CN102558326B

CN102558326B - Transcription factor MxIRO2, coding gene thereof, and application thereof

Info

Publication number: CN102558326B
Application number: CN201210029693.8A
Authority: CN
Inventors: 韩振海; 殷丽丽; 王忆; 张新忠; 吴婷; 许雪峰; 陈�峰
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2012-02-10
Filing date: 2012-02-10
Publication date: 2014-03-12
Anticipated expiration: 2032-02-10
Also published as: CN102558326A

Abstract

The invention discloses a transcription factor MxIRO2, a coding gene thereof, and an application thereof. The protein provided by the invention is a protein represented by the following (a) or (b): (a) a protein composed of an amino acid sequence represented by a sequence 1 in the sequence list; (b) a protein derived from (a) and related to plant iron-deficiency resistance, wherein the an amino acid residue sequence of the sequence 1 is subject to substitution and/or deletion and/or addition of one or more amino acid residues. The invention assists in providing a good theoretical basis for further studies on iron-deficiency resistance of malus xiaojinensis.

Description

A kind of transcription factor MxIRO2 and encoding gene thereof and application

Technical field

The present invention relates to a kind of albumen relevant to iron deficiency resistance of plants and encoding gene and application, particularly malus xiaojinensis (Malus xiaojinensis) MxIRO2 albumen and encoding gene and application.

Background technology

Ferro element is one of essential nutritive element of plant growth.Crop iron deficiency can cause the phenomenons such as blade yellow, early ageing, and too little iron can cause Crops production and quality to decline, and has a strong impact on agriculture production.Agricultural iron deficiency is a global problem, and there is the report of a large amount of relevant plant iron deficiencies countries in the world.

IRO2 is a bHLH transcription factor relevant to plant iron deficiency, in mechanism II plant paddy rice, be cloned at first, OsIRO2 is partly induced to express (Ogo et al. at paddy rice iron deficiency root and spire, Isolation and characterization of IRO2, a novel iron-regulated bHLH transcription factor in graminaceous plants, Journal of Experimental Botany, 2006,57 (11): 2867-2878).Subsequently, be found in barley with the gene HvIRO2 of OsIRO2 homology, and the aminoacid sequence of HvIRO2 and OsIRO2 has 64% similarity.All there is OsIRO2 binding sequence in the upstream of many grass iron deficiency expression profiles, therefore, OsIRO2 is an important regulatory factor.At wheat, Chinese sorghum, also finds and the aminoacid sequence of OsIRO2 open reading frame homology in some grasses such as corn, although the albumen of these helix-loop-helixs does not have the conserved domain of bHLH, they have high homology each other.Research shows, in two bHLH transcription factor AtbHLH38 that find in Arabidopis thaliana and AtbHLH39 and paddy rice, OsIRO2 belongs to same bHLH transcription factor member, and these tbHLH members and LeFER, AtFIT1, FRU and AtbHLH29 transcription factor belong to different bHLH family members, at AtbHLH38, in AtbHLH39 and paddy rice, in the bHLH structural domain of OsIRO2 albumen, contain Histidine, L-glutamic acid and arginine residues, this show IRO2 can and G-box (CACGTG) combination, and LeFER, AtFIT1, FRU, AtbHLH29 can and E-box (CANNTG-) in conjunction with (Li, X., Duan, X., Jiang, H.et al., Genome-wide analysis of basic/helix-loop-helix transcription factor family in rice and Arabidopsis, Plant Physiol, 2006, 141:1167-1184).Experiment is proof also, and the DNA region 5 of being combined with OsIRO2 '-CACGTGG-3 ' also contains G-box (Ogo et al., 2006), thereby makes plant can adapt to the environment of Fe Deficiency by the expression of being combined downstream gene with DNA.

Therefore, by can further understanding the molecular mechanism of Genes For Plant Tolerance Fe Deficiency to the research of upstream regulatory factor.

Summary of the invention

An object of the present invention is to provide albumen and encoding gene and application that a kind of iron deficiency resistance of plants is relevant.

The albumen that iron deficiency resistance of plants provided by the present invention is relevant, name is called MxIRO2, derives from Malus xiaojinensis, is following (a) or protein (b):

(a) protein that the aminoacid sequence shown in sequence 1 forms in sequence table;

(b) by the amino acid residue sequence of sequence in sequence table 1 through the replacement of one or several amino-acid residue and/or disappearance and/or interpolation and relevant to iron deficiency resistance of plants by (a) derivative protein.

The encoding gene of above-mentioned albumen is following 1)-3) in arbitrary described gene:

1) DNA molecular shown in sequence 2 in sequence table;

2) under stringent condition with 1) shown in DNA molecule hybridize and the DNA molecular of encoding said proteins;

3) with 1) or 2) gene there is more than 90% homology and the DNA molecular of encoding said proteins.

The expression cassette that contains above-mentioned arbitrary described encoding gene, recombinant expression vector, transgenic cell line or recombinant bacterium also belong to protection scope of the present invention.

Described recombinant expression vector is between the multiple clone site of pET-30a carrier, to insert the recombinant expression vector that the encoding gene described in claim 2 or 3 obtains.

The primer pair of above-mentioned arbitrary described encoding gene total length or its arbitrary fragment of increasing also belongs to protection scope of the present invention.

In described primer pair, a primer sequence is as shown in sequence in sequence table 3, and another primer sequence is as shown in sequence in sequence table 4.

Above-mentioned albumen also belongs to protection scope of the present invention in the application as in transcription factor.

The application of above-mentioned albumen in the tolerant to iron deficiency of regulating plant also belongs to protection scope of the present invention.

In above-mentioned application, described plant is malus xiaojinensis.

Wherein, the sequence in sequence table 1 is comprised of 253 amino-acid residues.

In order to make the MxIRO2 in (a) be convenient to purifying, the N-terminal of the protein that can form at the aminoacid sequence shown in sequence in sequence table 1 or C-terminal connect label as shown in table 1.

The sequence of table 1. label

MxIRO2 in above-mentioned (b) can synthetic, also can first synthesize its encoding gene, then carries out biological expression and obtain.The encoding gene of MxIRO2 in above-mentioned (b) can be by lacking the codon of one or several amino-acid residue by sequence in sequence table 2 in the DNA sequence dna shown in 5 ' end 1-762 bit base, and/or carry out the missense mutation of one or several base pair, and/or the encoding sequence that connects the label shown in table 1 at its 5 ' end and/or 3 ' end obtains.The replacement of described one or several amino-acid residue and/or disappearance and/or interpolation can be replacement and/or disappearance and/or the interpolation that is no more than 10 amino-acid residues.

MxIRO2 gene provided by the present invention has very important regulating effect for the plant of Fe Deficiency, the present invention's material malus xiaojinensis used is the plant that is a kind of resistance to iron deficiency, it is a kind of typical mechanism I plant, that is: plant is by the H+-ATPase on plasma membrane, make soil acidification, increase the solubility of iron, then under the effect of ferric iron reductase enzyme, ferric iron is reduced into ferrous iron, effect at ferrous iron translocator is transported ferrous iron in tenuigenin, increases the tolerant to iron deficiency of plant.In Arabidopis thaliana, having report ferric iron reductase enzyme and ferrous iron translocator is all the regulation and control that are subject to AtFIT1.The present invention is significant in the resistance to iron deficiency field of plant.

Accompanying drawing explanation

Fig. 1 is the pcr amplification product result of MxIRO2 gene

Fig. 2 is that the enzyme of prokaryotic expression carrier pET-MxIRO2 is cut qualification result

Fig. 3 is the protein expression situation that pET-MxIRO2 is transformed into full bacterium in e. coli bl21

Fig. 4 is that the enzyme of pGBKT7-MxIRO2 is cut qualification result

Fig. 5 is the PCR qualification result of the positive colony of AH109/pGBKT7-MxIRO2

Fig. 6 is growing state and the X-gal coloring test of transgenic yeast on different defective type substratum.

Embodiment

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

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

Embodiment 1, with the acquisition of iron deficiency resistance of plants associated transcription factor MxIRO2 and encoding gene thereof

(public Ke Cong China Agricultural University obtains malus xiaojinensis (Malus xiaojinensis), the non-patent literature of recording this malus xiaojinensis is: become bright Wu, Li Xiaolin, Zhang Yun is expensive, good rootstock resource-the malus xiaojinensis of apple, Agricultural University Of Southwest's journal, in October, 2000,22 (5): 383-386.)

One, the acquisition of MxIRO2 gene

Malus xiaojinensis (Malus xiaojinensis) tissue cultured seedling is cultivated in growth medium (MS+0.5mg/L IBA+0.2mg/L6-BA), after it grows into stem lignifying, be transferred to root media (1/2MS+1.0mg/L IBA), tissue cultured seedling bears after white root, moving to 1/2 pancebrin (forms in Table 2, initial pH value is adjusted to 6.0 with NaOH) in overlay film moisturizing cultivate 2 weeks, proceeding to afterwards pancebrin (forms in Table 1, initial pH value is adjusted to 6.0 with NaOH) cultivate one month, change weekly pancebrin one time, culture condition is: 16 hours (photon gamma flux density 250 μ molm of illumination cultivation ^-2s ^-1), temperature is 25 ± 2 ℃, dark culturing 8 hours, temperature is 17 ± 2 ℃.Carry out afterwards iron deficiency processing, method is: plant is gone to from pancebrin containing 4 μ M Fe nutritive mediums (form in Table 3, initial pH value is adjusted to 6.0 with NaOH), deionized water rinsing before shifting.

The formula of pancebrin is as following table 1:

The formula of table 1, pancebrin

The formula of 1/2 pancebrin is as following table 2:

The formula of table 2,1/2 pancebrin

Containing the formula of 4 μ MFe nutritive mediums as following table 3:

Table 3, containing the formula of 4 μ M Fe nutritive mediums

Take malus xiaojinensis as experiment material, extract respectively its iron deficiency and process 1 day and 7 days total RNA of root, is cDNA by its reverse transcription.Take this cDNA as template, take F1 and R1 as primer, carry out pcr amplification, i.e. MxIRO2 full length gene.

F1：5′-ATGTTAGCCTTGTCTCCTCCTAT-3′，

R1：5′-TTAATATTCTTGTCCATAGAAGGACAT-3′。

Get PCR product and carry out agarose gel electrophoresis, result as shown in Figure 1, wherein, the DNA molecular amount standard that swimming lane M is 2000bp,

swimming lane

1 and 2 is the PCR product of MxIRO2 gene, wherein swimming lane 1 is the iron deficiency product of 1 day, swimming lane 2 is the iron deficiency product of 7 days.Result shows to obtain the object band of 762bp.Cut object band, after purifying reclaims, according to pEASY-T1-simple Vector Kit support agent box (purchased from Quan Shijin biotech firm) specification sheets, PCR product is connected to pEASY-T1-simple upper, check order.Sequencing result shows, pcr amplification product sequence total length is 762bp, and its nucleotide sequence is as shown in sequence in sequence table 2, and coding has the protein of the amino acid residue sequence shown in sequence 1 in sequence table, and in sequence table, sequence 1 is comprised of 253 amino-acid residues.By this unnamed gene, be MxIRO2, by the albumen called after MxIRO2 of this genes encoding.

Two, the prokaryotic expression of MxIRO2 gene and protein purification

1, the structure of prokaryotic expression carrier pET-MxIRO2

The cDNA that the above-mentioned iron deficiency of take is processed the malus xiaojinensis root of 3d is template, use primers F 2 and R2 to carry out pcr amplification MxIRO2 gene, and clone again in pET-30a carrier (purchased from Bai Li bio tech ltd, Shanghai) after EcoRI and XhoI enzyme are cut, build and obtain prokaryotic expression carrier pET-MxIRO2.PET-MxIRO2 transforms escherichia coli DH5a bacterial strain, and the positive spot of picking carries out bacterium colony PCR, enzyme is cut and identified and sequence verification.Enzyme is cut qualification result as shown in Figure 2, wherein, the DNA molecular amount standard that swimming lane M1 is 2000bp, the DNA molecular amount standard that swimming lane M2 is 15000bp, swimming lane 1 is cut identification result figure for the enzyme of prokaryotic expression carrier pET-MIRO2.The bacterial plaque that qualification result is positive is shaken to bacterium, extract plasmid and carry out sequence verification.Sequencing result shows, between the EcoRI of carrier pET-30a and XhoI restriction enzyme site, inserted the nucleotide sequence shown in sequence 2 in sequence table, illustrates that prokaryotic expression carrier pET-MxIRO2 builds correct, can be for protein expression.Expression plasmid pET-MxIRO2 is transformed in escherichia coli expression bacterial strain BL21 (DE3) (purchased from Quan Shijin biotech firm, catalog number is CD901), and picking positive colony shakes bacterium and cultivates.

F2:5 '-CCG gAATTCaTGTTAGCCTTGTCTCCTCCTAT-3 ' (sequence 3);

R2:5 '-CCG cTCGAGaTATTCTTGTCCATAGAAGGACAT-3 ' (sequence 4);

2, the abduction delivering of target protein and purifying thereof

, in 10ml LB substratum, 37 ℃ of shaking culture are to OD to get 100ul incubated overnight bacterium liquid (turn and have the e. coli bl21 of pET-MxIRO2 bacterium liquid) ₆₀₀during for 0.4-0.6, adding final concentration is the IPTG of 0.5mM, in 30 ℃, carry out abduction delivering, respectively 1, 2, 3, during 4h, collect bacterium liquid 1ml, after the centrifugal 3min of 12000rpm, outwell upper strata substratum, after adding PBS suspension thalline, mix with appropriate 1 * SDS albumen sample-loading buffer, be placed in 100 ℃ and boil 5min, after centrifugal, get supernatant 20ul and carry out electrophoresis in 10% polyacrylamide gel, result is as Fig. 3 A, wherein swimming lane M is albumen maker,

swimming lane

1, 2, 3, 4 are respectively

induction

1, 2, 3, the band of whole bacterial protein during 4h, as can be seen from the figure the object band of pET-MIRO2 induction is below maker43KD, size is about 36KD.

The bacterium liquid that success is induced is forwarded in 100ml substratum, and 37 ℃ are cultured to OD ₆₀₀while reaching 0.4-0.6, add inductor IPTG to final concentration be 0.5mM, centrifugal after 30 ℃ of induction 4h, add the resuspended thalline of e. coli protein extracting solution, and to add final concentration be the N,O-Diacetylmuramidase of 1mg/ml, be placed in incubated at room 30min, flick during this time centrifuge tube for several times to accelerate cracking, ultrasonication 10min after cracking completes, 4 ℃ are transferred to supernatant in new centrifuge tube after centrifugal, supernatant is crossed to His-Bingding-Resin purification column (purchased from Yue Ke bio tech ltd, Shanghai) purifying target protein, imidazoles wash-out target protein with 300mM, every pipe is collected 1ml, collect altogether 5ml.1,2,3,4,5 albumen of collecting are carried out to SDS-PAGE electrophoresis detection, and result is as Fig. 3 B, and wherein swimming lane 1-5 is respectively the target protein that 1,2,3,4,5 pipes are collected, and size is about 36KD.

Three, functional verification

(1) MxIRO2 gene transformation yeast

This tests yeast expression system used is that a ripe proof albumen is the system of transcription factor.

Yeast strain AH109, yeast vector pGBKT7, defective type substratum SD-Trp and defective type substratum SD-Trp-His and yeast conversion test kit are all purchased from general Jino, Beijing Science and Technology Ltd..

With EcoRI and SalI double digestion MxIRO2 PCR product and yeast vector pGBKT7, glue reclaims object endonuclease bamhi respectively, obtains the carrier segment of MxIRO2 and the 7300bp of about 760bp, obtains recombinant expression vector pGBKT7-MxIRO2 after connection.Recombinant expression vector pGBKT7-MxIRO2 is transformed to bacillus coli DH 5 alpha competent cell, choose after spot shakes bacterium and carry out bacterium colony PCR checking, positive colony is shaken respectively after bacterium upgrading grain, double digestion checking recombinant plasmid, as shown in Figure 4, swimming lane 1 is cut result for the enzyme of pGBKT7-MxIRO2 to the result that agarose gel electrophoresis detects.The bacterial plaque that qualification result is positive is shaken to bacterium, extract plasmid and carry out sequence verification.Sequencing result shows that pGBKT7-MxIRO2 is the expression vector that contains MxIRO2.

Join yeast growth type substratum (YPD) and defective type substratum (SD-Trp and SD-Trp-His), utilize yeast conversion test kit that recombinant vectors pGBKT7-MxIRO2 and pGBKT7 are imported respectively in yeast strain AH109, the latter in contrast, obtain recombinant bacterium AH109/pGBKT7-MxIRO2 and AH109/pGBKT7, recombinant bacterium AH109/pGBKT7-MxIRO2 is coated on SD-Trp and SD-Trp-His substratum, AH109/pGBKT7 is also coated on respectively on SD-Trp and SD-Trp-His substratum, is inverted for 30 ℃ and cultivates.

Result shows, AH109/pGBKT7-MxIRO2 all has bacterium colony to grow on SD-Trp and SD-Trp-His, contrast AH109/pGBKT7 on SD-Trp, have colony growth and on SD-Trp-His substratum without colony growth.Picking list bacterium colony, with primers F 1 and R1, carry out the positive colony that bacterium colony PCR identifies AH109/pGBKT7-MxIRO2, as shown in Figure 5: wherein, M is the DNA molecular amount standard of DL2000bp Plus, 1 for turning the result of the MxIRO2 that increases in the recombinant bacterium that has AH109/pGBKT7-MxIRO2, and 2 for take the negative control of water as template amplification MxIRO2.Result shows, carrier pGBKT7-MxIRO2 successfully proceeds in yeast strain AH109.

Two, the self activation of bait plasmid and oxicity analysis

The positive bacteria of AH109/pGBKT7-MxIRO2 and AH109/pGBKT7 picking is joined in 50mlSD-Trp/Kan liquid nutrient medium, and 30 ℃, 16-24h is cultivated in 200rpm concussion, uses UV spectrophotometer measuring OD ₆₀₀value, be all greater than 0.8 and bacterial concentration close, illustrate that bait plasmid is to yeast strain nontoxicity.Because may causing AH109/pGBKT7-MxIRO2, the low-level leakage of HIS3 reporter gene forms low background in the substratum growth that lacks HIS3, for eliminating, disturb, by being grown in the line of AH109/pGBKT7-MxIRO2 bacterium on SD/-Trp plate, be transferred to the optimization of carrying out 3 '-AT concentration on the SD/-His/-Trp plate that 3 '-AT concentration is respectively 0mM, 0.2mM, 0.5mM and 1mM, take AH109/pGBKT7 as contrast simultaneously.Result shows, AH109/pGBKT7 containing 3 ' grow on SD/-His/-Trp substratum that-AT concentration is 0.2mM, and containing 3 ' can not grow on SD/-His/-Trp substratum that-AT concentration is 0.5mM; AH109/pGBKT7-MxIRO2 containing 3 ' grow on SD/-His/-Trp substratum that-AT concentration is 0.5mM, and containing 3 ' can not grow on SD/-His/-Trp substratum that-AT concentration is 1mM.

Get that 5ul bacterium liquid drops in respectively SD-Trp and containing 3 ' SD-Trp-His substratum that-AT concentration is 0.5mM on, be inverted for 30 ℃ and cultivate 4-6 days, the bacterium colony growing is done to X-gal coloring test, result as shown in Figure 6,1 bacterium colony growing for AH109/pGBKT7-MxIRO2,2 bacterium colonies that grow for AH109/pGBKT7, X-gal coloring test proof turns has the yeast strain of pGBKT7-MxIRO2 can show blue, and contrast can not develop the color, illustrate that bait plasmid has self activation ability to yeast AH109, MxIRO2 is a kind of activating transcription factor.

Claims

1. an albumen, is the protein that the aminoacid sequence shown in sequence 1 forms in sequence table.

2. the encoding gene of albumen described in claim 1.

3. encoding gene according to claim 2, is characterized in that: described encoding gene is the DNA molecular shown in sequence 2 in sequence table.

4. the expression cassette, recombinant expression vector, transgenic cell line or the recombinant bacterium that contain encoding gene described in claim 2 or 3.

5. recombinant expression vector according to claim 4, is characterized in that: described recombinant expression vector is between the multiple clone site of pET-30a carrier, to insert the recombinant expression vector that the encoding gene described in claim 2 or 3 obtains.

6. the primer pair of encoding gene total length described in the claim 2 or 3 that increases, in described primer pair, a primer sequence is as shown in sequence in sequence table 3, and another primer sequence is as shown in sequence in sequence table 4.

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