CN104862321B - Kalium ion transport GFP trkH, its encoding proteins and its application - Google Patents
Kalium ion transport GFP trkH, its encoding proteins and its application Download PDFInfo
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Abstract
The invention discloses a kind of kalium ion transport GFP trkH from marine microorganism, the gene has such as SEQ ID NO:Base sequence shown in 1.The gene trkH of the present invention derives from marine bacteria, and its albumen TrkH encoded has the function of kalium ion transport albumen, can improve transgenosis K+The K of native defect type saccharomyces cerevisiae+Absorption efficiency and to Na+Insensitivity, so as to improve its upgrowth situation under potassium deficiency and condition of salt stress.This is that the genoid identifies GAP-associated protein GAP function in eukaryotic system first, cultivate corresponding genetically modified plants, to there is very high application value in the efficient use aspects of crops potassium, while can be good at the cultivation for being applied to merit crops in potassium deficiency hypersaline environment.
Description
Technical field
The invention belongs to gene engineering technology field, and in particular to from the kalium ion transport albumen base of marine microorganism
Because of trkH, its encoding proteins and its application.
Background technology
K+It is one of nutrient necessary to plant and microorganism growth, is distributed widely in organism, participates in a variety of
Important physiology course.The universal potassium deficiency of China's soil, and with salination to a certain degree, intracellular excessive Na+Accumulation can produce
Toxigenicity, therefore maintain relatively stable in cytoplasm and higher K+Content and relatively low Na+Content is most important[1].Some researchs are
Clone obtains some Potassium Absorptions transhipment related gene from different plants, and these genes largely come from glycophyte,
The albumen majority of expression is to Na+It is sensitive so that they are in high Na+K can not be preferably exercised under environment+Absorption function.Existing document
Report, the K in certain micro-organisms+Transport protein is in Na+In the presence of still have higher K+Activity is absorbed, this thermophilic salt or resistance to them
Salt attribute is relevant[2].Ocean is hypersaline environment, the K in marine microorganism+Transport protein is likely to Na+Insensitivity.Due to
99% is not educable in marine environment microorganism[3], in order to obtain functional gene, from marine microorganism macro genome DNA
For experiment material, the macro genome DNA includes the hereditary information of nearly all microorganism in collected seawater.Pass through cloning approach
After obtaining target gene, gene is building up in Yeast expression carrier with seamless link technology, and will be recombinated with electric shock conversion method
Plasmid imports K+Function complementation experiment is carried out in native defect type saccharomyces cerevisiae CY162, this is also the trk bases of bacterial origin first
Because the expression in eukaryotic system is probed into.
Bibliography：
[1]VOLKOV V,WANG B,DOMINY PJ,et al.A salt-tolerant relative of
Arabidopsis thaliana,possesses effective mechanisms to discriminate between
potassium and sodium[J].Plant,Cell&Environment,2004,27(1):1-14.
[2]FAILLIE C.C,JABNOUNE M,ZIMMERMANN S,et al.Potassium and sodium
transport in non-animal cells:the Trk/Ktr/HKT transporter family[J].Cellular
and Molecular Life Sciences,2010,67(15):2511-2532.
[3]Joshi G K,Jugran J,Bhatt J P.Metagenomics:The Exploration of
Unculturable Microbial World[M].Advances in Biotechnology.Springer India,
2014:105-115.
The content of the invention
For the above-mentioned problems in the prior art, the present invention provides one kind from marine bacteria and absorbed with potassium
The kalium ion transport GFP of function, the gene are the DNA moleculars separated from marine microorganism macro genome DNA,
It is named as trkH.
Kalium ion transport GFP trkH provided by the invention from marine microorganism, there is such as SEQ ID NO:
Base sequence shown in 1.Its sequence is by 1392 base compositions, from the opening that 5 ' the 1st to the 1392nd, end residues are the gene
Reading frame sequence.
It is another aspect of the invention to provide with SEQ ID NO described above:Base sequence 95% shown in 1
Homology above, and encode and SEQ ID NO:The protein of base sequence coding shown in 1 has identical biological function
The base sequence of protein.
It is another aspect of the invention to provide the protein that kalium ion transport GFP TrkH described above is encoded,
With such as SEQ ID NO:Amino acid sequence shown in 2, it is the protein being made up of 463 amino acid residues.
It is another aspect of the invention to provide the protein that kalium ion transport GFP TrkH described above is encoded
Derived protein, it has such as SEQ ID NO:Amino acid sequence shown in 2 by the substitution of one or several amino acid residues,
Missing is added and caused amino acid sequence, and the derived protein is compiled with kalium ion transport GFP trkH described above
The protein of code has identical biological function.
It is another aspect of the invention to provide the recombinant expression carrier containing gene described above.It is provided by the present invention
From the kalium ion transport GFP trkH of marine microorganism, the expression vector that can be expressed with induction exogenous gene is connected to
TrkH gene recombinant vectors are prepared.In the case of preferable, the expression vector is plasmid pYES 2.0.Yeast expression
Carrier pYES2.0 is the shuttle plasmid with Amp and Ura selection markers, and in prokaryotes such as Escherichia coli, the carrier can be with
Start the expression of downstream gene using T7 promoters；In eukaryotic microorganisms such as saccharomyces cerevisiae, the carrier can be then utilized by half
The GAL promoters of the high induction of lactose start the expression of downstream gene.In Escherichia coli, carrier pYES2.0 with Amp due to resisting
Property so the screening of ammonia benzyl mycin can be carried out, and in eukaryotic microorganisms saccharomyces cerevisiae CY162, ammonia benzyl mycin grows to it to be made
Into influence, therefore can not be screened in yeast with this method.Saccharomyces cerevisiae CY162 genotype shows, its Ura gene mutation,
Uracil complex functionality is hindered, therefore can not be grown in the culture medium for lack Ura, can carry out Effective selection accordingly.
It is another aspect of the invention to provide the host cell containing trkH genes described above.Preferably, the host
Cell contains the recombinant expression carrier of trkH genes.In the inventive solutions, described host cell is preferably using wine
Brewer yeast K+Native defect type bacterial strain CY162 (Mat α, ura3-52, his3D200, his44-15, trkD1, trkD2::
pcK64).By having complementary functions, ion exhausts and the experiments such as growing state compares confirm that the gene exists in raising transgenic yeast
K under low potassium and salt stress environment+Application in absorption.Test result indicates that transgenic microorganism of the present invention improves K+Inhale
Receive speed and to Na+Insensitivity.
Remained to well it is another aspect of the invention to provide gene described above in the case where cultivating low potassium and salt stress environment
Application in the crops of growth.The TrkH albumen of the trkH coded by said gene of the present invention has K+Absorb and to Na+Insensitive
Characteristic, although the gene successfully obtains the transgenic eukaryotic microorganism (saccharomyces cerevisiae) of the gene from the bacterium present invention, and
Prove transgenic microorganism in low potassium and high salt (Na+) K under concentration+Absorption function, illustrate the present invention gene can be transferred to
In the eukaryotics such as plant cell, and applied to the cultivation for the crops for remaining to well grow under low potassium and salt stress environment.
Beneficial effects of the present invention:
The gene trkH of the present invention derives from marine bacteria, and its albumen TrkH encoded has the work(of absorption and transport potassium ion
Can, transgenosis K can be improved+The K of native defect type saccharomyces cerevisiae+Absorption efficiency and to Na+Insensitivity, so as to improve its
Upgrowth situation under potassium deficiency and condition of salt stress.This is that the genoid identifies GAP-associated protein GAP function in eukaryotic system first,
Corresponding genetically modified plants are cultivated, will there is very high application value in the efficient use aspects of crops potassium, can be good at simultaneously
Cultivation applied to merit crops in potassium deficiency hypersaline environment.
Brief description of the drawings
Fig. 1 be trkH genes clone and analysis in the electrophoretogram that carries out, wherein：Figure 1A is the grand genome of marine microorganism
DNA, wherein swimming lane M are λ-EcoT14DNA Marker, and swimming lane 1 and swimming lane 2 are macro genome DNA；Figure 1B is PCR amplifications
The Partial Fragment electrophoretogram of trkH genes, wherein swimming lane M are DNA marker DL 2000, and swimming lane 1 is the trkH genes of amplification
Partial Fragment；Fig. 1 C are that PCR detects electrophoresis after the trkH gene 5 ' end fragments of anchor PCR amplification connect pMD18-T cloning vectors
Figure, wherein swimming lane M are DNA marker DL 2000, and swimming lane 1-5 is the plasmid for containing 5 ' terminal sequences, and swimming lane 6 is blank control；
Fig. 1 D are that PCR detects electrophoretogram, wherein swimming lane M after the end fragment of trkH genes 3 ' of anchor PCR amplification connects pMD18-T cloning vectors
For DNA marker DL 2000, swimming lane 1 is blank control, and swimming lane 2-7 is the plasmid with 3 ' terminal sequences；Fig. 1 E obtain for PCR
The complete ORF of trkH genes is obtained, wherein swimming lane M is DNA marker DL 2000, and swimming lane 1 is blank control, and swimming lane 2 and 3 is
The complete ORF of trkH genes.
Fig. 2 is TrkH conservative domain；
The phylogenetic tree that Fig. 3 is TrkH is analyzed；
Fig. 4 is TrkH transmembrane structure domain analysis；
Fig. 5 is the function complementation experiment result of transgenic yeast；
Fig. 6 is transgenic yeast in 3mM K+Ion depletion experimental result under concentration；
Fig. 7 is transgenic yeast in different K+/Na+Growing state comparative experiments result under concentration.
Embodiment
With reference to specific embodiment, the present invention is expanded on further.It should be understood that these examples be merely to illustrate the present invention and
It is not used in the application of the limitation present invention.The method of unreceipted specific experiment condition in example below, generally according to routine
Condition described in condition or molecular cloning, or according to the condition provided on product description.Material used in example below
Material, reagent etc. unless otherwise specified, commercially obtain.
The kit and reagent used in experiment：It is seamlessly connected kit：Trangene companies, pEASY-Uni
Seamless Cloning and Aseembly Kit；Plasmid extraction kit：Sangon Biotech companies, SanPrep posts
The a small amount of extraction agent boxes of formula DNA.
The clone of embodiment 1trkH genes and analysis
1. marine microorganism group DNA extraction
Marine microorganism gathers：Seawater is derived from DaLian, China black stone reef marine site, obtains being directly used in by sand filter device
The clean seawater of cultivation.Clean seawater is filtered, microorganism is then stayed on filter membrane, then is gently scraped simultaneously with the blade of cleaning
Centrifuged with seawater flushing, obtain marine microorganism biased sample.The marine microorganism biased sample is stored in rapidly 4 DEG C, and
It is used for follow-up DNA extraction experiments as early as possible.With reference to physicochemical cell cracking process and a variety of enzyme digestions, from marine microorganism sample
Extracted in product and purify to obtain the high marine microorganism macro genome DNA (Figure 1A) of the good purity of quality.
2. the Trk subtribes gene design degenerate primer known to, sequence is respectively HYS and HYA in table 1.It is micro- with ocean
Biological macro genome DNA is template, and degenerate primer HYS and HYA is respectively as sense primer and anti-sense primer, PCR amplifications trkH
The part conservative fragments of gene, enter row agarose gel electrophoresis to PCR primer, reclaim 720bp DNA fragmentation (Figure 1B).It will return
The fragment of receipts is connected in carrier T, and with heat-shock transformed method by vector introduction DH5 α competent cells.Cell is coated on and contained
Have and screening and culturing carried out on the flat board of Amp antibiotic, it is to be generated grow positive single bacterium colony after, shaken in picking colony liquid medium within
Simultaneously upgrading grain send sequencing to bacterium, and the Partial Fragment information of trkH genes is can determine that after sequencing.According to the trkH bases for having cloned to obtain
Because of part known array, 3 specific nested primers are respectively designed to two side directions.The nested primer sequence at 5 ' ends is respectively table 1
In 5-A1,5-A2 and 5-A3, the nested primer sequences at 3 ' ends are respectively 3-S1,3-S2 and 3-S3 in table 1.The grappling of both sides
Experiment first carries out single primer amplification PCR reactions with outermost primer respectively, then carries out tailings reactions to product with dGTP, then with
Oilgo d(C)18Nest-type PRC is carried out with the nested primer of inner side, product enters row agarose gel electrophoresis.According on NCBI to
Know comparison and analysis that partial sequence is carried out, the preliminary size for judging gene both sides residue zone of ignorance.5 ' end about 650bp, 3 '
About 200bp is held, the blob of viscose of purpose size and area above is cut during gel extraction, recovery purifying is connected to pMD18-T clones and carried
On body, connection product conversion bacillus coli DH 5 alpha competent cell, 37 DEG C are incubated overnight；Picking single bacterium colony shakes bacterium, carries out bacterium solution
PCR, then the bacterium solution of positive findings is extracted into plasmid, carry out plasmid PCR detection and be sequenced (Fig. 1 C and Fig. 1 D).With Blast to upper
State the sequence information for being sequenced to obtain in experiment to be compared and splice, then determine that trkH genes are complete with NCBI ORF Finder
Whole ORF sequences, total length primer Trk-S and Trk-A are designed, using macro genome DNA as template, enters performing PCR, recovery purifying purpose piece
Section, is connected on pMD18-T cloning vectors, and connection product conversion bacillus coli DH 5 alpha competent cell, 37 DEG C are incubated overnight；Choose
Take single bacterium colony to shake bacterium, carry out bacterium solution PCR, then extract the plasmid of positive findings bacterium solution, carry out plasmid PCR detection (Fig. 1 E), it is determined that
Positive colony is simultaneously sequenced.Sequencing result shows that this full length sequence is identical with splicing sequence, is correct complete trkH genes.By
Degenerate pcr and anchor PCR, gained fragment is sequenced and spliced, the trkH mrna length 1392bp finally given, coding one
The individual albumen TrkH by 463 Amino acid profiles.
The amino acid sequence of TrkH albumen is analyzed with the Protein Blast on-line analyses softwares on NCBI, tied
Fruit display sequence has certain conservative, belongs to TrkH superfamilies (Fig. 2).Microorganism with partly having done functional analysis
TrkHs albumen carries out homology analysis, homology (such as table 2) between 22-36%.Using ClustalX, BioEdit and
The softwares such as MEGA5.0 are to TrkH albumen and had done other bacterium TrkHs amino acid sequences of functional verification and be compared and be
System development tree analysis, and outer group is with Escherichia coli EcTrkA (regulatory protein) sequence, to ensure the accuracy of result.As a result show
Show that affiliation is farther out (Fig. 3) between TrkH and other bacterium TrkHs for having done functional verification.Utilize SMART softwares
(http://smart.embl-heidelberg.de/) to TrkH carry out transmembrane structure analysis, the results showed that its contain 10 across
Spanning domain, it is consistent with typical prokaryotic micro-organisms TrkH protein transmembrane structures, it was demonstrated that to be the potassium transhipment cross-film egg of bacterial origin
(Fig. 4) in vain.
The primer in the clone of the trkH genes of table 1 and analysis
The TrkH of table 2 and other bacterium Trk family K that function is reported+Transport protein homology analysis
The function complementation experiment and depletion experimental of the transgenic yeast of embodiment 2
1. the screening of transgenic yeast：Linearize Yeast expression carrier pYES 2.0 by way of digestion, according to upper
State acquired trkH gene orders in cloning experimentation, design with homology arm seamless connection primer (in table 1 OmTrkH-tys and
OmTrkH-tya), using marine microorganism macro genome DNA as template, PCR obtains gene ORF sequences, is tried by being seamlessly connected
Agent box, it is directly connected on the Yeast expression carrier pYES 2.0 of linearisation, converts bacillus coli DH 5 alpha, and detected and surveyed
Sequence.Obtain being connected with the recombinant plasmid of trkH gene ORF sequences after sequencing is correct, be named as pYES2.0-trkH.At the same time,
The EctrkH of Escherichia coli is gene constructed on pYES 2.0, and the recombinant plasmid pYES2.0-EctrkH of acquisition is as positive right
According to, and empty carrier is as negative control.Using electric shock transformation method, by three kinds of plasmid pYES 2.0, recombinant plasmid pYES2.0-
TrkH and recombinant plasmid pYES2.0-EctrkH are transferred to K respectively+In native defect type yeast strain CY162, in selective training
Support and screened in base SC-Ura, obtain positive transformants.In the plasmid being transferred to due to containing coding URA genes, so bacterial strain
Can on screening and culturing medium normal growth, can not then be grown without converting successful bacterial strain, matter then is carried out to transformant
Grain PCR identifications.
The yeast strain for being transferred to recombinant plasmid pYES 2.0-trkH is represented in result figure with CY162-TrkH, is transferred to weight
Group plasmid pYES 2.0-EctrkH yeast strain represents that the yeast strain for being transferred to empty carrier is then used with CY162-EcTrkH
CY162-p is represented.
Due to K+Native defect type bacterial strain CY162 is difficult to be grown in low potassium (being less than 7mM) environment, and yeast has complementary functions reality
Test and can be used to identify K+Transport protein exercises Potassium Absorption transport function.Because the insertion gene in recombinant plasmid can be by carrier
Upper GAL1 promoters induced expression, therefore tested from galactolipin as glycogen.
2. the function complementation experiment of transgenic yeast
1. the yeast after above-mentioned three kinds conversions is inoculated in 50ml K containing 50mM respectively+YPD fluid nutrient mediums in, overnight
Concussion and cultivate 12-14h；
2. 1ml bacterium solutions are taken to be transferred to 100ml SC-Ura+Gal+50mM K+Liquid inducing culture in, 28 DEG C concussion
48h is cultivated, induces expression of recombinant proteins；
3. measure the OD of bacterium solution600Value, appropriate bacterium solution, low-speed centrifugal, with SC-Ura+Gal+3mM K are taken by calculating+Liquid
Body culture medium washs 2-3 times, and thalline is resuspended makes OD600Value is 1.0, then to bacterium solution carry out gradient dilution (10 times, 100 times and
1000 times)；
4. 1 μ l bacterium solution points are respectively taken in SC-Ura+Gal+3mM K+Solid medium on, just putting 1h absorbs bacterium solution；
3-4d is cultivated 5. being inverted in 28 DEG C of constant incubators, observes result.
As shown in figure 5, containing 50mM K+Culture medium in, yeast can grow (Fig. 5 A).Containing 3mM K+'s
In culture medium, being transferred to trkH and EctrkH yeast can grow, and the growth for turning empty carrier yeast is then inhibited (figure
5B).1,10 in Fig. 5 A and 5B-1、10-2With 10-3For the multiple of bacterium solution gradient dilution.Fig. 5 result shows that TrkH has K+
Absorption and transport ability, it is a kind of kalium ion transport albumen.
3. the K of transgenic yeast+Depletion experimental
1. the yeast after three kinds of conversions is inoculated in 50ml K containing 50mM respectively+YPD fluid nutrient mediums in, overnight concussion
Cultivate 12-14h；
2. 1ml bacterium solutions are taken to be transferred to 100ml SC-Ura+Gal+50mM K+Liquid inducing culture in, 28 DEG C concussion
48h is cultivated, induces expression of recombinant proteins；
3. 4 DEG C, 3000rpm centrifugations, 5min collects yeast cells, with SC-Ura+Gal+0mM K+Fluid nutrient medium wash
Wash and be resuspended, 28 DEG C of concussion and cultivate 5h；
4. the OD of bacterium solution is measured respectively600Value, appropriate bacterium solution, low-speed centrifugal, with SC-Ura+Gal+3mM K are taken by calculating+Fluid nutrient medium wash and be resuspended in the 100ml fluid nutrient mediums, make yeast OD600Value is 0.4, continues vibration training
Support；
5. taking 150 μ l bacterium solutions every 1h, 12000r/min centrifugation 10min, discard precipitation and retain supernatant, while cover 150 μ
l SC-Ura+Gal+3mM K+Fluid nutrient medium.The K in supernatant is determined with atomic absorption spectrophotometer+Content.
As shown in fig. 6, change over time, the K in culture medium+Concentration is gradually reduced.By contrast, yeast CY162-
TrkH and CY162-EcTrkH institutes K in the medium+Concentration declines faster, i.e. K+More efficiently absorbed.As a result show,
TrkH and EcTrkH have K+Absorption, it is kalium ion transport albumen.
Yeast is in different K after embodiment 3 converts+/Na+Growth analysis under concentration
The thalline of yeast after taking appropriate three kinds of conversions with oese respectively, is equably transferred to containing different K+Concentration
On the SC-Ura+Gal plating mediums of (0.1mM, 0.5mM, 1mM, 3mM and 50mM), in 28 DEG C of incubated carton upside down cultures
4d, observe growing state and compare, as a result as shown in Figure 7 A, in 1mM K+With 3mM K+Culture medium in, expression TrkH and
EcTrkH yeast shows obvious growth vigor than turning the yeast of zero load.In 0.5mM K+Condition of culture under, expression
TrkH and EcTrkH yeast shows weaker growth but still must be good than turning unloaded yeast growth.And in 0.1mM K+Training
Under the conditions of supporting, three primary yeasts can not grow.According to three primary yeast growing states, TrkH K is tentatively known+Absorption region is 1-
3mM, it was demonstrated that TrkH is a kind of low affine K+Transport protein.
The thalline of yeast, is equably transferred to containing different K after taking appropriate three kinds of conversions with oese respectively+/Na+Concentration
SC-Ura+Gal plating mediums on, in 28 DEG C of incubated carton upside down culture 4d, observation growing state simultaneously compares.As a result such as
Shown in Fig. 7 B and 7C, in 50mM K+Under sufficient condition of culture, three primary yeasts are containing different Na+Feelings are grown in concentration cultures
Condition is basically identical, and higher than 200mM Na+When, growth is substantially suppressed, and can hardly be grown；In 3mM K+Culture
Under the conditions of, three primary yeasts show a certain degree of growth differences：When containing 3mM K in culture medium+And 0m M Na+Or 50mM
Na+When, the yeast ratio for expressing TrkH and EcTrkH turns the more preferable of unloaded yeast growth, when containing 100mM Na in culture medium+When,
The upgrowth situation for expressing TrkH yeast is slightly better than the yeast for expressing EcTrkH.It is higher than 200mM Na when containing in culture medium+When,
The growth of three primary yeasts is substantially suppressed.It follows that in 3mM K+And 100mM Na+Condition of culture under, marine bacteria
The TrkH in source shows higher Na than the EcTrkH of Escherichia coli+Insensitivity.
Claims (6)
- A kind of 1. kalium ion transport GFP trkH from marine microorganism, it is characterised in that its base sequence such as SEQ ID NO:Shown in 1.
- 2. the protein TrkH of gene code as claimed in claim 1, its amino acid sequence such as SEQ ID NO:Shown in 2.
- 3. the recombinant expression carrier containing gene described in claim 1.
- 4. the host cell containing the recombinant expression carrier described in claim 3.
- 5. host cell according to claim 4, it is characterised in that the host cell is saccharomyces cerevisiae CY162.
- 6. application of the gene in the crops that can be grown under low potassium, salt stress environment are cultivated described in claim 1.
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Citations (4)
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|---|---|---|---|---|
| WO2008023264A2 (en) * | 2006-02-09 | 2008-02-28 | Ocean Nutrition Canada, Ltd. | Coenzyme q10 production from marine bacteria |
| CN101343316A (en) * | 2008-09-05 | 2009-01-14 | 中国科学院微生物研究所 | Kalium ion transport associated protein system, encoding gene cluster and application thereof |
| WO2011130725A3 (en) * | 2010-04-16 | 2012-04-19 | Myriant Technologies Inc | Production of organic acids from xylose rich hydrolysate by bacterial fermentation |
| CN104619838A (en) * | 2012-08-22 | 2015-05-13 | 诺维信公司 | Metalloprotease from exiguobacterium |
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|---|---|---|---|---|
| WO2008023264A2 (en) * | 2006-02-09 | 2008-02-28 | Ocean Nutrition Canada, Ltd. | Coenzyme q10 production from marine bacteria |
| CN101343316A (en) * | 2008-09-05 | 2009-01-14 | 中国科学院微生物研究所 | Kalium ion transport associated protein system, encoding gene cluster and application thereof |
| WO2011130725A3 (en) * | 2010-04-16 | 2012-04-19 | Myriant Technologies Inc | Production of organic acids from xylose rich hydrolysate by bacterial fermentation |
| CN104619838A (en) * | 2012-08-22 | 2015-05-13 | 诺维信公司 | Metalloprotease from exiguobacterium |
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