 mutant cross;

FIG. 4 is a graph of Δ SNP-index values calculated from DNA-bulk sequencing, where the value at Chr4.10486704 is one;

FIG. 5 shows that the bdr1 gene over-expression strain is less boron sensitive than Col-0 and bdr1 mutants in bdr1 mutant, and the bdr1 gene function complementation bdr1 phenotype is determined;

FIG. 6 shows the results of analysis of pectin components in roots of Col-0 and bdr1 mutants;

FIG. 7 shows the results of analysis of boron concentration in roots of Col-0 and bdr1 mutants.

Detailed Description

The invention provides a low boron tolerance gene bdr1 in Arabidopsis, which is obtained by mutating the 1165 th base from T to C on the basis of At4g 19180. The nucleotide sequence of gene bdr1 is preferably shown in SEQ ID No. 1. The At4g19180 has the sequence accession number NC-003075.7 shown in SEQ ID No.5 At the Gene ID:827656 At NCBI.

The invention provides a coding protein of a low boron tolerance gene bdr1 in arabidopsis thaliana, and on the basis of a protein APY7, the 389 th amino acid is mutated from cysteine to arginine. The amino acid sequence of the encoded protein is preferably shown as SEQ ID No. 2. The GenBank accession number of protein APY7 is NP _ 567579.2.

The invention provides a primer pair for cloning a low boron resistance gene bdr1 in arabidopsis thaliana, which comprises an upstream primer shown as SEQ ID No.3 and a downstream primer shown as SEQ ID No. 4. The source of the primer pair is not particularly limited in the present invention, and a primer synthesis method well known in the art may be used.

In the present invention, the method of cloning the low boron resistance gene bdr1 using the primer set may be a PCR method. The reaction program of the PCR is preferably 98 ℃, 10s, 55 ℃, 10s, 72 ℃, 2.5min, and 34 cycles. The present invention is not particularly limited to the PCR amplification system, and a PCR amplification system known in the art may be used.

In the present invention, the method for constructing a transgenic plant having a low boron tolerance preferably comprises the steps of:

cloning the low boron tolerance gene bdr1 by using the primer pair to obtain a PCR amplification product;

inserting the PCR amplification product into a plant expression vector to obtain a recombinant plant expression vector;

and introducing the recombinant plant expression vector into a plant by using agrobacterium to obtain a transgenic plant.

The present invention is not particularly limited in the kind of the plant expression vector, and a plant expression vector well known in the art may be used. The method for introducing a plant by using Agrobacterium of the present invention is not particularly limited, and a method for transforming Agrobacterium known in the art may be used.

In the present invention, the breeding is preferably performed based on the presence or absence of the low boron tolerance gene bdr1 or a protein encoding the same in the plant to be bred. The breeding method preferably adopts the primer pair to carry out PCR amplification.

In the present invention, the low boron means that the concentration of boron element in the environment is preferably not higher than 1 μ M, more preferably 0.5 to 0.001 μ M, and most preferably 0.1 μ M.

The invention provides a low boron tolerance gene bdr1 in arabidopsis thaliana, and a protein encoded by the gene and the application of the gene, which are described in detail below with reference to examples, but the invention is not limited to these.

Example 1

bdr1 mutant Low boron tolerance assay

(1) By formulating a liquid medium containing both 0.1. mu. M B and 30. mu. M B (Takano et al, 20006), (NaH) required for growth of Arabidopsis thaliana₂PO₄·2H₂O 1.51mM，Na₂HPO₄·12H₂O 0.26mM，MgSO₄·7H₂O 1.5mM，Ca(NO₃)₂·4H₂O 2.0mM，KNO₃3.0 mM，MnSO₄10.3μM，ZnSO₄·7H₂O 1.0μM，CuSO₄·5H₂O 1.0μM，CoCl₂·6H₂O 130nM，(NH₄)₆Mo₇O₂₄·4H₂O 24nM，FeSO₄·7H₂O 50μM，Na₂EDTA·2H₂O50 μ M) to compare the overground growth phenotype of the wild type (Col-0) and bdr1 mutants.

As shown in FIG. 1, the overground growth phenotype of the two was not greatly different under the condition of 30. mu. M B; whereas under 0.1 μ M B conditions, the bdr1 mutant had a larger leaf area, suggesting that the bdr1 mutant has a low boron tolerance phenotype.

(2) The growth phenotype of the roots of Col-0 and bdr1 mutants was analyzed by formulating a solid medium containing 0.1. mu. M B and 30. mu. M B (liquid medium with 1% Gellan gam) required for Arabidopsis growth.

The results are shown in FIG. 2. As can be seen from FIG. 2, the difference between the major root lengths of the two plants under the condition of 30 μ M B is not large; whereas under 0.1 μ M B conditions, the bdr1 mutant had significantly longer main roots, suggesting that the bdr1 mutant had a low boron tolerance phenotype.

Example 2

bdr1 identification of Gene

To determine which gene in the bdr1 mutant determines low boron tolerance, construction of hybrid materials of Col-0 and bdr1 mutants was performed

(1) Artificial pollination is carried out in flowering period by using Col-0 as male parent and bdr1 mutant as female parent, and the obtained F1 generation material is propagated again to obtain F2 generation. Phenotypic identification was performed with 0.1 μ M B low boron solid medium as in example 1.

The results are shown in FIG. 3. The results, using the length of the main root as the identification criterion, showed that the ratio of long to short roots was 1:3 (table 1), which complies with mendelian's law of inheritance, indicating that the low boron resistance phenotype is determined by a recessive single gene.

TABLE 1 statistical data for phenotypic segregation

(2) F2 long root 30 strain is selected as a DNA bulk, short root 20 strain is selected as another DNA bulk, and 10 Col-0 and bdr1 mutants are selected as control DNA bulk. The high-purity DNA is extracted by adopting a DNA extraction kit method. Taking 0.1g F2 young leaves, and grinding into powder with liquid nitrogen. 400 μ L of AP1Buffer containing 1 μ L of RNase (100mg/mL) was added in a 65 ℃ water bath for 10 min. Add 130. mu. L P3 Buffer, ice-wash for 5min, 14000rpm, and centrifuge for 5 min. The supernatant was filtered by QIAshredder Mini spin column (lilac), 14000rpm, and centrifuged for 5 min. To the filtrate was added AW 1Buffer (containing ethanol) in an amount of 1.5 times the volume of the filtrate, and the mixture was mixed. The mixture was transferred to a DNeasy Mini spin column at 8000rpm and centrifuged for 1 min. Add 500. mu.L AW2 Buffer, centrifuge twice at 8000rpm, centrifuge for 2min at 14000 rpm. Add 100. mu.L AE Buffer, and stand at room temperature for 5min, 8000rpm, 1min to recover DNA. The obtained DNA bulk is sent to a sequencing platform for sequencing, the machine is Illumina Hiseq 4000(San Diego, CA, USA), the sequencing mode is double-End sequencing (seed-End, PE), and the length of each read is 100 bp.

(3) And (6) analyzing the data.

Using SOAP2 software (Nielsen et al 2011,http://soap.genomics.org.cn/html) the sequenced reads were aligned to the arabidopsis reference genome (Tair 10). And calculating the sequencing depth and the coverage degree relative to the reference genome according to the alignment result. And (4) detecting SNP variation sites by using SNPsnp software. Since the control bdr1 low boron tolerant phenotype must be met, the corresponding SNP site screening criteria should be SNPs that are divergent and homozygous in both parents, consistent with homozygous SNPs in the low boron tolerant (long root) DNA bulk, and inconsistent with SNPs in the low boron sensitive (short root) DNA bulk. SNPs that meet this criteria are and only have one Chr4.10486704 (Table 2).

TABLE 2 SNP results of each DNA-bulk sequencing analysis

Its corresponding Δ SNP-index is 1, see fig. 4(Takagi et al 2015), demonstrating that this SNP (chr4.10486704) is highly likely to be the mutant gene controlling the bdr1 low boron resistance phenotype.

According to the reference genome annotation information, the SNP is located at 10,486,704bp on the fourth chromosome of Arabidopsis thaliana. As shown in Table 3, the nucleic acid of this site in Col-0 is adenine A, the nucleic acid in bdr1 is guanine G, the nucleic acid on the complementary strand thereof is thymine T in Col-0 and cytosine C in bdr 1. This mutation occurred in gene At4g19180 encoding a protein APY7, corresponding to the amino acid information cysteine (TGC) At position 389 in Col-0 and arginine (CGC) At position 389 in bdr 1.

Corresponding SNP nucleic acid information and corresponding encoded amino acid information in Table 3 Col-0 and bdr1

Example 3

bdr1 Gene cloning and functional complementation analysis

Designing primer clone gene by using open reading frame ORF sequence of At4g19180 coded protein in reference genome as template, and reintroducing the gene into bdr1 mutant to verify functional complementation

(1) Total RNA extraction and reverse transcription

Total RNA extraction reagent (A) is used for extracting total RNA of plants

Super Total RNA Extraction Kit, Promega, Madison, Wis., USA), please refer to Kit instructions. The brief steps are as follows: a Col-0 sample growing for seven days is taken, liquid nitrogen is ground into powder, and lysate is added to be uniformly dissolved. Mixing the sample with RNA diluent for 5min, centrifuging at 12,000g for 5min, collecting supernatant, adding anhydrous ethanol, mixing, and filtering with centrifugal column. Adding DNA enzyme, incubating for 15min, washing with RNA lotion twice, centrifuging at 12,000g, eluting with ribozyme-free water, and storing at-80 deg.C. Mu.g of RNA was reverse transcribed into first strand cDNA using M-MLV reverse transcriptase (Promega, Madison, Wis., US) and oligo (dT)15primers (Promega, Madison, Wis., USA) as follows: system I (Table 4) was prepared and then reverse transcribed at 65 ℃ for 15 min.

TABLE 4 System I formulated in reverse transcription of RNA

System II (Table 5) was prepared and the two systems were mixed and extended at 42 ℃ for 1 hour to obtain first strand cDNA.

TABLE 5 System II formulated in reverse transcription of RNA

(2) bdr1 Gene cloning and plant Material construction

Two primers were designed to contain two restriction sites XbaI and SacI for construction into the plant binary expression vector PBI 121. The primer pairs were 5'-GCTCTAGAATGGTTTTTGGTAGGATCAC-3' (SEQ ID No.3) and 5'-GCGAGCTCCTACATTTTGAGCATGTGTG-3' (SEQ ID No. 4). The PCR reaction system is as follows: PrimeSTAR HS (Premix) 25. mu.L, each 0.5. mu.L of primers, 0.5. mu.L of first strand template, and water to 50. mu.L. The PCR program was set at 98 deg.C, 10s, 55 deg.C, 10s, 72 deg.C, 2.5min for 34 cycles. Direct sequencing of the PCR product verified the correct bdr1 sequence. The PBI121 plasmid was treated with XbaI and SacI restriction endonucleases to recover the backbone ligation PCR product, yielding a 35s: bdr1 plasmid. Transgenic material which is transformed into 35s, bdr1 and resistant to kanamycin is obtained by a GV3101 mediated Arabidopsis bud transformation method, and the background is bdr1 mutant. On the basis of At4g19180 in the bdr1 mutant, the 1165 th base mutation site C can be realized by a conventional CRISPR/Cas9 gene editing tool, so that the bdr1 mutant is obtained by repeating the conventional molecular biology means.

(3) bdr1 functional verification

Considering whether the amino acid substitution by the point mutation in bdr1 indeed abolished the function of the At4g19180 gene, thereby allowing arabidopsis plants to acquire low boron tolerance, the following validation was performed. Another bdr1-2 mutant was selected to verify whether the functional deletion of the At4g19180 gene has application value in improving low boron tolerance. bdr1-2 mutant is the functional deletion of At4g19180 gene by inserting a T-DNA sequence.

FIG. 5 shows that the bdr1-2 mutant has a low boron tolerance phenotype comparable to the bdr1 mutant, indicating that loss of function of the At4g19180 gene has the function of increasing low boron tolerance in plants. The phenotype was identified with two mutant lines bdr1 and bdr1-2 of Col-0, two lines 35s: bdr1, low boron conditions (0.1. mu. M B). The results are shown in FIG. 5, which shows that the overexpression strain can restore the bdr1 mutant sensitivity to low boron, and has no significant difference with Col-0. Thus, point mutant bdr1 of the bdr1 gene is a determinant gene that results in the Arabidopsis low boron tolerance phenotype.

Since the content of RG-II diploids determines the sensitivity of plants to Boron deficiency, the pectin content of Col-0 and bdr1 roots was analyzed by mass spectrometry (FIG. 6), see the prior art (MiwaK, Wakuta S, Takada S, Ide K, Takano J, Naito S, Omori H, Matsunagat, Fujiwara T.2013. circles of BOR2, a Boron Exporter, in Cross Linking of Rhamnalactrontron II and Root Electron amplification in Boron Limitation. PLANT 163: 1699.). The results showed that the RG-II diplome content in the bdr1 mutant was significantly increased compared to Col-0, so that the bdr1 mutant showed higher low boron tolerance.

ICP-MS was further used to analyze the Boron content of Col-0 and bdr1 mutants under low Boron conditions, as found in the prior art (Wang S, Yoshinari A, Shimada T, Hara-Nishimura I, Mitani-Ueno N, Feng J, Feng Ma J, Naito S, Takano J.2017 polar Localization of the NIP 5; 1 Boricic Channel Is Main by endo enzymosis and facts Boron Transport in Arabidopsis roots.824 the Plant Cell29: 824) and showed no significant difference therebetween (FIG. 7), thus bdr1 mutant showed improved Boron utilization efficiency without affecting Boron absorption, thereby demonstrating low Boron tolerance for growth.

The foregoing is only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Sequence listing

<110> university of agriculture in Huazhong

<120> low boron tolerance gene bdr1 in arabidopsis thaliana and encoding protein and application thereof

<160> 5

<170> SIPOSequenceListing 1.0

<210> 1

<211> 2223

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 1

atggtttttg gtaggatcac tgaacttttt acagctgcgt caagtcgatt accagcgggt 60

tcacaatcat ctgttccata tatgcctact ggatcatcac cggatgtagg aacctcagtt 120

tcggattcaa taagtattgg gaatggtgga cggaagaatt gtttgagaca ctcagcgtct 180

ctccaagatt tttcttcgta ccatgggttt gatcctgaag aatccattct tcctcgggaa 240

gctatatcat ggggacaaaa tggatcaagt ttttccaagg aaaaggggag tgtccctaat 300

gggaccaatc catctacacg ccgtaaattg atacgcgcgg ttatgattgt tatgtgcctg 360

tttctgtttg cttttctggt gtatatagtc tccatgtata tttatacaaa ctggtcacgg 420

ggagcatcaa gatattatgt tgtgtttgac tgtggaagta ctgggactcg tgcatatgtt 480

tatcaggctt ccataaacta caagaaagat agtagtctcc ccattgttat gaaatctttg 540

acagaaggta tttctagaaa gagtaggggt cgtgcttatg atcggatgga gacagagcct 600

gggtttgata agctcgtcaa taacagaaca ggcctgaaaa ctgcaattaa accccttatt 660

cagtgggcag agaagcaaat cccaaagaat gctcatagaa ccacatctct ctttgtttac 720

gcaacagcag gcgtccggag gcttcgtcct gctgattcaa gctggatttt aggtaatgtg 780

tggtctatac tagcgaagtc tcccttcact tgccggaggg agtgggttaa gatcatcagt 840

ggtactgagg aagcttattt tggatggacc gcccttaatt accaaacaag tatgttggga 900

gccctaccaa agaaggcaac atttggtgca cttgacttag gtggctcatc attgcaggta 960

acctttgaga atgaggagcg gacgcataat gagaccaacc taaatctcag aattgggtct 1020

gttaaccatc atcttagtgc atattctctt gccggatatg gtcttaatga tgcgtttgat 1080

aggtctgttg ttcatctttt aaagaagcta ccaaatgtca acaaatctga tttgattgaa 1140

ggaaaattgg aaatgaaaca cccgcgctta aattctggat acaatggaca gtacatatgt 1200

tctcagtgtg cttccagtgt ccaaggaggt aaaaaaggaa agtcaggagt ttctatcaag 1260

ctcgttggtg ctccaaactg gggagagtgc agcgcacttg caaaaaatgc tgtaaatagc 1320

tctgaatggt caaacgcaaa acatggagtt gattgtgatt tgcagccttg tgctcttcct 1380

gatggttacc ctcgtcctca tggtcagttc tatgctgtat ccggattctt cgtggtgtac 1440

cgttttttca acttaagtgc tgaagcatcc cttgatgatg tattggaaaa gggtagggaa 1500

ttttgtgaca aggcttggca agtagccaga accagtgttt ctccacaacc cttcattgaa 1560

cagtactgct ttagagcgcc atacatagtc tccctgttac gagaaggttt atatattaca 1620

gataagcaaa tcataattgg gtctgggagt ataacttgga cacttggagt agctcttctg 1680

gaatcgggga aggctctatc ttctacattg ggactgaaga gctatgagac tctgagcatg 1740

aagataaacc caattgccct tatatctatc ttgatcttgt cactgcttct gcttctctgt 1800

gcactatcac gtgtcagcaa ttgtttgcca agattcttta gaaagtcata tctcccactt 1860

tttagacaca atagcacctc agcttcgtct gttcttaaca tcccgtctcc cttcagattc 1920

cagcgatgga gtcctatgag tacaggggta aagacaccat tgagtccaac agttcgggga 1980

tcaccgcgta ggccatttag ttttggaagc agcatccagc tcatggaatc ttcattgtac 2040

tcatcgagta gctgcgttat gcatagttgt tcttctgata gcttagggga tatacaatat 2100

gacagtactg gttccttctg gtcctcccct cgcaggagtc aaatgcgtct ccaaagccga 2160

agatcgcaat ctcgagaaga tcttagttca tcgctcgctg attcacacat gctcaaaatg 2220

tag 2223

<210> 2

<211> 740

<212> PRT

<213> Artificial Sequence (Artificial Sequence)

<400> 2

Met Val Phe Gly Arg Ile Thr Glu Leu Phe Thr Ala Ala Ser Ser Arg

1 5 10 15

Leu Pro Ala Gly Ser Gln Ser Ser Val Pro Tyr Met Pro Thr Gly Ser

20 25 30

Ser Pro Asp Val Gly Thr Ser Val Ser Asp Ser Ile Ser Ile Gly Asn

35 40 45

Gly Gly Arg Lys Asn Cys Leu Arg His Ser Ala Ser Leu Gln Asp Phe

50 55 60

Ser Ser Tyr His Gly Phe Asp Pro Glu Glu Ser Ile Leu Pro Arg Glu

65 70 75 80

Ala Ile Ser Trp Gly Gln Asn Gly Ser Ser Phe Ser Lys Glu Lys Gly

85 90 95

Ser Val Pro Asn Gly Thr Asn Pro Ser Thr Arg Arg Lys Leu Ile Arg

100 105 110

Ala Val Met Ile Val Met Cys Leu Phe Leu Phe Ala Phe Leu Val Tyr

115 120 125

Ile Val Ser Met Tyr Ile Tyr Thr Asn Trp Ser Arg Gly Ala Ser Arg

130 135 140

Tyr Tyr Val Val Phe Asp Cys Gly Ser Thr Gly Thr Arg Ala Tyr Val

145 150 155 160

Tyr Gln Ala Ser Ile Asn Tyr Lys Lys Asp Ser Ser Leu Pro Ile Val

165 170 175

Met Lys Ser Leu Thr Glu Gly Ile Ser Arg Lys Ser Arg Gly Arg Ala

180 185 190

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

195 200 205

Arg Thr Gly Leu Lys Thr Ala Ile Lys Pro Leu Ile Gln Trp Ala Glu

210 215 220

Lys Gln Ile Pro Lys Asn Ala His Arg Thr Thr Ser Leu Phe Val Tyr

225 230 235 240

Ala Thr Ala Gly Val Arg Arg Leu Arg Pro Ala Asp Ser Ser Trp Ile

245 250 255

Leu Gly Asn Val Trp Ser Ile Leu Ala Lys Ser Pro Phe Thr Cys Arg

260 265 270

Arg Glu Trp Val Lys Ile Ile Ser Gly Thr Glu Glu Ala Tyr Phe Gly

275 280 285

Trp Thr Ala Leu Asn Tyr Gln Thr Ser Met Leu Gly Ala Leu Pro Lys

290 295 300

Lys Ala Thr Phe Gly Ala Leu Asp Leu Gly Gly Ser Ser Leu Gln Val

305 310 315 320

Thr Phe Glu Asn Glu Glu Arg Thr His Asn Glu Thr Asn Leu Asn Leu

325 330 335

Arg Ile Gly Ser Val Asn His His Leu Ser Ala Tyr Ser Leu Ala Gly

340 345 350

Tyr Gly Leu Asn Asp Ala Phe Asp Arg Ser Val Val His Leu Leu Lys

355 360 365

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

370 375 380

Met Lys His Pro Arg Leu Asn Ser Gly Tyr Asn Gly Gln Tyr Ile Cys

385 390 395 400

Ser Gln Cys Ala Ser Ser Val Gln Gly Gly Lys Lys Gly Lys Ser Gly

405 410 415

Val Ser Ile Lys Leu Val Gly Ala Pro Asn Trp Gly Glu Cys Ser Ala

420 425 430

Leu Ala Lys Asn Ala Val Asn Ser Ser Glu Trp Ser Asn Ala Lys His

435 440 445

Gly Val Asp Cys Asp Leu Gln Pro Cys Ala Leu Pro Asp Gly Tyr Pro

450 455 460

Arg Pro His Gly Gln Phe Tyr Ala Val Ser Gly Phe Phe Val Val Tyr

465 470 475 480

Arg Phe Phe Asn Leu Ser Ala Glu Ala Ser Leu Asp Asp Val Leu Glu

485 490 495

Lys Gly Arg Glu Phe Cys Asp Lys Ala Trp Gln Val Ala Arg Thr Ser

500 505 510

Val Ser Pro Gln Pro Phe Ile Glu Gln Tyr Cys Phe Arg Ala Pro Tyr

515 520 525

Ile Val Ser Leu Leu Arg Glu Gly Leu Tyr Ile Thr Asp Lys Gln Ile

530 535 540

Ile Ile Gly Ser Gly Ser Ile Thr Trp Thr Leu Gly Val Ala Leu Leu

545 550 555 560

Glu Ser Gly Lys Ala Leu Ser Ser Thr Leu Gly Leu Lys Ser Tyr Glu

565 570 575

Thr Leu Ser Met Lys Ile Asn Pro Ile Ala Leu Ile Ser Ile Leu Ile

580 585 590

Leu Ser Leu Leu Leu Leu Leu Cys Ala Leu Ser Arg Val Ser Asn Cys

595 600 605

Leu Pro Arg Phe Phe Arg Lys Ser Tyr Leu Pro Leu Phe Arg His Asn

610 615 620

Ser Thr Ser Ala Ser Ser Val Leu Asn Ile Pro Ser Pro Phe Arg Phe

625 630 635 640

Gln Arg Trp Ser Pro Met Ser Thr Gly Val Lys Thr Pro Leu Ser Pro

645 650 655

Thr Val Arg Gly Ser Pro Arg Arg Pro Phe Ser Phe Gly Ser Ser Ile

660 665 670

Gln Leu Met Glu Ser Ser Leu Tyr Ser Ser Ser Ser Cys Val Met His

675 680 685

Ser Cys Ser Ser Asp Ser Leu Gly Asp Ile Gln Tyr Asp Ser Thr Gly

690 695 700

Ser Phe Trp Ser Ser Pro Arg Arg Ser Gln Met Arg Leu Gln Ser Arg

705 710 715 720

Arg Ser Gln Ser Arg Glu Asp Leu Ser Ser Ser Leu Ala Asp Ser His

725 730 735

Met Leu Lys Met

740

<210> 3

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 3

gctctagaat ggtttttggt aggatcac 28

<210> 4

<211> 28

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 4

gcgagctcct acattttgag catgtgtg 28

<210> 5

<211> 4600

<212> DNA

<213> Artificial Sequence (Artificial Sequence)

<400> 5

ttctcgtgga atcatttgac acaatttcat ctggtttttc ttctctctag ggagttatcg 60

catcagcact aaactatgga ctcttgactt ggtccaataa gattattggt cctgccttgg 120

ttgctctcta caatcctctt caacccgctg catcagcttt cctatcgaga atctttctcg 180

gtagccccat ttatctcgga aggttagata cattttctgg aatctttttc aaccatccac 240

aatcttcact tgcaaaatta aacactttca cttttcaaca tcttgttcca tttcacagtt 300

ttcacttgca aaattttcta actcggtttc ttttcctcta cgcagcgttg tgggtggatt 360

cttcatcatt ctaggactct atatggtcac ttgggcatcg tttagagagc gaaaaacagc 420

ggtttccggg attgggattg cgccccatgg tctaaaaacc tcagagcctc tgatatttaa 480

tgggacagtt aacagactag gacagttgtt ttctggatta cccagttcct ctgtgaaatc 540

tgcagactaa aaagatacca aaaagttaag attcagacga gatcccattt tacaacagct 600

ataacttttt gttaactttc atctcttatt aatatcttct tcgattacta ctaatcacag 660

attcacaagt acttgctccc ttgttaatta ctctttaatc ttgaagctca tctaattcgc 720

attggttata tatctaaacc atatattagc tatgaagatg acattttatc tttacctttg 780

aattttttaa caatagataa cattcagtgc aagatataca atatagtctt aggttaaaag 840

ggggatatca taaagttgga aaaagcaatc caagaaaaag ctgtgtctta tttggtaaga 900

acacataaca gtagtggcaa atgtacgaat tccaaaaaag aagttggttt tatcccaatc 960

tagtttaact ttttcgcaaa aaaatcacac gagttatcaa aaaaacgtgg aactttattc 1020

tttctttttg gcaactaaaa ttgtttttag ccttaggtaa tctcttaaag gtatacaaat 1080

tggatgagtg acacacacca ttagcatttt aatatcggcg ccattagcat tttttaacca 1140

acaaatagac gatttgttaa ataaattact aagaaagaaa taaacttatt tcatttacat 1200

ttccacccca aatgttttca aacattctaa atttcccctg gaaattgaaa attgagcaag 1260

acgttttctg ttgaagagag aaaataaaaa tcagagaaca aaaagggaaa gagtcttttc 1320

tgcacaaaac ggatctctct ccaacgtctt cgtcttcctc tggtctgagt cgtgtggttt 1380

tagccccgat actcatcaga tctgcttaag attctcgatt tttcttatac gaagacgacg 1440

attggttgat taactcatat atgtatatgt gcttttatac tccttagacg cagagcgaga 1500

tacgagatct aagagctgag gaagctttga cggtggctga agtttcgtct tcgttctatg 1560

ccaatgcgtg tttctggtat tgtaccctag ctatccaatt ttatgattga gtcgttgtgt 1620

tgttatcgca atgttgattt aactgcagat cgctcctttc attgtgtttg ttgatttgtg 1680

aatcactcaa gctcttttcc gagatttttg attgaagaca taaattgtcg atattcattg 1740

ttgaatgcat ttgatttgat ttgtgtagtt aacattttct gcagaagaat gaagaatctg 1800

aagagttcgt tgtgcttctg catttgagta aagacaacgg agaaggttgt gttgagtttg 1860

ttagtatatg taggcaagag aaatggtttt tggtaggatc actgaacttt ttacagctgc 1920

gtcaagtcga ttaccagcgg gttcacaatc atctgttcca tatatgccta ctggatcatc 1980

accggatgta ggaacctcag tttcggattc aataagtatt gggaatggtg gacggaagaa 2040

ttgtttgaga cactcagcgt ctctccaaga tttttcttcg taccatgggt ttgatcctga 2100

agaatccatt cttcctcggg aagctatatc atggggacaa aatggatcaa gtttttccaa 2160

ggaaaagggg agtgtcccta atgggaccaa tccatctaca cgccgtaaat tgatacgcgc 2220

ggttatgatt gttatgtgcc tgtttctgtt tgcttttctg gtgtatatag tctccatgta 2280

tatttataca aactggtcac ggggagcatc aagatattat gttgtgtttg actgtggaag 2340

tactgggact cgtgcatatg tttatcaggc ttccataaac tacaagaaag atagtagtct 2400

ccccattgtt atgaaatctt tgacagaagg tatttctaga aagagtaggg gtcgtgctta 2460

tgatcggatg gagacagagc ctgggtttga taagctcgtc aataacagaa caggcctgaa 2520

aactgcaatt aaacccctta ttcagtgggc agagaagcaa atcccaaaga atgctcatag 2580

aaccacatct ctctttgttt acgcaacagc aggcgtccgg aggcttcgtc ctgctgattc 2640

aagctggatt ttaggtaatg tgtggtctat actagcgaag tctcccttca cttgccggag 2700

ggagtgggtt aagatcatca gtggtactga ggaagcttat tttggatgga ccgcccttaa 2760

ttaccaaaca agtatgttgg gagccctacc aaagaaggca acatttggtg cacttgactt 2820

aggtggctca tcattgcagg taacctttga gaatgaggag cggacgcata atgagaccaa 2880

cctaaatctc agaattgggt ctgttaacca tcatcttagt gcatattctc ttgccggata 2940

tggtcttaat gatgcgtttg ataggtctgt tgttcatctt ttaaagaagc taccaaatgt 3000

caacaaatct gatttgattg aaggaaaatt ggaaatgaaa cacccgtgct taaattctgg 3060

atacaatgga cagtacatat gttctcagtg tgcttccagt gtccaaggag gtaaaaaagg 3120

aaagtcagga gtttctatca agctcgttgg tgctccaaac tggggagagt gcagcgcact 3180

tgcaaaaaat gctgtaaata gctctgaatg gtcaaacgca aaacatggag ttgattgtga 3240

tttgcagcct tgtgctcttc ctgatggtta ccctcgtcct catggtcagt tctatgctgt 3300

atccggattc ttcgtggtgt accgtttttt caacttaagt gctgaagcat cccttgatga 3360

tgtattggaa aagggtaggg aattttgtga caaggcttgg caagtagcca gaaccagtgt 3420

ttctccacaa cccttcattg aacagtactg ctttagagcg ccatacatag tctccctgtt 3480

acgagaaggt ttatatatta cagataagca aatcataatt gggtctggga gtataacttg 3540

gacacttgga gtagctcttc tggaatcggg gaaggctcta tcttctacat tgggactgaa 3600

gagctatgag actctgagca tgaagataaa cccaattgcc cttatatcta tcttgatctt 3660

gtcactgctt ctgcttctct gtgcactatc acgtgtcagc aattgtttgc caagattctt 3720

tagaaagtca tatctcccac tttttagaca caatagcacc tcagcttcgt ctgttcttaa 3780

catcccgtct cccttcagat tccagcgatg gagtcctatg agtacaggta actcaatatg 3840

cataatcttg agttatggcc agttcttctg ttctcagttc ttaacattgt ctttctttgt 3900

gtcgttaatt ttctttttgg gaagatattt attcttagag cttattttct ttcaggggta 3960

aagacaccat tgagtccaac agttcgggga tcaccgcgta ggccatttag ttttggaagc 4020

agcatccagc tcatggaatc ttcattgtac tcatcgagta gctgcgttat gcatagttgt 4080

tcttctgata gcttagggga tatacaatat gacagtactg gttccttctg gtcctcccct 4140

cgcaggagtc aaatgcgtct ccaaagccga agatcgcaat ctcgagaaga tcttagttca 4200

tcgctcgctg attcacacat gctcaaaatg taggagaaat ttttggtacg gcttttgtca 4260

cttccacacc agcctaattt tttctatttt ccggttatga gaatcacaat atagaaagga 4320

aaaaaatggg aaaacttcat gagcaaaaat ttccttgttt cagttgacat gtaattcttt 4380

ctatatacca tatacttata tatgattact aatttaacct cagttcaact accaatcact 4440

ctcttcctct ttaccctttt tgtgtatttc tccaaattct ttcttttaca gagctggtcc 4500

aagttctctt ttcatctcca agtgtcttgg gtttgagaga gaagaatcga ttcccagatt 4560

gaaaataaaa cctgtatgtg ctcattttct gctccttgct 4600

Claims

1. A low boron tolerance gene bdr1 in Arabidopsis thaliana is characterized in that the 1165 th base is obtained by mutating T to C on the basis of At4g 19180.

2. The Arabidopsis thaliana low boron tolerance gene bdr1 of claim 1, wherein the nucleotide sequence is represented by SEQ ID No. 1.

3. A coding protein of a low boron tolerance gene bdr1 in arabidopsis thaliana is characterized in that on the basis of a protein APY7, the 389 th amino acid is mutated from cysteine to arginine.

4. The Arabidopsis thaliana low boron tolerance-encoding protein according to claim 3, wherein the amino acid sequence thereof is represented by SEQ ID No. 2.

5. A primer pair for cloning the low boron resistance gene bdr1 in Arabidopsis thaliana as described in claim 1 or2, comprising an upstream primer shown by SEQ ID No.3 and a downstream primer shown by SEQ ID No. 4.

6. Use of the low boron tolerance gene bdr1 of claim 1 or2, the encoded protein of claim 2 or 4, or the primer pair of claim 5 for constructing a transgenic plant with low boron tolerance.

7. Use of the low boron tolerance gene bdr1 of claim 1 or2, the encoded protein of claim 2 or 4, or the primer pair of claim 5 for the cultivation or selection of a plant with low boron tolerance.

8. The use according to claim 6 or 7, wherein low boron means that the concentration of boron element in the environment is not higher than 1 μ M.