CN100500847C - Iron binding protein of small red beam, coding gene and application thereof - Google Patents
Iron binding protein of small red beam, coding gene and application thereof Download PDFInfo
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- CN100500847C CN100500847C CNB2005100683451A CN200510068345A CN100500847C CN 100500847 C CN100500847 C CN 100500847C CN B2005100683451 A CNB2005100683451 A CN B2005100683451A CN 200510068345 A CN200510068345 A CN 200510068345A CN 100500847 C CN100500847 C CN 100500847C
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Abstract
The present invention discloses a kind of small red bean iron binding protein and code gene and application. The purpose is that offer a kind of small red bean iron binding protein and code gene and application in breeding of iron rich plant. This protein has amino acid serials as followings: (1) The list of SEQ ID NO:1, (2) taking in the list of SEQ ID NO:1 amino acid from one to ten amino acid substitute, absence or add protein which iron enrichment action. The coging gene has one of following nucleotide acid series: (1) The list of SEQ ID NO:2 is DNA series, (2) on high preciseness condition can cross fertilize with the list of SEQ ID NO:2 restrained DNA series amino acid serials .The gene in this invention has an important merit in breeding of iron rich plant, offer an economic and effective solution of iron is common lack at the moment.
Description
Technical field
The present invention relates to vegetable-protein and encoding gene thereof and application, particularly relate to a kind of iron binding protein of small red beam and encoding gene thereof and its application in cultivating iron-dense plants.
Background technology
Iron is that human body carries out one of necessary element of physiological metabolism activity, and iron deficiency is grown by children's intelligence, muscle power and immunity, function such as digest and assimilate considerable influence is all arranged.30% population iron deficiency is arranged in the world wide approximately, wherein, serious with women and children.Can prevent and treat iron deficiency though eat the food that adds ferro element and eat of benefit iron medicine, the cost of this prevention and treatment iron deficiency is too high, is difficult to popularize in developing country.
Iron-binding protein is the protein that is present in a kind of special storage iron in the vegetable cell plastid.Goto et al agrobacterium-mediated transformation imports paddy rice, the transgenic paddy rice T that obtains with peculiar promotor GluB-1 of rice paddy seed storage protein and Soybean Ferritin Gene
1Iron level for seed is more than 3 times of non-transgenic rice paddy seed (GotoF Yoshihara T, Shigemoto N, Takaiwa F, 1999, Ironfortifiation of rice seedby the soybean ferritin gene.Nature Biotechology, 17:282-286).This ferritin transgenic paddy rice is also tested in the mouse of iron deficiency, the result shows that transgenic paddy rice source of iron that provides and the bioavailable iron source that provides with other form can reach identical result of treatment (Murray-Kolb LE, Takaiwa F GotoF et al.2002, Transgenic rice is a source of iron for iron for iron-depletedrats.J Nutr, 132 (5): 957-960).
Separation and clone about iron-binding protein gene in the plant have related to plants such as soybean, pea, cowpea, clover, corn, rape, Arabidopis thaliana, potato and orchid, but these gene China do not have independent intellectual property right.In addition, being cloned in of iron-binding protein gene still do not have report both at home and abroad in the relevant Semen Ormosiae Hosiei.
Summary of the invention
The purpose of this invention is to provide a kind of iron binding protein of small red beam and encoding gene thereof.
Iron binding protein of small red beam provided by the present invention, name is called A-Fer, derives from Vigna Semen Ormosiae Hosiei (Vignaangularis), and it has one of following amino acid residue sequences:
1) the SEQ ID № in the sequence table: 1;
2) with SEQ ID № in the sequence table: 1 amino acid residue sequence is through replacement, disappearance or the interpolation of one to ten amino-acid residue and have the iron acting protein of enrichment.
SEQ ID № in the sequence table: 1 is made up of 255 amino-acid residues.
The encoding gene of iron binding protein of small red beam provided by the present invention (A-Fer) also belongs to protection scope of the present invention.It can have one of following nucleotide sequence:
1) SEQ ID № in the sequence table: 2 dna sequence dna;
2) under the rigorous condition of height can with SEQ ID № in the sequence table: the nucleotide sequence of the 2 dna sequence dnas hybridization that limit.
The rigorous condition of described height be under 65 ℃ of conditions hybridization and wash film, film washing liquid is 0.1 * SSC, 0.1%SDS.
SEQ ID № in the sequence table: 2 by 1030 based compositions, and its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 81-848 bit base.
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 A-Fer.
Utilize plant expression vector,, can obtain transgenic cell line and transfer-gen plant that iron-holder improves iron binding protein of small red beam gene transfered plant cell of the present invention.
Described plant expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of plant micropellet bombardment.Described plant expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprise the polyadenylic acid signal and any other participation mRNA processing or the dna fragmentation of genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor, and the non-translational region of inducing (Ti) plasmid gene (as kermes synthetic enzyme Nos gene), plant gene (as soybean storage protein gene) 3 ' end to transcribe as the Agrobacterium crown-gall nodule all has similar functions.
When using A-Fer to make up plant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or inducible promoter, as cauliflower mosaic virus (CAMV) 35S promoter, root specific expression promoter 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, 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-weedkiller gene) etc.From the security consideration of transgenic plant, can not add any selected marker, directly with adverse circumstance screening transformed plant.
Carry A-Fer of the present invention 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.By the plant transformed host both can be monocotyledonss such as paddy rice, wheat, corn, also can be dicotyledonss such as Semen Ormosiae Hosiei, soybean, romaine lettuce, Arabidopis thaliana.
The encoding gene of iron binding protein of small red beam provided by the invention will have important use and be worth in cultivating iron-dense plants, provide an economy, valid approach for solving present ubiquitous sideropenia problem.
The present invention will be further described below in conjunction with specific embodiment.
Description of drawings
Fig. 1 is 1% an agarose gel electrophoresis detected result of 3 ' RACE amplified production
Fig. 2 is 1% an agarose gel electrophoresis detected result of 5 ' the RACE amplified production first time
Fig. 3 is 1% an agarose gel electrophoresis detected result of 5 ' the RACE amplified production second time
Fig. 4 is 1% an agarose gel electrophoresis detected result of 5 ' RACE amplified production for the third time
Fig. 5 is the part physical map of the plant expression vector pCambia1301-UbiN-Fer of iron binding protein of small red beam gene A-Fer
Fig. 6 is for taking turns the mature embryo callus that screens the upland rice 297 that obtains through two in the Totomycin substratum
Fig. 7 is the PCR detected result of iron binding protein of small red beam gene A-Fer transgenic paddy rice
Embodiment
Method therefor is ordinary method if no special instructions among the following embodiment.
The clone of embodiment 1, iron binding protein of small red beam gene A-Fer
One, adopts the 3 ' terminal sequence of 3 ' RACE amplification A-Fer
From gene pool, search the aminoacid sequence of the iron-binding protein of other plant, carry out sequence alignment with DNAssist software, obtain the conjugated protein conservative amino acid residue sequence of plant iron " AYFDRDN ", according to this amino acid residue sequence design degenerated primer, with DNAssist software the nucleotide sequence of above-mentioned iron-binding protein gene is carried out sequence alignment again, to reduce degeneracy, the primer sequence of 3 ' RACE of final design is: 5 '-TACTTNGACAGNGACAACG-3 '.Adopt 3 ' the RACE test kit and the reference reagent box specification sheets of TaKaRa company to operate, 3 ' terminal sequence of amplification iron binding protein of small red beam gene (called after A-Fer), after reaction finishes, amplified production is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is Marker to detected result as shown in Figure 1, swimming lane 1-4 is 3 ' RACE amplified production), the purpose band (arrow indication) that reclaims between the 600-700bp checks order, sequencing result shows that this 3 ' RACE amplified production has SEQ ID № in the sequence table: 3 nucleotide sequence, from 5 ' end 1-19 bit base is 3 ' RACE primer sequence, is 3 ' end connector primer sequence (TaKaRa company) from 5 ' end 615-636 bit base.NCBI Blastn analytical results shows this sequence and plant iron binding-protein gene homology, wherein, can reach more than 90% with the homology of the ferritin gene partial sequence of Kidney bean and soybean, show that this amplified fragments is a 3 ' terminal sequence of iron binding protein of small red beam gene.
Two, adopt the 5 ' terminal sequence of 5 ' RACE amplification A-Fer
Adopt TaKaRa company's 5 ' RACE test kit and reference reagent box specification sheets to operate, 5 ' the terminal sequence of amplification A-Fer, because the segment with the each amplification of this 5 ' RACE test kit is shorter, so present embodiment carried out three times 5 ' RACE altogether and increased, and detailed process is as follows:
1,5 ' RACE amplification for the first time
3 ' terminal sequence of the Semen Ormosiae Hosiei ferritin gene that amplifies according to step 1, design reverse transcription primer and two pairs of inverse PCR primers, primer sequence is as follows:
The CTGCCACACTGT-3 ' of reverse transcription primer 5-P-1:5 '-(P) (5 ' end phosphorylation)
5-A1(1)：5’-TGTGGAAAAGGGGGATGC-3’
5-S1(1)：5’-TCTTCCACCACGAGTGTTCTG-3’
5-A2(1)：5’-AGAAGTTGGTGAATGAG-3’
5-S2(1)：5’-TTGGAAAGCTACGTTGTC-3’
Adopt the nest-type PRC method to increase, except that amplimer with annealing temperature is different, reaction system and reaction conditions are all with reference to 5 ' RACE test kit specification sheets, first round amplimer is 5-A1 (1) and 5-S1 (1), second to take turns amplimer be 5-A2 (1) and 5-S2 (1), and annealing temperature is 58 ℃.After reaction finishes, amplified production is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is Marker to detected result as shown in Figure 2, swimming lane 1 is a first round PCR product stoste, swimming lane 2 is 10 times of diluents of first round PCR product, swimming lane 3 is 100 times of diluents of first round PCR product), reclaim the purpose band (arrow indication) of about 200bp, check order, sequencing result shows that this 5 ' RACE amplified production has SEQ ID № in the sequence table: 4 nucleotide sequence, from 5 ' end 1-17 bit base is primer 5-A2 (1) sequence, from 5 ' end 33-45 bit base is reverse transcription primer 5-P-1 primer sequence, from 5 ' end 175-193 bit base is primer 5-S2 (1) sequence, is that length is the new amplification region of 118bp from 5 ' end 46-163 bit base.NCBI Blastn analytical results shows that this sequence is the partial sequence of plant iron binding-protein gene.
2,5 ' RACE amplification for the second time
3 ' terminal sequence of the iron binding protein of small red beam gene that amplifies according to step 1 and 5 ' RACE amplified fragments sequence for the first time, design is 5 ' RACE reverse transcription primer and two pairs of inverse PCR primers for the second time, and primer sequence is as follows:
The CAAATCCCTTG-3 ' of reverse transcription primer 5-P-2:5 '-(P)
The pcr amplification primer:
5-A1(2)：5’-ACCACTCCTTGTTTGCATAC-3’
5-S1(2)：5’-TCGCAATCATCAGCGTAATAC-3’
5-A2(2)：5’-TGACAGGGACAACGTAGCTTTC-3’
5-S2(2)：5’-ACGAAACCTGTGGAGCAG-3’
Adopt the nest-type PRC method to increase, except that amplimer with annealing temperature is different, reaction system and reaction conditions are all with reference to 5 ' RACE test kit specification sheets, first round amplimer is 5-A1 (2) and 5-S1 (2), second to take turns amplimer be 5-A2 (2) and 5-S2 (2), and annealing temperature is 56 ℃.After reaction finishes, amplified production is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is Marker to detected result as shown in Figure 3, swimming lane 1 is a first round PCR product stoste, swimming lane 2 is 10 times of diluents of first round PCR product, swimming lane 3 is 100 times of diluents of first round PCR product), reclaim the purpose band (arrow indication) between the 200-300bp, check order, sequencing result shows that this 5 ' RACE amplified production has SEQ ID № in the sequence table: 5 nucleotide sequence, from 5 ' end 1-21 bit base is primer 5-A2 (2) sequence, from 5 ' end 22-33 bit base is reverse transcription primer 5-P-2 sequence, from 5 ' end 206-225 bit base is primer 5-S2 (2) sequence, is that length is the new amplification region of 171bp from 5 ' end 34-203 bit base.NCBI Blastn analytical results shows that this sequence is the partial sequence of plant iron binding-protein gene.
3,5 ' RACE amplification for the third time
3 ' terminal sequence of the Semen Ormosiae Hosiei ferritin gene that amplifies according to step 1 and twice 5 ' sequence of RACE amplified fragments, design is 5 ' RACE reverse transcription primer and two pairs of inverse PCR primers for the third time, and primer sequence is as follows:
Reverse transcription primer 5-P-(2): 5 '-(P) CAAATCCCTTG-3 '
The pcr amplification primer:
5-A1(3)：5’-ACCACTCCTTGTTTGCATAC-3’
5-S1(3)：5’-CAGTGAGAGGCGCAGTTGAGG-3’
5-A2(3)：5’-TGACAGGGACAACGTAGCTTTC-3’
5-S2(3)：5’-TCTGCTTTCCCCTTTCTTATTC-3’
Adopt the nest-type PRC method to increase, except that amplimer with annealing temperature is different, reaction system and reaction conditions are all with reference to 5 ' RACE test kit specification sheets, first round amplimer is 5-A1 (3) and 5-S1 (3), second to take turns amplimer be 5-A2 (3) and 5-S2 (3), and annealing temperature is 60 ℃.After reaction finishes, amplified production is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is Marker to detected result as shown in Figure 4, swimming lane 1 is 100 times of diluents of first round PCR product, swimming lane 2 is 10 times of diluents of first round PCR product, swimming lane 3 is a first round PCR product stoste), reclaim the purpose band (arrow indication) between the 200-300bp, check order, sequencing result shows that this 5 ' RACE amplified production has SEQ ID № in the sequence table: 6 nucleotide sequence, from 5 ' end 1-21 bit base is primer 5-A2 (3) sequence, from 5 ' end 22-32 bit base is reverse transcription primer 5-P-2 sequence, is that length is the new amplification region of 127bp from 5 ' end 33-159 bit base.NCBI Blastn analytical results shows that this sequence is the partial sequence of plant iron binding-protein gene.
The sequence assembly of 3 ' RACE and 5 ' RACE amplification is got up, obtain the cDNA sequence of Semen Ormosiae Hosiei ferritin gene A-Fer, has SEQ ID № in the sequence table: 2 nucleotide sequence, by 1030 based compositions, its encoding sequence is that coding has SEQ ID № in the sequence table: the protein of 1 amino acid residue sequence from 5 ' end 81-848 bit base.Carry out the homology comparison of sequence, comparison result is as shown in table 1, the protein sequence and the other plant iron-binding protein gene that show this iron binding protein of small red beam coded by said gene have higher homology, wherein, the homology with the CDS encoded protein matter sequence of the iron-binding protein gene of Kidney bean and soybean can reach more than 90%.
Table 1 iron binding protein of small red beam gene C DS encoded protein matter sequence homology comparison result
| Botanical name | Semen Ormosiae Hosiei CDS sequence homology result relatively |
| Phaseol μ s v μ lgaris-kidney bean (Kidney bean) | 94.5％ |
| Mal μ s xiaojinensis (malus xiaojinensis) | 90.4％ |
| Glycine max-soybean (soybean) | 90.0％ |
| Garden pea (pea gardens) | 80.4％ |
| Solan μ m t μ beros μ m (potato) | 76.5％ |
| Cowpea (cowpea) | 75.5％ |
| Arabidopsis thaliana (Arabidopis thaliana) | 66.2％ |
| Nicotiana tabac μ m (common tobacco) | 67.5％ |
| Rape (bird rape) | 67.5％ |
| Oryza sativa (paddy rice) | 65.0％ |
| Zea mays (corn) | 65.0％ |
One, the acquisition of A-Fer transgenic paddy rice and PCR thereof detect
1, the structure of Semen Ormosiae Hosiei ferritin gene plant expression vector
According to the CDS sequences Design primer of Semen Ormosiae Hosiei ferritin gene, restriction enzyme site is introduced at the primer two ends:
CDS-P-ATG:5 '-
TCCCCCGGGATGGCCCTTGCTCCATCTAAAG-3 ' (band underscore base is Restriction enzyme Sma I recognition site and protection base)
CDS-P-TGA:5 '-
GACTAGTTCAAGCAGCATGTCCATC-3 ' (band underscore base is restriction enzyme SpeI recognition site and protection base)
Genomic dna with Semen Ormosiae Hosiei is a template, under the guiding of primer CDS-P-ATG and primer CDS-P-TGA, the CDS sequence of pcr amplification Semen Ormosiae Hosiei ferritin gene, again this sequence clone is gone into plant expression vector pCambia1301-UbiN (GenBank number: AF234296) between the SmaI of multiple clone site and the SpeI restriction enzyme site, obtain the plant expression vector of Semen Ormosiae Hosiei ferritin gene A-Fer, called after pCambia1301-UbiN-A-Fer, this carrier part physical map as shown in Figure 5.
2, Semen Ormosiae Hosiei ferritin gene plant expression vector rice transformation
The plant expression vector pCambia1301-UbiN-A-Fer of the Semen Ormosiae Hosiei ferritin gene A-Fer that step 1 is made up transforms upland rice 297 mature embryo callus with particle bombardment, carry out 2 with the NB substratum that contains the 50mg/L Totomycin and take turns screening, the every wheel screened 20-30 days, the resistant calli that obtains obtains resistant plant through pre-differentiation, differentiation as shown in Figure 6 again.
3, the PCR of resistant plant detects
The genomic dna of extraction step 2 resistant plants, as template, at primer 1:5 '-TCAACTGCGCCTCTCACTG-3 ' and primer 2: the PCR of 5 '-TTGCGATCTGCCACACTG-3 ' resistant plant detects, and the PCR reaction conditions is: 94 ℃ of 3min earlier; 94 ℃ of 30sec again, 60 ℃ of 30sec, 72 ℃ of 90sec, totally 30 circulations; Last 72 ℃ of 10min.After reaction finishes, amplified production is carried out 1% agarose gel electrophoresis to be detected, (swimming lane M is Marker to detected result as shown in Figure 7, swimming lane 1-12 is the pcr amplification product of different transgenic lines), the transgenic positive plant can amplify the band of about 500bp, shows the transgenic paddy rice that has obtained iron binding protein of small red beam gene A-Fer.
Two, the iron level of A-Fer transfer-gen plant is measured
The blade of the transgenic paddy rice of iron binding protein of small red beam gene A-Fer that the not homophyletic of getting the step 1 acquisition is carries out iron level and measures (is contrast with the wild-type plant), measurement result is as shown in table 2, the iron level of transfer-gen plant is apparently higher than contrast, compare with the wild-type plant, the iron level of transfer-gen plant is the highest to increase by 2 times.
The measurement result of iron level in the table 2 Transferrins,iron complexes trans-genetic hybrid rice blade
Sequence table
<160>6
<210>1
<211>255
<212>PRT
＜213〉Vigna Semen Ormosiae Hosiei (Vigna angularis)
<400>1
<210>2
<211>1030
<212>DNA
＜213〉Vigna Semen Ormosiae Hosiei (Vigna angularis)
<400>2
<210>3
<211>636
<212>DNA
＜213〉Vigna Semen Ormosiae Hosiei (Vigna angularis)
<400>3
<210>4
<211>192
<212>DNA
＜213〉Vigna Semen Ormosiae Hosiei (Vigna angularis)
<400>4
<210>5
<211>223
<212>DNA
＜213〉Vigna Semen Ormosiae Hosiei (Vigna angularis)
<400>5
<210>6
<211>241
<212>DNA
＜213〉Vigna Semen Ormosiae Hosiei (Vigna angularis)
<400>6
Claims (2)
1, a kind of method of cultivating iron-dense plants is to utilize plant expression vector with the iron binding protein of small red beam gene transfered plant cell, obtains transgenic cell line and transfer-gen plant that iron-holder improves; Wherein, described iron binding protein of small red beam gene is one of following nucleotide sequence:
1) nucleotide sequence of the iron binding protein of small red beam of encoding amino acid sequence shown in SEQ ID NO:1;
2) nucleotide sequence shown in SEQ ID NO:2.
2, method according to claim 1 is characterized in that: the vegetable cell that is imported into described plant expression vector is the cell of Semen Ormosiae Hosiei, paddy rice, corn, wheat, soybean or romaine lettuce.
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| Title |
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| . Liu,Y., Sun,C. and Fu,Y.GENEBANK,Vol.AY683197 . 2004 |
| . Liu,Y., Sun,C. and Fu,Y.GENEBANK,Vol.AY683197 . 2004 * |
| 大豆铁结合蛋白基因在旱稻中的转化. 董阔.中国优秀博硕士学位论文全文数据库. 2004 |
| 大豆铁结合蛋白基因在旱稻中的转化. 董阔.中国优秀博硕士学位论文全文数据库. 2004 * |
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