CN104480078A - Atyipiex canexcen cytochrome P-450 gene clone and application thereof - Google Patents

Abstract

An atyipiex canexcen cytochrome P-450 gene clone and its application belong to the technical field of plant genetic engineering. A plant stress resistance related protein AcCYP450 is a protein formed by an amino acid sequence as shown in the SEQ ID NO:2 in the sequence table. A coding sequence of an encoding gene AcCYP450 of the plant stress resistance related protein AcCYP450 is one of the following sequences: 1) a nucleotide sequence of the encoding sequence, as shown at the 164th-781st sites of deoxynucleotide at 5' end of SEQ ID NO: 1 in the sequence table; and 2) a nucleotide molecule of SEQ ID NO:2 protein sequence in the coding sequence table. A recombinant expression vector contains a coding gene AcCYP450 of the plant stress resistance related protein AcCYP450. A recombinant strain contains the coding gene AcCYP450 of the plant stress resistance related protein AcCYP450. Through qRT-PCR detection on the origin plant atyipiex canexcen, expression of the gene is up-regulated by the stress of NaCl and PEG. Meanwhile, AcCYP450 is introduced into brewer's yeast to obtain transgenetic brewer's yeast Saccharomyces cerevisiae, and Saccharomyces cerevisiae undergoes stress treatment. It can show through results that AcCYP450 can raise drought-resistant capacity (sorbitol) and salt tolerance (NaCl) of the yeast.

Description

Atyiplex canescen cytochrome P450 gene clone and application thereof

Technical field

The invention belongs to field of plant genetic, particularly relate to and use Saccharomyces Cerevisiae in S accharomyces cerevisiaeINVSc1 bacterial strain to express the albumin A cCYP450 relevant to plant stress-resistance (being specially salt and arid), adopt real-time fluorescence quantitative PCR (PCR in real time simultaneously, Real-Time-PCR) method study the function of this gene pairs abiotic stress NaCl and PEG.

Background technology

In current agriculture production, environment stress affects one of the most important factor of grain yield and challenge.Farm crop are in the whole season of growth, the impact of various stress factors can be subject to, as the disease and pest etc. of biotic, and the salt of abiotic stress, alkali, high temperature, low temperature, arid etc., these are coerced and limit growing of plant, crop yield finally can be caused to reduce, quality declines, direct impact even threatens grain security, have a strong impact in the adverse circumstance factor of crop production at these, extreme temperature, arid, high salt, nutritional trouble (comprise mineral substance poisoning and mineral deficiency) is the main limiting factor affecting crop yield, direct from salt every year, alkali, arid, the crop production reduction that the adverse circumstances such as low temperature stress cause still exceedes 20% of ultimate production, and Drought and salt to coerce be topmost restraining factors.Therefore, study the gene relevant with abiotic stress, and imported crop and carry out molecular breeding, cause the broad interest of researcher, and carry out the clone of adverse circumstance genes involved and the parsing of function thereof by engineered method, valuable information and material can be provided for plant stress-resistance breeding.But, from the angle directly solving key issue agriculture production, the genetically modified crops kind that agriculture production can rise remarkable effect is also few, the research of the mankind to plant adverse circumstance adaptability molecule mechanism is also nowhere near, far can not meet the demand of plant molecular breeding, compare with World Developed Countries, it is relative with the gene with important utility value less that China has independent intellectual property right.Meanwhile, current research also mainly concentrates on herbaceous plant, and also relative less with the Molecular and Genetic Study of arid to xylophyta and salt tolerant thereof.But at structure, growth, growth, physiology, and there is larger otherness in reply biology and abiotic stress etc. in draft and xylophyta.So, from xylophyta, find degeneration-resistant relevant gene and study, more candidate gene can be provided for the biology and this means of abiotic stress tolerance being improved crop by genetically engineered.

Atyiplex canescen (Atriplex canescens [Pursh] Nute) is a kind of saltbush, classification is Chenopodiaceae, atriplex, be prevalent in arid, semiarid zone, there is the characteristic of stronger drought-resistant, anti-saline and alkaline, cold resistant and resistance to heavy metal, especially drought-resistant and salt characteristic.Atyiplex canescen is at the Arid&semi-arid area of every annual rainfall>=180mm, average temperature of the whole year is only the low temp area of about 5 DEG C, even still well-grown under extreme low temperature is the severe environment such as-40 DEG C of areas and saltings, also be applicable to growth in the area of height above sea level about 2000 meters simultaneously, according to having been reported, Atyiplex canescen is called " biological demineralizer " by some country, and its one-year age just can absorb the salinity of more than 1 ton from the soil of 6.07 mu, Bush biomass also can reach 15t/hm ²crude protein content in branches and leaves, more than 12%, high yield and high quality, is rich in nutrition, and related science men think, Atyiplex canescen has and abundant nutritive value and considerable feed applications, therefore becomes desert, the very valuable excellent food shrub species in half-desert arid area.Meanwhile, Atyiplex canescen also has the ability of accumulation selenium, be therefore also widely used in soil conservation and slope, road is fixed in the U.S., but range improvement remains its main application.In recent years, Atyiplex canescen successively in the Xinjiang of China, Gansu, Ningxia, Qinghai and other local plantation, by large quantity research, fully demonstrated the anti-salt that Atyiplex canescen is stronger, alkali, arid, low temperature, barren good characteristic such as grade, becomes the fine tree species of greening and soil conservation gradually.

S. cervisiae (Saccharomyces cerevisiae) has typical eukaryotic system, it is diploid, it can form disulfide linkage in protein translation post-treatment process, there is the feature such as glycosylation and protein folding, make its gene expression pattern more close to the expression pattern in eukaryote, it also has the advantage that growth is quick, genetic manipulation is easy and be easy to cultivation etc., so in the correlative study to plant function gene, with yeast expression system screening, there is the irreplaceable dominance of prokaryotic expression system.Wine brewing yeast strain INVSc1 (genotype is MATa his 3D1 leu2 trp1-289 ura3-52) be through artificial reconstructed after uracil auxotrophy bacterial strain, when the yeast transformant of this bacterial strain uses SC-U to lack Screening of Media, false positive is low, transformation efficiency is high, is therefore desirable yeast expression bacterial strain.Utilize yeast nutrition deficient strain to the research of plant cDNA libraries and excavation, in plant, receive more and more concern.Frommer etc. and Hsu etc. (1993) are cloned into the NAT2/AAP1 gene of amino acid through enzyme in screening Arabidopis thaliana cDNA library, and this gene can the amino acid transport defect mutant of complementary yeast; The functional study of Yeast system research plant salt endurance expressing gene is also widely used, Yamada etc. (2002) just utilize yeast transformant to verify that propadiene cyclisation oxydase (AOC) gene can improve salt resistance greatly, infer in plant, also may have anti-salt functional to AOC gene further.Tang Yulin etc. (2007) utilize yeast to have studied soybean SALI3-2 albumen the salt resistance ability of yeast transformant can be made to be significantly improved, thus infer the anti-adversity ability that SALI3-2 gene has.As can be seen here, utilize yeast expression foreign protein and study its function and be subject to widespread use.

Real-Time PCR (RT-PCR) is the technology of the general gene expression detection of a kind of current application, it is by adding fluorophor, and utilize fluorescent signal to accumulate the process of the whole PCR of Real-Time Monitoring, finally by typical curve, quantitative analysis is carried out to unknown template, the method can avoid the deviation produced during the quantitative end product of normal PCR, thus the repeatability of experiment is improved, this technology has now been widely used in the expression amount change of monitoring cell mRNA.SYBR green is one of common method of Real-time PCR, advantages such as this method is highly sensitive, and versatility is good, and method is simple and generally being used by domestic and international scientific research.

Cytochrome P450 (cytochromeP450, be called for short CYP450) be the superfamily having the heme-thiolate proteins of absorption peak after a class is combined with CO with its reduction-state at wavelength 450nm place, it participates in the metabolism of endogenous material and exogenous material.CYP450 at Late Cambrian in 1958 in the hepatomicrosome of rat, 1969 in plant (cotton) be also found first.After this, the report that plant CYP450 studies also occurs in succession.On the endoplasmic reticulum that CYP450 is mainly distributed in cell and mitochondrial inner membrane, be a kind of terminal oxygenase, participate in the processes such as the sterols hormone sensitive lipase gene in organism.In recent years, found by research in the structure to CYP450, function and drug metabolism, in CYP450 and higher plant, many metabolism with the secondary metabolite of defense function are closely related.Removing toxic substances as the weedicide in xenobiotics, sterilant, pollutent etc. is reacted, and has the building-up reactions of phylactic sterol, alkaloid, flavonoid and terpene etc.Current research shows CYP450 not only as the key enzyme in drug metabolism processes, has material impact to cytokine and thermoregulation simultaneously.Can infer thus, this gene may participate in biotic and abiotic stress effect in plant.Therefore, to clone and the research of plant CYP450 gene, can be revert in crop by transgenic technology, and provide new material foundation for cultivating the genetically modified crops with the function such as Resistant, weedicide.But in current numerous researchs, also without about the research report of Atyiplex canescen AcCYP450 gene in the physiological function such as degeneration-resistant.

Summary of the invention

The present invention is by building low temperature (-10 DEG C) and salt stress (400mM NaCl) cDNA library of Atyiplex canescen (Atriplex canescens), and utilize Gateway technology to be recombinated to by cDNA library LR in Yeast expression carrier (pYES-DEST52), mixing plasmid vector is through being transformed in yeast strain INVSc1, and by simulation environment stress (NaCl, sorbyl alcohol) filter out the gene relevant to environment stress, utilize RT-PCR technology to detect this gene at environment stress (NaCl simultaneously, PEG6000) the expression amount change under condition, with the respective action of this gene of preliminary study environment stress in Atyiplex canescen.The present invention not only provides new foundation to the research of the drought-resistant of Atyiplex canescen and salt tolerant molecular mechanism, has the transgenic crop of anti-adversity ability and forest (salt tolerant, arid) lays the foundation for obtaining simultaneously.In the present invention, the recombination yeast transformant turning pYES-AcCYP450 shows the ability of significantly drought-resistant and salt stress-resistant.Simultaneously in origin plant Atyiplex canescen, the expression of AcCYP450 gene is also coerced salt stress and drought and is shown obvious response.

The invention provides a kind of albumen relevant to plant stress-resistance (salt and drought) containing CYP450 functional domain and coding gene sequence thereof in Atyiplex canescen, by utilizing Yeast system to carry out functional verification, and the method detected by RT-PCR in origin plant is resolved further to this gene and application in plant stress-resistance:

Cytopigment enzyme (P450) provided by the invention, name is called AcCYP450, is the nucleotide sequence shown in SEQ ID NO:1 in sequence table.AcCYP450 gene is obtained by the order-checking of Atyiplex canescen cDNA library.This gene is altogether by 1000 based compositions, and complete open reading frame contains 615bp, and simultaneously also containing 5 ' non-translational region and 3 ' non-translational region sequence, the initiator codon of open reading frame is ATG, and terminator codon is TAG.

Drought-resistant and the salt stress of albumen provided by the present invention, name is called AcCYP450, plant anti-adversity associated protein AcCYP450, the protein be made up of the aminoacid sequence shown in SEQ ID NO:2 in sequence table.

The encoding sequence of the encoding gene AcCYP450 of plant adversity resistance related protein AcCYP450 is one of following:

1) nucleotide sequence of encoding sequence holds 164-781 position deoxynucleotide as 5 ' of SEQ ID NO:1 in sequence table;

2) nucleic acid molecule of SEQ ID NO:2 protein sequence in polynucleotide.

A kind of recombinant expression vector, the encoding gene AcCYP450 containing plant adversity resistance related protein AcCYP450.

A kind of recombinant bacterium, the encoding gene AcCYP450 containing plant adversity resistance related protein AcCYP450.

Sequence 2 in sequence table is made up of 205 amino-acid residues altogether, analyzes further according to DNAMAN, this albumen iso-electric point 7.57, Tot Prot 23.3KDa.Generally, containing three trans-membrane region, Protein secondary structure is curling containing 103,83 spirals, and 20 fold, have 6 antigen peptide sections, leucine (Leu) content 12.64%.Albumen is containing 66 hydrophilic amino acids, and 137 hydrophobic amino acids, analyze the possible surf zone of sphaeroprotein at middle part and N end according to the Hphob./Kyte & Doolittle of Expasy protscale.PSORT Prediction predicted protein location may be microbody.

The Yeast expression carrier pYES-DEST52 (purchased from Invitrogen company) that the present invention relates to and AcCYP450 gene recombination are pYES-AcCYP450, and host's competent cell that this recombinant yeast expression vector transforms is yeast saccharomyces cerevisiae INVSc1.This Yeast expression carrier is except containing except the DNA sequences encoding of AcCYP450, also carry GAL1 promotor, T7 promotor, URA3 gene, ampicillin resistance marker's gene, CYC1 stop transcription signal, LR recombination site attR1 and attR2, lethal gene ccdB for reverse screening, the various controlling elements of exogenous gene albumen in yeast needed for high expression such as the 6xHis Tag of protein purification, the V5 epitope that protein expression detects.

The present invention passes through RT-PCR method, have detected the response (PEG that this gene is being coerced for Drought and salt, NaCl process), gene shows gene upregulation under two kinds of Stress treatments, this gene of further supposition participates in stress response plant, especially Drought and salt plays an important role in coercing, and the up-regulated expression of this gene contributes to the ability of the drought-resistant and anti-salt improving Atyiplex canescen.

The present invention is passing through yeast (NaCl, sorbyl alcohol) and RT-PCR (NaCl, PEG) about after the research of Drought and salt, the application of AcCYP450 gene in adverse circumstance: this gene overexpression may improve the resistance of plant, described stress resistance of plant specifically can be the resistance to abiotic stress and biotic, abiotic stress as arid, extreme temperature, saline and alkaline etc., the particularly ability of the drought-resistant and salt stress of plant.

Accompanying drawing explanation

Fig. 1 is Atyiplex canescen Total RNAs extraction picture

Fig. 2 is 1KbPlus DNA Ladder

Fig. 3 is that cDNA library Insert Fragment length PCR detects picture

Fig. 4 is for detecting picture for cDNA library Insert Fragment length PCR

Fig. 5 is 1Kb Plus DNA Ladder

Fig. 6 is pYES-AcGSK3 recombinant plasmid pcr amplification gel electrophoresis figure

M:DL2000 DNA Marker; M:2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 1,2: amplified fragments

Fig. 7 is AcCYP450 functional domain compare of analysis result in NCBI

Fig. 8 is the homologous protein phylogenetic analysis of AcCYP450 and other species

Fig. 9 is pYES-AcCYP450 positive recombination yeast transformant PCR gel electrophoresis figure

M:DL2000 DNA Marker; M:2000bp, 1000bp, 750bp, 500bp, 250bp, 100bp; 1,2: positive yeast transformant PCR electroresis appraisal

The result of Figure 10 under to be that recombination yeast pYES-AcCYP450 and pYES-DEST52 is non-coerce

Figure 11 is the result of recombination yeast pYES-AcCYP450 and pYES-DEST52 under 3M sorbyl alcohol is coerced

Figure 12 is the result of recombination yeast pYES-AcCYP450 and pYES-DEST52 under 2M NaCl coerces

Figure 10--in 12: pYES-AcCYP450 yeast conversion is obviously being better than empty carrier pYES-DEST52 yeast transformant to the anti-adversity ability of 2M NaCl and 3M sorbyl alcohol.

Figure 13 is the expression amount variation diagram of AcCYP450 gene after 400mM NaCl Stress treatment

Figure 14 is the expression amount variation diagram of AcCYP450 gene after 20%PEG6000 Stress treatment

In Figure 13 and 14: AcCYP450 gene starts up-regulated expression after NaCl process 6h, and when 12h, expression amount is the highest;

Meanwhile, after PEG process 6h, this gene starts up-regulated expression.

Embodiment

The acquisition of embodiment one: AcCYP450 full length gene cDNA and sequential analysis

1. Stress treatment Atyiplex canescen:

Grow trimestral Atyiplex canescen plant under-10 DEG C of conditions, be transplanted to artificial climate incubator, normal cultivation is after 10 days, and plant to be planted recovery survival, sends young leaves, robust growth.Plant is transferred to containing under nutritious water planting condition, normal growth two days later, with 500mL containing the saline solution water planting process Atyiplex canescen plant of NaCl400mM, Stress treatment, after 4 days, plucks blade, and tender stem and young root clean up and liquid nitrogen flash freezer,-80 DEG C frozen, in order to building storehouse.

2. construction cDNA library:

The Atyiplex canescen sample of NaCl process after 4 days (blade, tender stem and young root) is extracted total serum IgE (Fig. 1, Fig. 2), then use Invitrogen company ' the strand cDNA containing complete cap sequence is carried out enrichment by cap antibody module ', and according to full-Length cDNA Library Construction Kit test kit builds cDNA library, and by Gateway technology, this cDNA library LR is recombinated in Yeast expression carrier pYES-DEST52, at random PCR detection (Fig. 3, Fig. 4, Fig. 5) is carried out to cDNA library, the fragment of more than length 500bp is checked order, utilize the BlastX of NCBI to carry out comparison and the relevant biological information Epidemiological Analysis of functional domain, obtain the expressed sequence tag (ESTs) of Atyiplex canescen salt stress.

The method of 2.1 Atyiplex canescen Total RNAs extraction:

2.1.1 get 100mg plant sample, with the rapid grinding powder of liquid nitrogen in the mortar of precooling, be quickly transferred to and meet in cold 2mL centrifuge tube.(ensure that whole process is in ventilating kitchen, prevents RNA from degrading.)

2.1.2 add 1mlTrizol reagent, thermal agitation mixing 15s, leaves standstill the abundant lysing cell of 10min.

2.1.3 add 0.2mL chloroform (trichloromethane), concussion mixing 15s, leaves standstill 5min rapidly.

2.1.4 high-speed low temperature (12,000rpm, 4 DEG C) centrifugal 15min.

2.1.5 supernatant is carefully transferred to new 1.5mL centrifuge tube, add 0.5mL Virahol, put upside down mixing, leave standstill 10min.

2.1.6 high-speed low temperature (12,000rpm, 4 DEG C) centrifugal 10min again.

2.1.7 remove supernatant, add the ethanol of 0.5mL75%, 7,500rpm low-temperature centrifugation 5min is with washing precipitation.

2.1.8 repeating step 2.1.7 once, further washing precipitation, removes ion.

2.1.9 carefully supernatant is drawn, and dry RNA precipitation in ventilating kitchen, and with the DEPC water dissolution RNA of 50 μ L.

3. screen the clone of AcCYP450 gene fragment:

According to sequencing analysis, obtain Atyiplex canescen ESTs, therefrom obtain the cDNA complete sequence of a coding AcCYP450, the Yeast expression carrier pYES-AcCYP450 obtained that recombinated by LR is converted into (DH5 α) in competent escherichia coli cell, after incubated overnight, picking list bacterium colony, and in LB (Amp containing 100mg/L) liquid nutrient medium incubated overnight, extract plasmid by plasmid extraction kit (Ding Guo company).And according to the universal sequence PCR primer on carrier, and carry out plasmid PCR amplification checking, to identify the cDNA sequence of acquisition.

Upstream (T7): 5 '-TAATACGACTCACTATAGGG-3 '

Downstream (R): 5 '-AGGGTTAGGGATAGGCTTACCTTC-3 '

PCR reaction system (25 μ L): each 0.5 μ L, Tag enzyme 0.5 μ L of Buffer2.5 μ L, upstream and downstream primer (T7, R), dNTP0.5 μ L, template 0.2 μ L, ddH ₂o20.8 μ L.The condition of PCR reaction: 94 DEG C of denaturations 5 minutes; 94 DEG C of sex change 30 seconds; Be cooled to rapidly 58 DEG C of annealing 30 seconds; 72 DEG C extend 2 minutes, 30 circulations; 72 DEG C extend 10 minutes; 4 DEG C of cooling preservation.Amplified production detects through the agarose gel electrophoresis of 1%, and the position of about 1100bp has specific object band (Fig. 6).

The sequential analysis of embodiment two: AcCYP450 gene

After sequencing analysis, the full length cDNA sequence of AcCYP450 gene comprises the open reading frame of 1000bp, 205 amino-acid residues of encoding altogether, and analyze according to DNAMAN, albumen is made up of 203 amino acid, iso-electric point 7.57, Tot Prot 23.3KDa.Generally, containing three trans-membrane region, Protein secondary structure is curling containing 103,83 spirals, and 20 fold, have 6 antigen peptide sections, leucine (Leu) content 12.64%.Albumen is containing 66 hydrophilic amino acids, and 137 hydrophobic amino acids, analyze the possible surf zone of sphaeroprotein at middle part and N end according to the Hphob./Kyte & Doolittle of Expasy protscale.PSORT Prediction predicted protein location may be microbody, and this is the principal character of CYP450 family protein.Showing by analyzing, obtain the full length cDNA sequence of 1000 genes, and the accession number on GenBank being KJ027023.

Utilize NCBI (http://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi) to carry out conservative region analysis (Fig. 7) to this encoding gene, the albumen of this genes encoding belongs to CYP450 family protein.By NCBI, BLASTX is carried out to the aminoacid sequence of AcCYP450 genes encoding, by carrying out homology analysis and phylogenetic analysis with the aminoacid sequence of other species determining function, utilize MEGA4 software to carry out phylogenetic analysis, result shows: the evolution of amino acid relation that the CYP450 of AcCYP450 and potato encodes is nearest.(Fig. 8).

Embodiment three: the conversion of recombination yeast and detection

Wine brewing yeast strain INVSc1 is urinary ammonia acid auxotroph (Ura ^-) type strain, lacking the yeast minimal medium (SC-Ura of urinary ammonia acid ^-) on, this yeast strain almost can not Growth and reproduction.And Yeast expression carrier (pYES2-DEST52) is upper containing URA3 gene, and the expression of this gene can make yeast transformant at SC-Ura ^-normal growth on substratum.Therefore, SC-Ura ^-selective agar medium can carry out screening that is positive and non-positive yeast transformant.By Li-acetate method, vector plasmid pYES-AcCYP450 and pYES-DEST52 is transformed in competent yeast bacterial strain INVSc1 respectively, transformed yeast bacterium liquid is coated in SC-Ura ^-on solid selection medium, contrast with unconverted yeast, cultivate, after 2d except empty yeast is completely not long, the yeast transforming two kinds of plasmids all can grow and have bacterium colony to grow, and yeast conversion success is described, picking yeast list bacterium colony, carry out after incubated overnight abolishing cell walls, and carry out the further qualification of positive transformant by bacterium liquid PCR method.

1. use Lithium Acetate chemical transformation transformed saccharomyces cerevisiae INVSc1, its concrete step of converting:

A yeast strain mono-clonal (INVSc1) joins in 10mL YPD liquid nutrient medium by 1.1, and 30 DEG C are spent the night and shake training.

The OD of 1.2 detection yeast liquid ₆₀₀value, utilizes 50mL YPD liquid nutrient medium, the yeast liquid of incubated overnight is diluted to OD ₆₀₀be 0.4,30 DEG C to continue to shake training 2-4h.

After 1.3 low-temperature centrifugations (4 DEG C, 2500rpm) 5min, remove supernatant and collect thalline, with the resuspended thalline of 40mL 1xTE damping fluid.

1.4 again after low-temperature centrifugation (4 DEG C, 2500rpm) 5min, collects thalline, with the resuspended thalline of 2mL 1xLiAc/0.5xTE damping fluid.

1.5 by the resuspended thalline that obtains according to the system of every pipe 100 μ L, be dispensed into 1.5mL centrifuge tube.

The yeast cell of packing is placed in incubated at room 10min by 1.6.

1.7, in each transformation system (100 μ L), add 1 μ g plasmid DNA, 100 μ g denaturated salmon essence DNA, mixing.

700 μ L 1x LiAc/40%PEG-3350/1xTE are added, mixing in 1.8 every individual system.

Mixed solution 30min in 1.930 DEG C of incubation step 1.8.

1.10 DMSO adding 88 μ L in every individual system again, after mixing, 42 DEG C of Water Unders bath thermal shock 7min.

1.115000rpm low-temperature centrifugation 1min, remove supernatant.

1.12 with the resuspended thalline of damping fluid of the 1mL 1xTE got ready, and 5000rpm low-temperature centrifugation 1min, removes supernatant further.

1.13 is resuspended by thalline with the damping fluid of 100 μ L 1xTE, and be applied on yeast Selective agar medium, cultivates 24h for 30 DEG C.

2. the qualification of positive transformant

Single bacterium colony of random picking 5 yeast transformants (pYES2-AcCYP450) from yeast Selective agar medium, 30 DEG C of shaking culture spend the night (200rpm), collect incubated overnight thalline, boiling water boiling 5min, be placed in rapidly on ice 5min with smudge cells, repeat several times, bacterium liquid PCR is carried out as template using cytoclasis liquid centrifugal concentrating sample, carry out pcr amplification with primer T7 and R, PCR reaction system is (25 μ L): each 0.5 μ L, Tag enzyme 0.5 μ L of Buffer2.5 μ L, upstream and downstream primer (T7, R), dNTP 0.5 μ L, template 5 μ L, ddH ₂o15.5 μ L.

PCR primer is electrophoresis detection on the sepharose of 1%, and amplified fragments size is 1000bp, coincide (Fig. 9) with target length.Prove that recombinant plasmid pYES2-AcCYP450 has been transformed in yeast strain.

Embodiment four: positive transformants yeast environment stress process

1. prepare before environment stress process

Get appropriate positive yeast transformant (pYES-DEST52, pYES2-AcCYP450) bacterium liquid, be inoculated in the SC-U liquid nutrient medium containing 2% glucose, 200rpm shaking culture 24h under 30 DEG C of conditions, survey OD ₆₀₀value, and with SC-U liquid nutrient medium (2% glucose) by bacterium liquid OD ₆₀₀unification is adjusted to 0.4, cumulative volume 5mL, the centrifugal 1min of 8000rpm, Aspirate supernatant, add the yeast inducing culture resuspended thalline of 2mL containing 2% semi-lactosi, receive enlarged culturing in the inducing culture of 5mL with the ratio of 1:50,30 DEG C of shaking culture 24h, detect the OD of yeast INVSc1 (pYES-DEST52) and INVSc1 (pYES2-AcCYP450) ₆₀₀value, and OD is adjusted in unification ₆₀₀value is 2, for subsequent use.Different abiotic stress process is carried out to this two primary yeasts transformant, compares the anti-salt of two primary yeast transformants and drought stress ability, experiment repetition 3 times.

2. simulate environment stress process

The plant being present in occurring in nature in the face of various biology and abiotic stress, most importantly salt stress (NaCl, KCl etc.) and drought stress in abiotic stress.

Carry out the simulation of salt and arid in laboratory conditions, coerce with 2MNaCl analog salt, the sorbyl alcohol Drought stress simulation of 3M.Use above two kinds of simulated conditions process yeast transformants, contrasted by the growing state of yeast, thus evaluate this gene in yeast after abduction delivering on the impact of yeast resistance, to carry out pre-test (Figure 10, Figure 11, Figure 12) to the degeneration-resistant function of this gene in yeast.

2.1NaCl process: by above-mentioned thalline for subsequent use respectively with the thalline not diluting and dilute 10,100,1000,10000 times,

Drawing 2 μ L bacterium liquid is inoculated on the SC-U solid medium containing 2% semi-lactosi, cultivates two days later, compares the colony growth state of two primary yeast transformed bacterias for 30 DEG C.

2.23M sorbyl alcohol arid simulation process: by above-mentioned thalline for subsequent use respectively with the thalline not diluting and dilute 10,100,1000,10000 times, drawing 2 μ L bacterium liquid is inoculated on the SC-U solid medium containing 2% semi-lactosi, cultivate two days later, compare the colony growth state of two primary yeast transformed bacterias for 30 DEG C.

The response of embodiment five: AcCYP450 gene under Atyiplex canescen PEG and NaCl coerces

1. Atyiplex canescen Stress treatment:

By Atyiplex canescen seed in Nutrition Soil, after normal condition grows 50 days, choose well-grown Atyiplex canescen, transfer in MS nutritive medium and carry out water planting, every day changes nutritive medium, and water planting 3 days is after Atyiplex canescen robust growth, respectively with 400mMNaCl and 20%PEG6000 process, and 0,6,12,24, get root, stem and leaf, liquid nitrogen flash freezer ,-80 DEG C of preservations after 48h.

2.RNA extracts and reverse transcription:

Adopt Trizol method to extract total serum IgE (as embodiment one, 2.1), utilize Takara company rTreagentKit With gDNA Eraser (Perfect Real Time) test kit carries out reverse transcription to prepare cDNA, for quantityReal-time pcr analysis.

3. quantitative fluorescent PCR analysis:

Utilize green Reagent test kit, uses 7500 fluorescent quantitation systems axiol-ogy AcCYP450 gene (F:AGCCAAAGGATGACTTGCTGA; R:TTGAAGTTGTGCCTTGGCCT) time dependent expression amount difference under different treatment.The eucaryon elongation growth factor (EF1-α) as reference gene (F:5 '-CCCCAGTTCTCGACTGTCAC-3 '; R5 '-TGGTGGGAACCATCTTCACG-3 ').Reaction conditions is: 95 DEG C of denaturations 30 seconds; 95 DEG C of sex change 5 seconds, 60 DEG C of annealing extensions 34 seconds, carry out 40 circulations; 95 DEG C of sex change 15 seconds, 60 DEG C of annealing extensions 1 minute, 95 DEG C of sex change 15 seconds; 60 DEG C extend 15 seconds.Adopt 2 ^{-△ △ Ct}method analyzes expression amount difference (Figure 13, Figure 14).

Claims (4)

1. a plant anti-adversity associated protein AcCYP450, is characterized in that the protein be made up of the aminoacid sequence shown in SEQ ID NO:2 in sequence table.

2., by the encoding gene AcCYP450 of plant adversity resistance related protein AcCYP450 described in claim 1, it is characterized in that the encoding sequence of described encoding gene AcCYP450 is one of following:

1) nucleotide sequence of encoding sequence holds 164-781 position deoxynucleotide as 5 ' of SEQ ID NO:1 in sequence table;

2) nucleic acid molecule of SEQ ID NO:2 protein sequence in polynucleotide.

3. a recombinant expression vector, is characterized in that the encoding gene AcCYP450 containing plant adversity resistance related protein AcCYP450 described in claim 2.

4. a recombinant bacterium, is characterized in that the encoding gene AcCYP450 containing plant adversity resistance related protein AcCYP450 described in claim 2.