CN102559631A

CN102559631A - Malus xiaojinensis Cheng et Jiang MxHA5 protein, and coding gene and application thereof

Info

Publication number: CN102559631A
Application number: CN2012100124999A
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: 2012-07-11
Anticipated expiration: 2032-01-16
Also published as: CN102559631B

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 sox thereof and application

Technical field

The present invention relates to a kind of malus xiaojinensis MxHA5 albumen and encoding sox 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 influence human healthy.Show that 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.

Research shows that plant can only absorb the soluble iron-Fe (II) in the 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 iron deficiency and coerces symptom.The slight iron deficiency of plant can cause synthetic the minimizing with photosynthetic rate of chlorophyll to reduce, and too little iron causes chlorophyll to synthesize stopping, the young leaves flavescence, and living weight descends significantly.Understand fully that iron absorbs and the molecule mechanism of homeostasis, be significant with human nutrient imbalance improving plant.

In order to absorb effectively and to utilize the iron in the soil, plant has formed flexibility widely to iron deficiency in the evolution of long period of time process.Romheld and Marschner professor have at first proposed higher plant and have coerced formed two kinds of flexibility mechanism---machine-processed I and machine-processed II in the process at the long-term iron deficiency that adapts 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 that these two kinds of variations can increase the surface-area that redox reaction takes place plant, and improve the transport efficacy of plant to iron.Physiological variation is made up of three parts: a Fe (III) reductase enzyme system, a strong proton are secreted pump (H outward ⁺-ATPase) with 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 the form, and iron deficiency are coerced react effectively.Mechanism I plant must be reduced to Fe (II) with Fe (III) earlier and just can be absorbed and used.Supplying under the competent condition of iron, with Fe (III)-inner complex reduction, the Fe (II) that obtains reduction transports through cytoplasmic membrane plant roots earlier; Under the iron deficiency stress conditions, mechanism I plant is through activating a kind of special H ⁺-ATPase makes soil acidification, thereby increases the solubility of iron; Secrete some organic acids (intercalating agent of iron) simultaneously, with the form dissolving of the Fe in the soil (III) with Fe (III)-inner complex; Through a kind of special root reductase enzyme on the cytoplasmic membrane Fe (III)-inner complex is reduced then.This reductase enzyme catalysis electronics is gone back ortho states from kytoplasm pyridine nucleotide (NADH) is striden film and is passed to outside the born of the same parents as Fe (the III)-inner complex of electron acceptor(EA), makes its reduction become Fe (II), and then gets into root cells by the transhipment of the Fe on the plasma membrane (II) translocator.

Mechanism II plant only refers to grass.The iron deficiency chlorosis symptom of comparing generation with non-grass with dicotyledons when grass grows in alkaline soils seldom; Reason is the grass secretion and discharges high siderophore; High siderophore combines to form carrier complexes with iron; And on plasma membrane, produce and a kind of the phytosiderophore mixture is had the absorption and transport system of high affinity, thereby grass can be absorbed effectively and the iron that utilizes ferric iron to resist in the environment is coerced.High siderophore is mugineic acid compound (MAs or Ps); This is one type of low-molecular-weight nonprotein amino acid; Fe (III) there is the intensive affinity and can forms stable, octahedral ferric iron huge legendary turtle compound; And on plasma membrane, produce and a kind of Ps or MAs are had the translocator YSL of high affinity, by YSL Fe (III)-Ps is transported in the kytoplasm, thereby grass can be absorbed effectively and the iron that utilizes ferric iron to resist in the environment is coerced.

Plant plasma membrane serves H ⁺-ATPase belongs to P type ATPase, is the insertion albumen on the plasma membrane, has the energy that utilizes hydrolysising ATP to produce, the H that cytoplasmic membrane is inboard ⁺Pump abbreviates proton pump as to the characteristic in the plasma membrane outside.It has important regulatory role 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 physiological processs such as seed germination, cell polarity growth.Plant plasma membrane serves H+-ATPase is plant vital activity " a 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 received widely and having paid attention to.

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 root CsHA1 gene transcription level that research draws the iron deficiency cucumber has improved, and can descend for iron deficiency plant CsHA1 gene transcription level after supplying iron again.At present, 12 H from Arabidopis thaliana, have been found ⁺-ATPase gene family (AHA1～AHA12).Discover the influence that wherein has 5 expression of gene coerced by iron deficiency.Coerce down at iron 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 about 2 times, and the expression amount of AHA11 is reduced about 2 times.

Malus xiaojinensis (Malus xiaojinensis Cheng et Jiang) is an apple iron high efficiency gene type, belongs to mechanism I plant.Though a lot of research proof H have been arranged ⁺The patience that-ATPase gene pairs improves plant has great role, but does not also see about plasma membrane H in the 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 sox and application.

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

(a) protein of forming by the aminoacid sequence shown in the sequence in the sequence table 1;

(b) with the amino acid residue sequence of sequence in the 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) deutero-protein.

In order to make the protein in (a) be convenient to purifying, proteinic N-terminal or C-terminal that can the aminoacid sequence shown in the sequence 1 is formed 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 synthesize its encoding sox earlier, carry out biology again and express and to obtain.Proteinic encoding sox in above-mentioned (b) can be through the codon with one or several amino-acid residue of disappearance in the dna sequence dna shown in the sequence in the 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 the table 1.

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

Said gene can be following 1)-4) in arbitrary described dna molecular:

1) in the sequence table sequence 2 from the dna molecular shown in 5 ' terminal the 1st to 2862 Nucleotide;

2) dna molecular shown in the sequence 2 in the 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 and the dna molecular of encoding said proteins more than 90%.

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

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

Available existing expression vector establishment contains the recombinant expression vector of said gene.Said expression vector comprises double base agrobacterium vector and the carrier etc. that can be used for micropellet bombardment.Said expression vector also can comprise 3 ' end untranslated zone of foreign gene, promptly comprises the dna fragmentation of polyadenylic acid signal and any other participation mRNA processing or genetic expression.Said polyadenylic acid signal can guide polyadenylic acid to join 3 ' end of mRNA precursor.When using said gene constructed recombinant expression vector, before its transcription initiation Nucleotide, can add any enhancement type promotor or constitutive promoter, they can use separately or be used in combination 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 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 said translation wave 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 identifying and screening, can process used expression vector, can produce enzyme or the gene of luminophor, antibiotic marker thing or the anti-chemical reagent marker gene etc. of colour-change as adding to encode with resistance.Also can not add any selected marker, directly according to phenotypic screen.

Said recombinant expression vector can be as follows (I) or (II):

(I) between the MCS of pEZS-NL carrier, insert the recombinant expression vector that said gene obtains;

(II) between the MCS of pYES2.0 plasmid, insert the recombinant expression vector that said gene obtains.

Said recombinant expression vector specifically can be said gene is connected the recombinant plasmid A that obtains with carrier pEASY-T1sample.Said 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 among the said recombinant plasmid A and the ApaI restriction enzyme site.Said recombinant expression vector specifically can be the recombinant plasmid C that obtains between the KpnI of the insertion of the small segment between KpnI and XbaI enzyme cutting site pYES2.0 plasmid among the said recombinant plasmid A and the XbaI enzyme cutting site.

Said reorganization bacterium specifically can be (II) said recombinant expression vector or recombinant plasmid C is imported the reorganization bacterium that obtains in the yeast.Said yeast specifically can be yeast saccharomyces cerevisiae, like yeast strain BJ2168.

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

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

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

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

Said biology is a yeast, and said gene imports the said biology that sets out through (II) said recombinant expression vector or recombinant plasmid C.Said yeast specifically can be yeast saccharomyces cerevisiae, like yeast strain BJ2168.

More than the metals ion of arbitrary said metals ion in coercing can be Mn ²⁺, Cu ²⁺And Zn ²⁺In at least a.

More than the metals ion of arbitrary said metals ion in lacking can be Fe ²⁺

Malus xiaojinensis MxHA5 albumen provided by the present invention and encoding sox MxHA5 thereof have very important regulatory role for the plant that iron deficiency is coerced; The used material malus xiaojinensis of the present invention 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 through activating the H on the plasma membrane ⁺-ATPase makes soil acidification, increases the solubility of iron.Under the effect of ferric iron reductase enzyme, ferric iron is reduced into ferrous iron then, transhipment is advanced in the tenuigenin under the effect of the ferrous iron translocator on plasma membrane, increases the tolerant to iron deficiency of plant.The plant that MxHA5 gene provided by the present invention is coerced for iron deficiency has very important regulatory role, 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 induces the fluorescent quantitative PCR result of back MxHA5 gene in the malus xiaojinensis Different Organs for iron deficiency.

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 the 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 among the following embodiment like no specified otherwise, is ordinary method.Used test materials among the following embodiment like no specified otherwise, is to buy from routine biochemistry reagent shop and obtains.Quantitative test in following examples all is provided with 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..

The 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 sox thereof

One, the acquisition of MxHA5 full length gene

The total RNA and the reverse transcription of extracting the malus xiaojinensis blade are cDNA.With cDNA is template, right with the primer that F1 and R1 form, and uses the HiFi mix of high-fidelity to carry out pcr amplification, obtains 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: 94 ℃ of preparatory sex change 5min of elder generation; 94 ℃ of sex change 30s, 50 ℃ of annealing 30s, 72 ℃ of extensions 3min, totally 33 circulations then; 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 is a pcr amplification product with swimming lane 2).The result shows, has obtained the dna fragmentation of about 2862bp.

(also directly the dna molecular shown in the composition sequence 1) is connected to the PCR product on the 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 behind this dna fragmentation of purifying and recovering; The T carrier pEASY-MxHA5 that obtains recombinating carries out enzyme successively 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 is cut product with swimming lane 2 for enzyme).Sequencing result shows that pcr amplification product sequence total length is about 2862bp, its nucleotide sequence like the sequence 2 of sequence table from shown in the Nucleotide of 5 ' terminal 1-2862 position, the protein (forming) shown in the sequence 1 in the code sequence tabulation by 954 amino-acid residues.

With the protein called after MxHA5 albumen shown in the sequence 1 of sequence table, the proteic unnamed gene of MxHA5 of will encoding is the MxHA5 gene.

Two, the MxHA5 expression of gene is analyzed 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), treat that it grows into the stem lignifying after, be transferred to root media (1/2MS+1.0mg/L IBA); After tissue cultured seedling bears white root, move to the middle overlay film of 1/2 pancebrin (composition is seen table 3, and original ph transfers to 6.0 with NaOH) and preserve moisture 2 weeks of cultivation; Change pancebrin (composition is seen table 2, and original ph transfers to 6.0 with NaOH) afterwards over to and cultivated one month, change pancebrin weekly one time; Culture condition in the 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 iron deficiency afterwards and induce, method is: plant is gone to from pancebrin contain 4 μ M Fe nutritive mediums (composition is seen table 4, and original ph transfers to 6.0 with NaOH), deionized water rinsing before shifting.

The prescription of table 2 pancebrin

The prescription of table 31/2 pancebrin

Table 4 contains the prescription 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.

With this strand cDNA is template, through the consumption (making pcr amplification product brightness unanimity) of pcr amplification 18s gene adjustment template, analyzes the expression of MxHA5 gene and 18s gene (internal control gene) then through quantitative fluorescent PCR earlier.

The primer of amplification 18s gene is following:

18SR-F：5′-ACACGGGGAGGTAGTGACAA-3′，

18SR-R：5′-CCTCCAATGGATCCTCGTTA-3′。

The primer of amplification MxHA5 gene is following:

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 preparatory sex change 30s; 95 ℃ of sex change 5s, 55 ℃ of annealing 30s, 72 ℃ of extensions 30s, totally 40 circulations then; Last 95 ℃ of 15s, 60 ℃ of 1min, 95 ℃ of 15s.

The relative expression quantity of target gene passes through 2- ^{Δ Δ CT}Method confirms that (concrete operations: the ratio of MxHA5 expression of gene amount and 18s expression of gene amount is as the relative expression quantity of MxHA5 gene in the same template; In mapping, be regarded as 1 to 0 moment expression of gene amount; Thereby observe out different time expression of gene amount more intuitively), the result sees Fig. 2.The MxHA5 gene all 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, MxHA5 expression of gene amount was minimum in the blade, and the increase MxHA5 expression of gene amount along with the treatment time strengthens afterwards, reaches the highest during by the 3rd day, descends afterwards.After iron deficiency was induced, MxHA5 expression of gene amount obviously strengthened in the root, when 24h, descend during than 12h (but strengthening) than before handling, afterwards again gradually amount strengthen.Can also observe, no matter the MxHA5 gene all can up-regulated expression in the iron deficiency inductive time length root, and iron deficiency induces the MxHA5 gene in the 1d rear blade can up-regulated expression.Explain that if coerced by iron deficiency plant increases the flexibility of resistance to iron deficiency through the MxHA5 gene of regulating root and leaf portion.

Three, the Subcellular Localization of MxHA5 gene

1, the structure of the transient expression carrier of the fusion gene of expression MxHA5 and eGFP

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

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

(3) small segment of step (1) and the carrier framework of step (2) are connected, obtain transient expression carrier pEZS-MxHA5.

(4) with transient expression carrier pEZS-MxHA5 transformed into escherichia coli DH5 α bacterial strain, the positive spot of picking carries out bacterium colony PCR successively, enzyme is cut and identified and sequence verification.Sequencing result shows; Between the KpnI of pEZS-NL carrier (available from full Shi Jin Bioisystech Co., Ltd) and ApaI restriction enzyme site, inserted in the sequence table sequence 2 from the dna fragmentation shown in the 5 ' terminal 1-2862 position, the eGFP gene fusion on this dna fragmentation and the pEZS-NL carrier.

2, adopt particle bombardment to carry out of the conversion of transient expression carrier to 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 petridish that contains the MS substratum, wraps petridish subsequent use with masking foil then.

(2) preparation of bronze suspension-s

Take by weighing the 60mg bronze, put into the centrifuge tube of 1.5ml sterilization, add the 1ml absolute ethyl alcohol, concussion 1min, the centrifugal 10s of 10000rpm abandons supernatant, adds the 1ml absolute ethyl alcohol again, concussion 1min, the centrifugal 10s of 10000rpm; Abandon supernatant, bronze is suspended from the 1ml sterilized water, obtain bronze suspension-s ,-20 ℃ of preservations are subsequent use.

(3) the fit preparation of bronze-plasmid dna complex

Draw 8.5 μ l bronze suspension-s, 3.5 μ l 0.1M NSC 223080,8.5 μ l 2.5M CaCl ₂2H ₂O and 5 μ l transient expression carrier pEZS-MxHA5 are with the 3min that vibrates on the mixture earthquake device; The centrifugal 20s of 10000rpm; Abandon supernatant, with absolute ethyl alcohol rinsing 3 times; Add 30 μ l absolute ethyl alcohols and suspend, it is fit to obtain bronze-plasmid dna complex.

(4) bombardment receptor material

Select the pressure membrane of certain pressure for use, soaked 1-2 hour in 70% absolute ethyl alcohol 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; It is fit to get 10 μ l bronzes-plasmid dna complex, evenly coats on the mid-way of bombardment film, and the area of coating is consistent with the pore diameter range on the carrier solid circle; After film dried, be installed on the launching device; Pressure membrane is installed to the lower end of gas accelerator; Onion epidermis cell places the middle part of petridish, puts into Vakuumkammer, takes off the petridish lid; Vacuumize pointer to 26; Exit on gas accelerator, pressure reaches in the time of can splitting the pressure that film can bear in pipe, 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

Petridish is sealed with Parafilm, cultivated 24h for 28 ℃, under fluorescent microscope, observe the expression of green fluorescence, adopting uses the same method handles pEZS-NL carrier (control plasmid).Result's (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.The result shows that MxHA5 albumen is positioned on the 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,, reclaims enzyme and cut product (about 2965bp) with the reorganization T carrier pEASY-MxHA5 that makes up among restriction enzyme KpnI and the XbaI enzyme cutting embodiment 1.

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

3, the carrier framework of the enzyme of step 1 being cut product and step 2 is connected, and obtains 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 (about 109bp).

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

(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 the primer of F1 and R1 composition (adopt to).The result is as shown in Figure 4, and (M is the dna molecular amount standard of DL 15000bp; Swimming lane 1 is transgenic yeast BJ2168/pYES2.0-MxHA5 with swimming lane 2; 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 the yeast ⁺The mensuration of-ATPase enzymic activity

H ⁺-ATPase enzyme activity determination method is with reference to 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 tested as follows:

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

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

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

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

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

6, detect P type H ⁺The preceding system 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.In preceding system, add 2 μ g plasmalemma proteins; Hatch 30min under 30 ℃, add the SDS aqueous solution termination reaction of 3mL 1g/100mL afterwards, detect the absorbance value (A) under the 820nm with spectrophotometer; (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 the different light absorption values, the inorganic phosphorus that the high more explanation of absorbance value discharges is many more, thereby confirms 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).

The result is as shown in Figure 5, and the absorption value of transgenic yeast BJ2168/pYES2.0-MxHA5 is higher than transgenic yeast BJ2168/pYES2.0, explains that more inorganic phosphorus discharges.The result shows, after importing and expressing the MxHA5 gene, and zymic P type H ⁺-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 (cultivating 18h usually) under 30 ℃, 200rpm condition, and this moment, goal gene was suppressed expression.

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

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 result sees Fig. 6.The result shows that behind importing and the expression 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, adopts the defective type substratum, the OD of culturing yeast to bacterium liquid under 30 ℃, 200rpm condition ₆₀₀Be about 1 (cultivating 1-2d usually), this moment, goal gene was suppressed expression.

3, the thalline of step 2 is seeded to 50mL and contains different metal ionic substratum (substratum first, substratum second or substratum third), under 30 ℃, 200rpm condition, cultivate, 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 is as shown in Figure 7.Under the condition that each metals ion is coerced, the H of transgenic yeast BJ2168/pYES2.0-MxHA5 ⁺-ATPase enzymic activity is higher than transgenic yeast BJ2168/pYES2.0, has explained that more inorganic phosphorus discharges.The result shows: behind importing and the expression MxHA5 gene, yeast is to Mn ²⁺, Cu ²⁺And Zn ²⁺The tolerance of coercing has strengthened.

Claims

1. protein is (a) or (b) as follows:

2. coding claim 1 said proteic gene.

3. gene according to claim 2 is characterized in that: said gene is following 1)-4) in arbitrary described dna molecular:

2) dna molecular shown in the sequence 2 in the sequence table;

4. contain claim 2 or 3 said expression of gene boxes, recombinant expression vector, transgenic cell line or reorganization bacterium.

5. recombinant expression vector according to claim 4 is characterized in that: said recombinant expression vector is (I) or (II) as follows:

(I) between the MCS of pEZS-NL carrier, insert the recombinant expression vector that claim 2 or 3 said genes obtain;

(II) between the MCS of pYES2.0 plasmid, insert the recombinant expression vector that claim 2 or 3 said genes obtain.

6. reorganization bacterium according to claim 4 is characterized in that: said reorganization bacterium is for importing the reorganization bacterium that obtains in the yeast with (II) of claim 5 said recombinant expression vector.

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

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

2. claim 2 or 3 said genes are imported in the biology that sets out the genetically modified organism that the resistance of reverse that obtains that metals ion is coerced increases;

3. claim 2 or 3 said genes are imported in the biology that sets out the genetically modified organism that the resistance of reverse that obtains that metals ion is lacked increases.

8. method according to claim 7 is characterized in that: said biology is a yeast, and claim 2 or 3 said genes import the said biology that sets out through (II) of claim 5 said recombinant expression vector.

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