CN102559631B

CN102559631B - Malus xiaojinensis Cheng et Jiang MxHA5 protein, and coding gene and application thereof

Info

Publication number: CN102559631B
Application number: CN 201210012499
Authority: CN
Inventors: 韩振海; 槐心体; 王忆; 张倩; 张新忠; 许雪峰
Original assignee: China Agricultural University
Current assignee: China Agricultural University
Priority date: 2012-01-16
Filing date: 2012-01-16
Publication date: 2013-11-06
Anticipated expiration: 2032-01-16
Also published as: CN102559631A

Abstract

The invention discloses a malus xiaojinensis Cheng et Jiang MxHA5 protein, and a coding gene and application thereof. The protein provided by the invention is protein (a) or protein (b), wherein the protein (a) has amino acid sequences shown as a sequence 1 in a sequence table; and the protein (b) has an amino acid residue sequence which is derived from the sequence 1 by substituting and/or losing and/or adding one or more amino acid residues and is related with the P type H<+>-ATPase enzymatic activity in plant. The MxHA5 protein provided by the invention has an important effect of adjusting ion-deficient stressed plant, and the invention further provides good theoretical basis for the research on the ion-deficiency resistance of malus xiaojinensis Cheng et Jiang, and has significance to cultivation of stress-tolerant plant.

Description

Malus xiaojinensis MxHA5 albumen and encoding gene thereof and application

Technical field

The present invention relates to a kind of malus xiaojinensis MxHA5 albumen and encoding gene and application.

Background technology

Iron is the necessary mineral element of human body.Because the plant edible iron that to be world population main source, thus the plant iron-holder low directly can affect the healthy of the mankind.Show according to World Health Organization's investigation, nearly 2,000,000,000 populations are suffering the torment of anaemia.Premature labor, birth defects disease, MR and health index descend and are mainly caused by anaemia.

Studies show that, plant can only absorb the soluble iron-Fe (II) in soil, but in alkaline soil (pH7.4～8.5), the concentration of soluble iron is less than 10 ^-10MolL-1 causes many plants to show the Fe Deficiency symptom.The slight iron deficiency of plant can cause that chlorophyll is synthetic to be reduced and photosynthetic rate reduces, and too little iron causes chlorophyll to synthesize stopping, the young leaves flavescence, and biomass significantly descends.Understand fully that iron absorbs and the molecule mechanism of homeostasis, the nutrient imbalance that improves plant and the mankind is significant.

In order effectively to absorb and to utilize iron in soil, plant has formed adaptability widely to iron deficiency in long-term evolutionary process.At first Romheld and Marschner professor are proposing higher plant formed two kinds of adaptability mechanism---machine-processed I and machine-processed II in long-term adaptation Fe Deficiency process on the basis of summing up former works.

Mechanism I plant refers to dicotyledons and non-Gramineae monocotyledons.They are divided into morphological change and physiology variation to the adaptation reaction of iron deficiency.Morphologic variation comprises and forms a large amount of lateral roots and special transhipment cell, and these two kinds of variations can increase the surface-area of plant generation redox reaction, and improve plant to the transport efficacy of iron.Physiological variation is comprised of three parts: a Fe (III) reductase enzyme system, a strong proton are secreted pump (H outward ⁺-ATPase) and the excretory system of organic substance.These three parts all are positioned on the cytoplasmic membrane of tip of a root epidermic cell of specialization on form, and effectively Fe Deficiency are reacted.Mechanism I plant must first be reduced to Fe (II) with Fe (III) and just can be absorbed and used.Under the condition for the iron abundance, first with Fe (III)-inner complex reduction, the Fe (II) that reduction is obtained transports by cytoplasmic membrane the root of plant; Under the Fe Deficiency condition, mechanism I plant is by activating a species specific H ⁺-ATPase makes soil acidification, thereby increases the solubility of iron; Secrete simultaneously some organic acids (intercalating agent of iron), with the form dissolving of the Fe in soil (III) with Fe (III)-inner complex; Then by a species specific root reductase enzyme on cytoplasmic membrane, Fe (III)-inner complex is reduced.Pyridine nucleotide (NADH) cross-film of this reductase enzyme catalysis electronics reduction-state from kytoplasm passes to outside born of the same parents Fe (III) as electron acceptor(EA)-inner complex, make its reduction become Fe (II), and then enter root cells by the transhipment of the Fe on plasma membrane (II) translocator.

Mechanism II plant only refers to grass.When growing in alkaline soils, grass compares the Iron Deficiency Chlorosis shape of generation with non-grass with dicotyledons seldom, reason is the grass secretion and discharges siderophore, siderophore is combined with iron and is formed carrier complexes, and produce a kind of absorption and transport system that the phytosiderophore mixture is had high affinity on plasma membrane, thereby make grass can effectively absorb and utilize ferric iron to come the iron in Antagonistic Environment to coerce.Siderophore is mugineic acid compound (MAs or Ps), this is a low-molecular-weight nonprotein amino acid of class, Fe (III) there is strong affinity and can forms stable, octahedral ferric iron huge legendary turtle compound, and produce a kind of translocator YSL that Ps or MAs is had high affinity on plasma membrane, by YSL, Fe (III)-Ps is transported in kytoplasm, thereby make grass can effectively absorb and utilize ferric iron to come the iron in Antagonistic Environment to coerce.

Plant plasma membrane serves H ⁺-ATPase belongs to P type ATPase, is the insertion albumen on plasma membrane, has the energy that utilizes hydrolysising ATP to produce, with the H of cytoplasmic membrane inboard ⁺Pump is to the feature in the plasma membrane outside, referred to as proton pump.It has important regulating effect to the adjusting of the absorption of vegetable cell nutritive substance, intracellular pH, the adjusting of stomatal movement, the adjusting of cell elongation growth and the physiological processs such as adjusting of environment-stress.In addition, plasmalemma of plant H ⁺-ATPase also participates in the adjusting of the physiological processs such as seed germination, cell polarity growth.Plant plasma membrane serves H+-ATPase is plant vital activity " dominant force enzyme ".Since confirming the ATPase activity in the plasma membranes that exsomatizing such as Hodges, plasma membrane H ⁺The research of-ATPase has been subject to paying attention to widely.

There are some researches show, under the iron deficiency condition, cucumber root H ⁺-ATPase is active to raise, also can inducing cucumber root system apical cell H ⁺-ATPase albumen and H ⁺The increase of the mRNA of-ATPase.And the transcriptional level of the root CsHA1 gene that research draws the iron deficiency cucumber has improved, and can descend for the transcriptional level of iron deficiency plant CsHA1 gene after again supplying iron.At present, 12 H have been found from Arabidopis thaliana ⁺-ATPase gene family (AHA1～AHA12).The research discovery wherein has the expression of 5 genes to be subjected to the impact of Fe Deficiency.Under Fe Deficiency, wherein the expression amount of AHA2 and AHA7 raises and is respectively 3.3 and 4.0 times, and the expression amount of AHA3 and AHA4 raises approximately 2 times, and the expression amount of AHA11 is lowered approximately 2 times.

Malus xiaojinensis (Malus xiaojinensis Cheng et Jiang) is an apple iron efficient fruit genotype, belongs to mechanism I plant.Although had and much studies have shown that H ⁺The patience that-ATPase gene pairs improves plant has great role, but yet there are no about plasma membrane H in malus xiaojinensis ⁺The report of-ATPase gene.

Summary of the invention

The purpose of this invention is to provide a kind of malus xiaojinensis MxHA5 albumen and encoding gene and application.

Protein provided by the invention available from malus xiaojinensis, is following (a) or (b):

(a) protein that is formed by the aminoacid sequence shown in sequence in sequence table 1;

(b) with 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 with plant in P type H ⁺-ATPase enzymic activity relevant by (a) derivative protein.

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

The sequence of table 1 label

Label	Residue	Sequence
			Poly-Arg	5-6 (being generally 5)	RRRRR

Poly-His	2-10 (being generally 6)	HHHHHH
			FLAG	8	DYKDDDDK
Strep-tag II	8	WSHPQFEK
			c-myc	10	EQKLISEEDL

Above-mentioned (b) but in the protein synthetic, also can first synthesize its encoding gene, then carry out biological expression and obtain.The encoding gene of the protein in above-mentioned (b) can be by the codon with one or several amino-acid residue of disappearance in the DNA sequence dna shown in sequence in sequence table 2, and/or carry out the missense mutation of one or several base pair, and/or obtain at the encoding sequence that its 5 ' end and/or 3 ' end connects the label shown in table 1.

The gene of encoding said proteins also belongs to protection scope of the present invention.

Described gene can be following 1)-4) in arbitrary described DNA molecular:

1) in sequence table sequence 2 from the DNA molecular shown in the 1st to 2862 Nucleotide of 5 ' end;

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

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

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

Above-mentioned stringent condition can be 0.1 * SSPE (or 0.1 * SSC), in the solution of 0.1%SDS, hybridize under 65 ℃ of conditions and wash film.

The recombinant expression vector, expression cassette, transgenic cell line or the recombinant bacterium that contain described gene all belong to protection scope of the present invention.

Available existing expression vector establishment contains the recombinant expression vector of described gene.Described expression vector comprises the double base agrobacterium vector and can be used for the carrier etc. of micropellet bombardment.Described expression vector also can comprise 3 ' of foreign gene and hold untranslated regional, namely comprises the DNA fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.The bootable polyadenylic acid of described polyadenylic acid signal joins 3 ' end of mRNA precursor.When using described gene constructed recombinant expression vector, can add any enhancement type promotor or constitutive promoter before its transcription initiation Nucleotide, they can use separately or be combined with other promotor; In addition, when using gene constructed recombinant expression vector of the present invention, also can use enhanser, comprise translational enhancer or transcriptional enhancer, these enhansers zone 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 synthesize.Translation initiation region can be from transcription initiation zone or structure gene.For the ease of identifying and screening, can process expression carrier used thereof, as add coding can produce enzyme or the gene of luminophor, the antibiotic marker thing with resistance or the anti-chemical reagent marker gene etc. of colour-change.Also can not add any selected marker, directly according to phenotypic screen.

Described recombinant expression vector can be following (I) or (II):

(I) insert the recombinant expression vector that described gene obtains between the multiple clone site of pEZS-NL carrier;

(II) insert the recombinant expression vector that described gene obtains between the multiple clone site of pYES2.0 plasmid.

Described recombinant expression vector specifically can be and described gene is connected the recombinant plasmid A that obtains with carrier pEASY-T1sample.Described recombinant expression vector specifically can be the recombinant plasmid B that obtains between the KpnI of the insertion of the small segment between KpnI and ApaI restriction enzyme site pEZS-NL carrier in described recombinant plasmid A and ApaI restriction enzyme site.Described recombinant expression vector specifically can be the small segment between KpnI in described recombinant plasmid A and XbaI enzyme cutting site is inserted the recombinant plasmid C that obtains between the KpnI of pYES2.0 plasmid and XbaI enzyme cutting site.

Described recombinant bacterium specifically can be (II) described recombinant expression vector or recombinant plasmid C is imported the recombinant bacterium that obtains in yeast.Described yeast specifically can be yeast saccharomyces cerevisiae, as yeast strain BJ2168.

The present invention also protects a kind of method that obtains genetically modified organism, for 1. following or 2. or 3.:

1. described gene is imported in the biology that sets out, obtain P type H ⁺The genetically modified organism that-ATPase enzymic activity increases;

2. described gene is imported in the biology that sets out the genetically modified organism that the resistance of reverse that obtains that metal ion is coerced increases;

3. described gene is imported in the biology that sets out the genetically modified organism that the resistance of reverse that obtains that metal ion is lacked increases.

Described biology is yeast, and described gene imports the described biology that sets out by (II) described recombinant expression vector or recombinant plasmid C.Described yeast specifically can be yeast saccharomyces cerevisiae, as yeast strain BJ2168.

Metal ion during above arbitrary described metal ion is coerced can be Mn ²⁺, Cu ²⁺And Zn ²⁺In at least a.

Metal ion during above arbitrary described metal ion lacks can be Fe ²⁺

Malus xiaojinensis MxHA5 albumen provided by the present invention and encoding gene MxHA5 thereof have 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 anti-iron deficiency, it is a kind of typical mechanism I plant, that is: plant must be by activating the H on plasma membrane ⁺-ATPase makes soil acidification, increases the solubility of iron.Then under the effect of ferric iron reductase enzyme, ferric iron is reduced into ferrous iron, under the effect of the ferrous iron translocator on plasma membrane, transhipment is advanced in tenuigenin, increases the tolerant to iron deficiency of plant.MxHA5 gene provided by the present invention has very important regulating effect for the plant of Fe Deficiency, and the present invention provides good theoretical foundation for the tolerant to iron deficiency of further studying malus xiaojinensis.The present invention has great value for cultivating plant with adverse resistance.

Description of drawings

Fig. 1 is the MxHA5 gene of pcr amplification and the agarose gel electrophoresis figure that enzyme is cut product thereof.

Fig. 2 is that iron deficiency is induced the fluorescent quantitative PCR result of rear MxHA5 gene in the malus xiaojinensis Different Organs.

Fig. 3 is the Subcellular Localization detected result of MxHA5 gene.

Fig. 4 is the PCR qualification result of transgenic yeast.

Fig. 5 is P type H in transgenic yeast ⁺The mensuration that-ATPase enzyme is lived.

Fig. 6 is the growing state of gene yeast under 30 μ M Ferrozine dispositions.

Fig. 7 is transgenic yeast H under manganese, copper and zinc disposition respectively ⁺The mensuration that-ATPase enzyme is lived.

Embodiment

Following embodiment is convenient to understand better the present invention, but does not limit the present invention.Experimental technique in following embodiment if no special instructions, is ordinary method.Test materials used in following embodiment if no special instructions, is and purchases available from routine biochemistry reagent shop.Quantitative test in following examples all arranges repeated experiments three times, results averaged.

Malus xiaojinensis (Malus xiaojinensis Cheng et Jiang): the public can obtain from China Agricultural University; Put down in writing the non-patent literature of this malus xiaojinensis: Cheng Minghao, Li Xiaolin, Zhang Yun is expensive, the good rootstock resource-malus xiaojinensis of apple, Agricultural University Of Southwest's journal, in October, 2000,22 (5): 383-386.

Yeast strain BJ2168 (belonging to yeast saccharomyces cerevisiae): the public can obtain from China Agricultural University, the non-patent literature of putting down in writing this material is: Hong Ding, Lihong Duan, Huilan Wu, et al.Regulation of AhFR01, an Fe (III)-chelate reductase of peanut, during iron deficiency stress and intercropping with maize.Physiologia Plantarum.2009,136:274-283..

Defective type substratum: Ura Minus Media (general Jino, Beijing Science and Technology Ltd.) 8g/L, glucose 20g/L, all the other are distilled water.

Inducing culture: Ura Minus Media (general Jino, Beijing Science and Technology Ltd.) 8g/L, semi-lactosi 20g/L, all the other are distilled water.

The acquisition of embodiment 1, MxHA5 albumen and encoding gene thereof

One, the acquisition of MxHA5 full length gene

Total RNA and the reverse transcription of extracting the malus xiaojinensis blade are cDNA.Take cDNA as template, with the primer pair that F1 and R1 form, use the HiFi mix of high-fidelity to carry out pcr amplification, obtain pcr amplification product.

F1：5’-ATGGCCGGCGATAAGG-3’；

R1：5’-AACTGTATAATGTTGTTGAATTGTATCG-3’。

Pcr amplification system (20 μ l): ddH ₂O 7.0 μ l, 2 * HiF Mix (0.5U/ μ l) 10 μ l, F1 (10mM) 1.0 μ l, R1 (10mM) 1.0 μ l and template 1.0 μ l.

Pcr amplification program: first 94 ℃ of denaturation 5min; Then 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ of extensions 3min, totally 33 circulations; Last 72 ℃ are extended 10min.

Pcr amplification product is carried out 1% agarose gel electrophoresis, see Figure 1A (swimming lane M is the DNA molecular amount standard of 15000bp, and swimming lane 1 and swimming lane 2 are pcr amplification product).Result shows, has obtained the approximately DNA fragmentation of 2862bp.

After purifying reclaims this DNA fragmentation, (also directly the DNA molecular shown in composition sequence 1) is connected to the PCR product on carrier pEASY-T1 sample (available from full Shi Jin biotech firm) according to pEASY-T1sample-Vector Kit support agent box (available from full Shi Jin biotech firm) specification sheets, the T carrier pEASY-MxHA5 that obtains recombinating carries out successively enzyme and cuts evaluation (KpnI and ApaI) and order-checking.Enzyme is cut qualification result and is seen Figure 1B (swimming lane M is the DNA molecular amount standard of 15000bp, and swimming lane 1 and swimming lane 2 are cut product for enzyme).Sequencing result shows, pcr amplification product sequence total length is about 2862bp, its nucleotide sequence as the sequence 2 of sequence table from as shown in the Nucleotide of 5 ' end 1-2862 position, the protein shown in sequence 1 in the code sequence list (being formed by 954 amino-acid residues).

With the protein called after MxHA5 albumen shown in the sequence 1 of sequence table, be the MxHA5 gene with the unnamed gene of coding MxHA5 albumen.

Two, the expression analysis of MxHA5 gene in the malus xiaojinensis Different Organs

The malus xiaojinensis tissue cultured seedling is cultivated in growth medium (MS+0.5mg/L IBA+0.2mg/L 6-BA), after it grows into the stem lignifying, be transferred to root media (1/2MS+1.0mg/L IBA); After tissue cultured seedling bears white root, move to overlay film moisturizing 2 weeks of cultivation in 1/2 pancebrin (composition sees Table 3, and initial pH value transfers to 6.0 with NaOH); Change afterwards pancebrin (composition sees Table 2, and initial pH value transfers to 6.0 with NaOH) over to and cultivated one month, change weekly pancebrin one time; Culture condition in whole process is: 16 hours (photon gamma flux density 250 μ EM of illumination cultivation ^-2S ^-1), temperature is 25 ± 2 ℃; Dark culturing 8 hours, temperature are 17 ± 2 ℃.Carry out afterwards iron deficiency and induce, method is: plant is gone to from pancebrin contain 4 μ M Fe nutritive mediums (composition sees Table 4, and initial pH value transfers to 6.0 with NaOH), deionized water rinsing before shifting.

The formula of table 2 pancebrin

The formula of table 31/2 pancebrin

Table 4 contains the formula of 4 μ M Fe nutritive mediums

Get respectively that iron deficiency induces 0, the young root of the malus xiaojinensis of 12h, 24h, 3d and 6d or total RNA of mature leaf, ultraviolet spectrophotometer is measured the content of RNA, the above-mentioned RNA of quality such as gets, reverse transcription becomes strand cDNA.

Take this strand cDNA as template, first adjust the consumption (make pcr amplification product brightness consistent) of template by pcr amplification 18s gene, then analyze the expression of MxHA5 gene and 18s gene (reference gene) by quantitative fluorescent PCR.

The primer of amplification 18s gene is as follows:

18SR-F：5′-ACACGGGGAGGTAGTGACAA-3′，

18SR-R：5′-CCTCCAATGGATCCTCGTTA-3′。

The primer of amplification MxHA5 gene is as follows:

RT-F1：5′-CTTTCAACCCCGTAGACAA-3′，

RT-R1：5′-CCCAGAGAACGAAGTCCAC-3′。

What the consumption of adjustment template adopted is regular-PCR.Reaction system (20 μ l): ddH ₂O 7.0 μ l, Taq Mix (0.5U/ μ l) 10 μ l, 18SR-F (10mM) 1.0 μ l, 18SR-R (10mM) 1.0 μ l, template 1.0 μ l.Reaction conditions: 95 ℃ of sex change 5s, 55 ℃ of annealing 30s, 72 ℃ of extensions 30s, totally 40 circulations.

Quantitative fluorescent PCR is adopted in the genetic expression component analysis.The step of quantitative fluorescent PCR three-step approach is: 95 ℃ of denaturation 30s; Then 95 ℃ of sex change 5s, 55 ℃ of annealing 30s, 72 ℃ of extensions 30s, totally 40 circulations; Last 95 ℃ of 15s, 60 ℃ of 1min, 95 ℃ of 15s.

The relative expression quantity of target gene passes through 2- ^{Δ Δ CT}Method determines that (concrete operations: in same template, the ratio of the expression amount of the expression amount of MxHA5 gene and 18s gene is as the relative expression quantity of MxHA5 gene, in mapping, the expression amount of 0 moment gene is considered as 1, thereby observe out the expression amount of different time gene more intuitively), the results are shown in Figure 2.The MxHA5 gene has expression in root and blade, and the expression amount in root is slightly higher than the expression amount in blade.After iron deficiency was induced 12h, in blade, the expression amount of MxHA5 gene was minimum, and the expression amount along with the increase MxHA5 gene in treatment time strengthens afterwards, reaches the highest during by the 3rd day, descends afterwards.After iron deficiency was induced, in root, the expression amount of MxHA5 gene obviously strengthened, descend during than 12h when 24h (but strengthening than before processing), afterwards again gradually amount strengthen.Can also observe, in the time length root no matter iron deficiency is induced, the MxHA5 gene all can up-regulated expression, and iron deficiency induces the MxHA5 gene in the 1d rear blade can up-regulated expression.Illustrate if be subjected to for a long time Fe Deficiency, plant increases the adaptability of resistance to iron deficiency by the MxHA5 gene of regulating root and leaf section.

Three, the Subcellular Localization of MxHA5 gene

1, express the transient expression Vector construction of the fusion gene of MxHA5 and eGFP

(1) the T carrier pEASY-MxHA5 that will recombinate cuts and reclaims fragment (approximately 2971bp) with restriction enzyme KpnI and ApaI enzyme.

(2) cut the pEZS-NL carrier with restriction enzyme KpnI and ApaI enzyme, reclaim carrier framework (approximately 5680bp).

(3) small segment of step (1) is connected 2 with step) carrier framework connect, obtain transient expression carrier pEZS-MxHA5.

(4) transient expression carrier pEZS-MxHA5 is transformed e.colistraindh5α, the positive spot of picking carries out bacterium colony PCR successively, enzyme is cut and identified and sequence verification.Sequencing result shows, inserted sequence 2 in the sequence table from the DNA fragmentation shown in 5 ' end 1-2862 position, the eGFP gene fusion on this DNA fragmentation and pEZS-NL carrier between the KpnI of pEZS-NL carrier (available from full Shi Jin Bioisystech Co., Ltd) and ApaI restriction enzyme site.

2, adopt particle bombardment to carry out the transient expression carrier to the conversion of onion epidermis cell

(1) preparation of onion epidermis to be transformed

Under aseptic condition, the entocuticle of the onion of tearing is tiled in the culture dish that contains the MS substratum, then wraps culture dish standby with masking foil.

(2) preparation of bronze suspension

Take the 60mg bronze, put into the centrifuge tube of 1.5ml sterilization, add the 1ml dehydrated alcohol, concussion 1min, the centrifugal 10s of 10000rpm abandons supernatant, then adds the 1ml dehydrated alcohol, concussion 1min, the centrifugal 10s of 10000rpm; Abandon supernatant, bronze is suspended from the 1ml sterilized water, obtain bronze suspension ,-20 ℃ save backup.

(3) preparation of bronze-plasmid dna complex zoarium

Draw 8.5 μ l bronze suspension, 3.5 μ l 0.1M nitrosamine, 8.5 μ l 2.5M CaCl ₂2H ₂O and 5 μ l transient expression carrier pEZS-MxHA5 are with the 3min that vibrates on mixture earthquake device; The centrifugal 20s of 10000rpm; Abandon supernatant, with dehydrated alcohol rinsing 3 times; Add 30 μ l dehydrated alcohols to suspend, obtain bronze-plasmid dna complex fit.

(4) bombardment receptor material

Select the pressure membrane of certain pressure, soaked 1-2 hour in 70% dehydrated alcohol together with the bombardment film, taking-up is dried; The metal plate washer is sterilized on spirit lamp after with 70% soaked in absolute ethyl alcohol; Get 10 μ l bronzes-plasmid dna complex fit, evenly coat on the mid-way of bombardment film, the area of coating is consistent with the pore diameter range on carrier solid circle; After film is dried, be installed on launching device; Pressure membrane is installed to the lower end of gas accelerator; Onion epidermis cell is placed in the middle part of culture dish, puts into vacuum chamber, takes off the culture dish lid; Vacuumize pointer to 26; Exit on gas accelerator, until in pipe, pressure reaches in the time of can splitting the pressure that film can bear, can split film and break, gas is flushed on the bombardment film, and carrier moves downward, and is blocked by the metal plate washer, and following metallic particles sees through the mesh of metal plate washer, directive target cell.

(5) Fluirescence observation

Culture dish is sealed with Parafilm, cultivated 24h for 28 ℃, in the expression of fluorescence microscopy Microscopic observation green fluorescence, adopting uses the same method processes pEZS-NL carrier (control plasmid).Result is (A1 is the bright field photo of pEZS-NL carrier, and A2 is the dark field photo of pEZS-NL carrier, and B1 is the bright field photo of transient expression carrier pEZS-MxHA5, and B2 is the dark field photo of transient expression carrier pEZS-MxHA5) as shown in Figure 3.Result shows, the MxHA5 protein localization is on plasma membrane.

The acquisition of embodiment 2, transgenic yeast and the analysis of anti-heavy metal toxicity thereof

One, the acquisition of transgenic yeast

1, with the restructuring T carrier pEASY-MxHA5 that builds in restriction enzyme KpnI and XbaI enzyme cutting embodiment 1, reclaim enzyme and cut product (approximately 2965bp).

2, with restriction enzyme KpnI and XbaI enzyme cutting pYES2.0 plasmid (Yeast expression carrier, Beijing DingGuo ChangSheng Biology Technology Co., Ltd), reclaim carrier framework (approximately 5800bp).

3, the enzyme of step 1 is cut the carrier framework connection that product is connected with step, obtained recombinant expression vector pYES2.0-MxHA5.

4, utilize yeast conversion test kit (general Jino, Beijing Science and Technology Ltd.) that recombinant expression vector pYES2.0-MxHA5 is imported yeast strain BJ2168, obtain transgenic yeast BJ2168/pYES2.0-MxHA5.

5, the structure of control plasmid

(1) with pEASY-T1 sample with restriction enzyme KpnI and XbaI enzyme cutting and reclaim small segment (approximately 109bp).

(2) with restriction enzyme KpnI and XbaI enzyme cutting pYES2.0 plasmid, reclaim carrier framework (approximately 5800bp).

(3) small segment with step (1) is connected with the carrier framework of step (2), obtains control plasmid BJ2168/pYES2.0.

6, utilize yeast conversion test kit (general Jino, Beijing Science and Technology Ltd.) that control plasmid is imported yeast strain BJ2168, obtain transgenic yeast BJ2168/pYES2.0.

7, transgenic yeast BJ2168/pYES2.0-MxHA5 is carried out bacterium colony PCR and identify (primer pair that adopts F1 and R1 to form).(M is the DNA molecular amount standard of DL 15000bp to result as shown in Figure 4, swimming lane 1 and swimming lane 2 are transgenic yeast BJ2168/pYES2.0-MxHA5, swimming lane 3, swimming lane 5 and swimming lane 6 are recombinant expression vector pYES2.0-MxHA5, and swimming lane 4 is transgenic yeast BJ2168/pYES2.0).

Two, P type H in yeast ⁺The mensuration of-ATPase enzymic activity

H ⁺-ATPase enzyme activity determination method is with reference to the people's such as E.Nso yeast cell plasma membrane H ⁺-ATPase activity determination method (E.Nso, A.Goffeau and J.-P.Dufour.Fluctuations during growth of the plasma membrane H+-ATPase activity ofSaccharomyces cerevisiae andSchizosaccharomyces pombe.Folia Microbiol.2002.47:401-406.).

Yeast strain BJ2168 can't grow on the defective type substratum.

Respectively transgenic yeast BJ2168/pYES2.0-MxHA5 and transgenic yeast BJ2168/pYES2.0 are carried out following experiment:

1, adopt the defective type substratum, culturing yeast spend the night (usually cultivating 18h) under 30 ℃, 200rpm condition.

2, with the centrifugal 5min of bacterium liquid 2000rpm of 3mL step 1, collect thalline and wash thalline (do not contain glucose in substratum used, other component is with the defective type substratum).

3, the thalline with step 2 is seeded to the 50mL inducing culture, cultivates under 30 ℃, 200rpm condition, until bacterium liquid OD ₆₀₀Be 1.

4, utilize fungi/yeast cell film vesica (RSOV and IOV) preparation test kit (Shanghai outstanding U.S. gene Pharmaceutical Technology Co., Ltd) to extract the plasma membrane total protein of yeast.

5, with bovine serum albumin be the concentration of the plasmalemma protein of standard substance determination step 4 extractions.

6, detect P type H ⁺The precursor of-ATPase enzymic activity is 1ml, comprises following material: 6mmol/L ATP, 9mmol/L MgCI ₂6H ₂O, 50mmol/L MES; PH6.0 (regulating with NaOH).Add 2 μ g plasmalemma proteins in front system, hatch 30min under 30 ℃, the SDS aqueous solution termination reaction that adds afterwards 3mL 1g/100mL, with the absorbance value (A) under spectrophotometer detection 820nm, (y=0.8434x-0.0269 is x wherein: the burst size of inorganic phosphorus according to the inorganic phosphorus typical curve, y: light absorption value) calculate the burst size of the inorganic phosphorus under different light absorption values, the inorganic phosphorus that the higher explanation of absorbance value discharges is more, thereby determines P type H ⁺-ATPase enzymic activity.

Revision test is set three times, results averaged.The absorbance value of transgenic yeast BJ2168/pYES2.0-MxHA5 is 0.172, and enzymic activity is 0.118 (μ mol/min P per mg).The absorbance value of transgenic yeast BJ2168/pYES2.0 is 0.155, and enzymic activity is 0.104 (μ mol/min P per mg).

Result as shown in Figure 5, the absorption value of transgenic yeast BJ2168/pYES2.0-MxHA5 illustrates that higher than transgenic yeast BJ2168/pYES2.0 more inorganic phosphorus discharges.Result shows, after importing and express the MxHA5 gene, and the P type H of yeast ⁺-ATPase enzymic activity strengthens greatly.

Three, the existence situation of transgenic yeast in the iron deficiency environment

Yeast strain BJ2168 can't grow on the defective type substratum.

1, adopt the defective type substratum, culturing yeast spends the night (usually cultivating 18h) under 30 ℃, 200rpm condition, the suppressed expression of goal gene this moment.

3, the thalline with step 2 is seeded to the inducing culture that 50mL contains 30 μ M Ferrozine, cultivates under 30 ℃, 200rpm condition, until bacterium liquid OD ₆₀₀Be to begin timing at 1 o'clock, measure respectively the OD600 value of 0h, 12h, 24h, 48h, 60h, 72h.

Revision test is set three times, results averaged.Transgenic yeast BJ2168/pYES2.0-MxHA5 is than fast many of the growth of transgenic yeast BJ2168/pYES2.0.The results are shown in Figure 6.Result shows, after importing and express the MxHA5 gene, yeast strengthens the tolerance of iron deficiency.

Four, the more anti-Mn of transgenic yeast ²⁺, Cu ²⁺And Zn ²⁺Coerce

Yeast strain BJ2168 can't grow on the defective type substratum.

1, adopt the defective type substratum, culturing yeast is to the OD of bacterium liquid under 30 ℃, 200rpm condition ₆₀₀Be 1 left and right (usually cultivating 1-2d), the suppressed expression of goal gene this moment.

3, the thalline with step 2 is seeded to the substratum (substratum first, substratum second or substratum third) that 50mL contains the different metal ion, cultivates under 30 ℃, 200rpm condition, until bacterium liquid OD ₆₀₀Be 1, sampling detects H ⁺-ATPase enzymic activity (the same step 2 of method).

The substratum first (contains 200 μ M MnCl ₂The semi-lactosi substratum): MnCl ₂All the other are distilled water for 200 μ mol/L, Ura Minus Media (general Jino, Beijing Science and Technology Ltd.) 8g/L, semi-lactosi 20g/L.

Substratum second (contains 200 μ M CuCl ₂The semi-lactosi substratum): CuCl ₂All the other are distilled water for 200 μ mol/L, Ura Minus Media (general Jino, Beijing Science and Technology Ltd.) 8g/L, semi-lactosi 20g/L.

Substratum third (contains 200 μ M ZnCl ₂The semi-lactosi substratum): ZnCl ₂All the other are distilled water for 200 μ mol/L, Ura Minus Media (general Jino, Beijing Science and Technology Ltd.) 8g/L, semi-lactosi 20g/L.

Revision test is set three times, results averaged.Transgenic yeast BJ2168/pYES2.0-MxHA5 is at Mn ²⁺, Cu ²⁺And Zn ²⁺Absorbance value under coercing is respectively 0.395,0.444 and 0.357, and enzymic activity is respectively 0.306,0.347 and 0.275 (μ mol/min P per mg).Transgenic yeast BJ2168/pYES2.0 is at Mn ²⁺, Cu ²⁺And Zn ²⁺Absorbance value under coercing is respectively 0.162,0.304 and 0.155, and enzymic activity is respectively 0.110,0.230 and 0.103 (μ mol/min P per mg).

Partial results as shown in Figure 7.Under the condition that each metal ion is coerced, the H of transgenic yeast BJ2168/pYES2.0-MxHA5 ⁺-ATPase enzymic activity has illustrated that higher than transgenic yeast BJ2168/pYES2.0 more inorganic phosphorus discharges.Result shows: after importing and express the MxHA5 gene, yeast is to Mn ²⁺, Cu ²⁺And Zn ²⁺The tolerance of coercing has strengthened.

Claims

1. protein, the protein that is formed by the aminoacid sequence shown in sequence in sequence table 1.

2. the gene of coding claim 1 described albumen.

3. gene according to claim 2, it is characterized in that: described gene is following 1) or 2) described DNA molecular:

2) 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 claim 2 or 3 described genes.

5. recombinant expression vector according to claim 4, it is characterized in that: described recombinant expression vector is following (I) or (II):

(I) inserts the recombinant expression vector that the described gene of claim 2 or 3 obtains between the multiple clone site of pEZS-NL carrier;

(II) inserts the recombinant expression vector that the described gene of claim 2 or 3 obtains between the multiple clone site of pYES2.0 plasmid.

6. recombinant bacterium according to claim 4, is characterized in that: the recombinant bacterium of described recombinant bacterium for obtaining in (II) of claim 5 described recombinant expression vector importing yeast.

7. method that obtains genetically modified organism, for 1. following or 2. or 3.:

1. the described gene of claim 2 or 3 is imported in the biology that sets out, obtain P type H ⁺The genetically modified organism that-ATPase enzymic activity increases;

2. the described gene of claim 2 or 3 is imported in the biology that sets out the genetically modified organism that the resistance of reverse that obtains that metal ion is coerced increases;

3. the described gene of claim 2 or 3 is imported in the biology that sets out the genetically modified organism that the resistance of reverse that obtains that metal ion is lacked increases;

Described biology is yeast.

8. method according to claim 7 is characterized in that: the described gene of claim 2 or 3 imports the described biology that sets out by (II) of claim 5 described recombinant expression vector.

9. the application of the described gene of the described albumen of claim 1, or claim 2 or 3 in cultivating genetically modified organism; The P type H of described genetically modified organism ⁺-ATPase enzymic activity and/or the resistance of reverse that metal ion is coerced and/or resistance of reverse that metal ion is lacked are higher than the biology that sets out;

Described biology is yeast.