CN108192916A - Turn the initiative and application of the heavy metal super-enriched transgenic engineering rice of S.plumbizincicola SpNramp5 genes - Google Patents
Turn the initiative and application of the heavy metal super-enriched transgenic engineering rice of S.plumbizincicola SpNramp5 genes Download PDFInfo
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Abstract
The present invention discloses a kind of initiative for the heavy metal super-enriched transgenic engineering rice for turning S.plumbizincicola SpNramp5 genes, including:Clone SpNramp5 genes in S.plumbizincicola;Based on plant expression vector, the SpNramp5 genes are inserted into the conversion carrier by methods of homologous recombination, obtain overexpression binary vector by conversion carrier of the structure comprising strong promoter, screening-gene;Gained overexpression binary vector is transferred to by Agrobacterium tumefaciems in Rice Callus, cultivates transgenic seedling and screening transgenic positive plant;Wherein, transgene receptor plant is rice Nipponbare kind.Breeding method provided by the invention can solve the problems such as wild heavy metal accumulation plant patience is limited, growth is slow, biomass is small, growing environment is special and heavy metal returns to.In addition, the present invention also provides in a kind of initiative for the heavy metal super-enriched transgenic engineering rice for turning S.plumbizincicola SpNramp5 genes gained heavy metal super-enriched transfer-gen plant heavy metal-polluted soil reparation in application.
Description
Technical field
The invention belongs to transfer-gen plants and heavy metal pollution regulation field, and in particular to turn S.plumbizincicola SpNramp5 bases
The initiative and application of the heavy metal super-enriched transgenic engineering rice of cause.
Background technology
Heavy metal pollution problem has become a worldwide great environmental problem, heavy metal pollution not only threaten animals and plants,
Micro-organisms are developed, and also threaten human health or even life by food chain etc..Lead to the heavy metal of soil pollution mainly to have
As, Cd, Co, Cr, Cu, Hg etc., the combined pollution of generally several heavy metal species.Heavy metal mining area, the weight in rapid economic development area
Metallic pollution situation is particularly acute, and many agricultural product in China is caused to be denied access to international market due to heavy metals exceeding standard.
Various countries increasingly pay attention to the ecological effect of heavy metal pollution and the research of prevention, have explored a variety of improvement skills
Art.However traditional heavy metal-polluted dyeing technique such as soil solidification, vitrifying, leaching, wash local method, heavy metal pollution of soil, electrochemical process etc.
Physico-chemical process, it is not only with high costs, it is difficult to large-scale use, and frequently result in soil texture destruction, geobiont work
Property decline and soil fertility degeneration.Therefore, it is inexpensively permanent effective to seek one kind, and can be with the replacement method of maintenance of soil fertility
The difficult point and hot spot always studied in the world.
The strategy to be cleared the pollution off using green plants, i.e. phytoremediation technology have it is at low cost, it is environmental-friendly and can be again
The features such as utilization is a kind of very promising environmental pollution original position governance way.The phytoremediation of broad sense refers to be extracted by plant
It takes, convert, fixing, volatilizing, the process that degradation removes the pollutants such as soil and Organic Pollutants In Water, heavy metal.
By plant of the plantation with heavy metal accumulation ability, by plant harvesting and deal carefully with and (be such as ashed), to reach a huge sum of money
Belong to and remove soil environment, remove the purpose of heavy metal pollution of soil.
Gene is to determine most important factor of the plant to heavy metal-polluted soil absorption and accumulation characteristic, and heavy metal accumulation plant is resistance to heavy
In terms of the mechanism of metal mainly includes three, first, Rejection mechanism hinders the absorption or transport in vivo of heavy metal, after absorption again
It excretes;Second, localization mechanism makes heavy metal be accumulated in privileged sites such as the vacuoles, epidermal hair, cell wall of plant, from
And be isolated with the other components in cell, achieve the effect that removing toxic substances;Mechanism, plant substance in vivo and heavy metal network is complexed in third
It closes, including being complexed to form sulfide with inorganic matter, after small organic molecule such as GSH, oxalic acid, histidine and citric acid complexing
Assemble in vacuole, be complexed with the metal chelating albumen of macromolecular.But at present, there are patience to have for wild heavy metal accumulation plant
The problems such as limit, growth are slowly, biomass is small, growing environment is special and heavy metal returns to.Technique for gene engineering is current soil
Therefore the most High biotechnology of prospect in heavy metal repairing research, by the heavy metal super-enriched gene cloning of plant and turns
Move on to biomass is big, on the rapid plant of production to improve the ability of phytoremediation, be to solve the problems, such as heavy metal pollution of soil
Effective ways.
Invention content
The purpose of the present invention is to provide a kind of heavy metal super-enriched transgenosis works for turning S.plumbizincicola SpNramp5 genes
The initiative and application of journey rice, solving wild heavy metal accumulation plant, there are patience is limited, growth is slow, biomass is small, growth
Environment is special and the problems such as heavy metal returns to.
To achieve the above object, the present invention, which provides, a kind of turn the heavy metal super-enriched of S.plumbizincicola SpNramp5 genes and turns base
Because of the initiative of engineering rice, the construction method of heavy metal super-enriched transgenic engineering rice is referred specifically to, including:
(1) clone of heavy metal super-enriched gene clones SpNramp5 genes in S.plumbizincicola.S.plumbizincicola (Sedum
Plumbizincicola be) a kind of heavy metal super-enriched plant for being found in south China mining area, because its have to heavy metal it is super
Strong resistance and accumulation ability are a kind of main rehabilitation plants of current heavy-metal contaminated soil repairing test.SpNramp5
Albumen has very strong turn-over capacity to heavy metal ion such as cadmium, manganese, and result of study shows that SpNramp5 is S.plumbizincicola pair
The heavy metal adsorptions such as cadmium, manganese and the key gene of accumulation, to plant in containing the heavy-metal contaminated soils environment such as cadmium normal growth
And removing toxic substances plays an important role;
(2) structure of overexpression binary vector, based on plant expression vector, structure includes strong promoter, screening
The SpNramp5 genes that step (1) clone obtains are inserted into the conversion by methods of homologous recombination and carried by the conversion carrier of gene
Body obtains overexpression binary vector;
(3) heavy metal super-enriched transfer-gen plant structure, passes through crown gall by the overexpression binary vector obtained by step (2)
Agrobacterium is transferred in Rice Callus, and screening and culturing, the transgenic resistance rice callus group of acquisition are carried out with screening and culturing medium
It knits by inducing, breaking up, taking root, the exercise and transplanting of transgenic seedling, screens heavy metal super-enriched transgenic positive plant;
Wherein, the SpNramp5 gene nucleotide series such as SEQ ID NO:Shown in 1, the rice varieties are Japan
It is fine.
Preferably, in the step (1), the method for cloning SpNramp5 genes in S.plumbizincicola is:
Using the cDNA of S.plumbizincicola as template, with such as SEQ ID NO:2 and SEQ ID NO:Sequence shown in 3 for primer into
Row PCR amplification obtains SpNramp5 genes.
Preferably, the plant expression vector is pCAMBIA1301 carriers, the strong promoter starts for Ubiquitin
Son, the screening-gene are Bar screening-genes, and the conversion carrier further includes 3 × Flag labels.Using including Ubiquitin
The conversion carrier of promoter, destination gene expression amount is high, and genetic stability is good, and actual application prospect protrudes, and enhances target
The high conversion of gene is expected to obtain the transgenic line of target gene Efficient Conversion.In addition, 3 × Flag labels of addition, it can be square
Just transformation efficiency and genetic stability analysis are carried out in protein level, is expected to filter out with lasting stability heredity for application practice
Transgenic line.
Preferably, the construction method of conversion carrier described above includes the following steps:
(a) 3 × Flag marks are added between pCAMBIA1301 vector multiple cloning sites Kpn I and the Sac I
Label;
(b) the Bar screening-genes on the pCAMBIA3301 carriers are cloned using pcr amplification reaction;
(c) using the carrier obtained in restriction endonuclease Xho I digestion steps (a), and will be after the Bar screening-genes and digestion
Carrier carry out homologous recombination;
(d) using the Ubiquitin promoters on Hind III and BamH I digestion pUN1301 carriers;
(e) using the recombinant vector obtained in restriction endonuclease Hind III and BamH I digestion steps (c), and by described in
Ubiquitin promoters are connect with the recombinant vector after digestion.
Specifically, the nucleotide sequence of the Ubiquitin promoters such as SEQ ID NO:Shown in 4, the Bar screens base
Because of nucleotide sequence such as SEQ ID NO:Shown in 5, the nucleotide sequence such as SEQ ID NO of 3 × Flag labels:Shown in 6.
Preferably, in step (b), the PCR primer of the Bar screening-genes on clone's pCAMBIA3301 carriers
Sequence such as SEQ ID NO:10 and SEQ ID NO:Shown in 11.
Preferably, the screening and culturing medium includes glufosinate-ammonium.
Preferably, the plant expression vector is pCAMBIA1301 carriers, the strong promoter starts for 2 × CaMV35S
Son, the screening-gene are HYG screening-genes.Using the conversion carrier for including 2 × CaMV35S promoters, destination gene expression
Amount is high, and genetic stability is good, and actual application prospect protrudes, and enhances the high conversion of target gene, is expected to obtain target gene
The transgenic line of Efficient Conversion.
Preferably, the HYG screening-genes are the screening-gene that the pCAMBIA1301 carriers carry, the conversion carries
The construction method of body includes the following steps:
(a) using pCAMBIA1301 carriers described in restriction endonuclease Hind III and Pst I digestions;
(b) it using pCAMBIA1301 carriers described in restriction endonuclease Hind III and BamH I digestions, obtains CaMV35S and starts
Son;
(c) restriction endonuclease Hind III and Pst I restriction enzyme sites are contained according to the design of the nucleotide sequence of CaMV35S promoters
Primer, and carry out pcr amplification reaction by template of CaMV35S promoters, obtain containing Hind III and Pst I restriction enzyme sites
CaMV35S promoters, and the CaMV35S promoters progress digestion obtained using restriction endonuclease Hind III and Pst I to PCR;
(d) digestion products obtained in step (c) are recycled, and the digestion carrier with being obtained in step (a) is connect.
Specifically, the nucleotide sequence of 2 × CaMV35S promoters such as SEQ ID NO:Shown in 7, the HYG screenings
The nucleotide sequence of gene such as SEQ ID NO:Shown in 8.
Preferably, the screening and culturing medium contains hygromycin.
Preferably, in step (2), the structure of the overexpression binary vector includes the following steps:
SpNramp5 genes, the homologous recombination PCR primer of clone's SpNramp5 genes are cloned using pcr amplification reaction
Include BamH I restriction enzyme sites.
Preferably, the homologous recombination PCR primer sequence such as SEQ ID NO of clone's SpNramp5 genes:12 and SEQ
ID NO:Shown in 13.
Preferably, the Agrobacterium tumefaciems is EHA105.
The present invention also provides a kind of heavy metal super-enriched transgenic engineering rice for turning S.plumbizincicola SpNramp5 genes
Application of the heavy metal super-enriched transfer-gen plant of gained in heavy metal-polluted soil reparation in initiative.
Compared with prior art, the heavy metal super-enriched transgenosis provided by the invention for turning S.plumbizincicola SpNramp5 genes
The initiative of engineering rice has the advantages that following several respects:Firstth, the target gene definite functions of selection conversion, come from wild
Heavy metal super-enriched plant S.plumbizincicola, coding albumen transport the efficient of absorption, therefore be expected to obtain to the heavy metals such as cadmium
It obtains to put into practice the super enrichment transfer-gen plant used;Secondth, structure includes the conversion carrier of strong promoter, destination gene expression
Amount is high, and genetic stability is good, and actual application prospect protrudes;Third starts target gene using strong promoter, enhances target base
The high conversion of cause, therefore it is expected to obtain the transgenic line of target gene Efficient Conversion;4th, acceptor material variety selection is special
Very, the rice varieties selected of the present invention are Nipponbare rice, and genetic transformation effect is good, and biomass is big, wide adaptability, can be
Southern and northern wide geographic area uses, therefore having a extensive future in soil remediation.
Description of the drawings
Fig. 1 is SpNramp5 gene electrophoretograms;
Fig. 2 is clone's SpNramp5 gene bacterium colony PCR electrophoretograms;
Fig. 3 is SpNramp5 gene overexpression binary vector digestion verification figures;
Fig. 4 is pUb-SpNramp5-Bar overexpression binary vector figures;
Fig. 5 is p2 × 35S-SpNramp5-HYG overexpression binary vector figures;
Fig. 6 schemes for the SpNramp5 transgene genetic transformation stages;
Fig. 7 is SpNramp5 transgenic positive plant qualification figures;
Fig. 8 is the horizontal qualification figure of SpNramp5 transfer-gen plant heavy metal adsorptions.
Specific embodiment
For the purpose of the present invention, technical solution and advantageous effect is better described, below in conjunction with attached drawing and specific implementation
The invention will be further described for example.It should be noted that following the methods of implementing are explained further to what the present invention was done
It is bright, it should not be taken as limitation of the present invention.If material used in the embodiment of the present invention, reagent all can be from without specified otherwise
Commercial sources obtain.
1 SpNramp5 gene clonings of embodiment
S.plumbizincicola seedling is extracted into its RNA into powdered using liquid nitrogen grinding using Trizol methods, it is anti-using TOYOBO
Transcript reagent box into cDNA, sets its reverse transcription according to the cDNA sequence of the NCBI SpNramp5 genes provided using Primer5
Its upstream and downstream primer, and synthetic primer are counted, the nucleotides sequence for designing primer is classified as:
Upstream sequence:5 ' TTCCGGCCTTCGACTTTGACG, 3 ' (such as SEQ ID NO:Shown in 2);
Downstream sequence:5 ' AACCGTTAACGCGCATTCGCACA, 3 ' (such as SEQ ID NO:Shown in 3).
After primer synthesis, dissolved using 1 × TE, then carry out PCR amplification, using S.plumbizincicola cDNA as template,
Use high-fidelity enzymeGXL Premix expand it, and amplified production is used to 1% Ago-Gel
Electrophoresis is carried out, target stripe is cut, is recycled using plastic recovery kit, amplified production electrophoretogram is as shown in Figure 1, arrow
Signified band is purpose band.Then recovery product and PMD19-T (simple) carrier are carried out staying overnight connection, by connection product
E. coli competent Top10 is converted using heat shock method, monoclonal is chosen and is bacterium solution PCR, after amplification, amplified production is carried out
Agarose gel electrophoresis, electrophoretogram is as shown in Fig. 2, arrow meaning 1-12 bacterium solutions are positive bacterium solution in figure.By positive strain into
Bacterium solution access LB-Amp culture mediums are shaken bacterium by row sequencing after sequencing is correct, and upgrading grain is spare.
The structure of the conversion carrier of 2 heavy metal super-enriched genetically modified plants of embodiment
(1) structure comprising Ubiquitin promoters, Bar screening-genes and the label converting carriers of 3 × Flag is based on experiment
PCAMBIA1301-3 × Flag carriers that room is had by oneself, obtain after being transformed by following steps:
1. obtain Bar screening-genes:PCAMBIA3301 carriers contain Bar screening-genes and CaMV35S promoters, at this
In embodiment, start Bar screening-genes using the CaMV35S promoters that pCAMBIA3301 carriers carry, target gene can be enhanced
Efficient Conversion, the nucleotide sequence such as SEQ ID NO of CaMV35S promoters:Shown in 9.Use Xho I inscribe cleavages
PCAMBIA3301 carriers, the Ago-Gel that digestion products are carried out to 1% carry out electrophoresis, recycle small band (608bp).According to
PCAMBIA1301 carrier sequences and Bar gene orders design homologous recombination primer, the nucleotides sequence for designing primer are classified as:
Upstream sequence:5‘ACAAATCTATCTCTCTCGAGTCTACCATGAGCCCAGAACG 3’;
Downstream sequence:5‘TTATTATGGAGAAACTCGAGTCAAATCTCGGTGACGGGCA 3’.
Wherein, upstream sequence such as SEQ ID NO:Shown in 10, downstream sequence such as SEQ ID NO:Shown in 11.Then with recycling
Segment for template, carries out PCR amplification with Bar homologous recombinations primer, it is spare after recycling segment.With Xho I inscribe cleavages
PCAMBIA1301-3 × Flag carriers, the Ago-Gel that digestion products are carried out to 1% carry out electrophoresis, recycle big band.By it
Before the small band that is recovered to and big band carry out homologous recombination, the product after homologous recombination is converted into Escherichia coli using heat shock method
Competence Top10 chooses monoclonal and is bacterium solution PCR, then sequencing company sent to be sequenced positive strain, the nucleotides sequence of Bar genes
Row such as SEQ ID NO:Shown in 5, bacterium solution access LB-Kan culture mediums are shaken into bacterium after sequencing is correct, upgrading grain is spare, intermediate carrier
PCAMBIA1301-3 × Flag-Bar is built successfully.
2. obtain Ubiquitin promoters:By in the culture medium containing the bacterial strain of pUN1301 carriers access LB-Kan, shake
Bacterium extracts plasmid.Using Hind III and BamH I digestion pUN1301, the Ago-Gel that digestion products carry out 1% carries out electricity
Swimming, recycles small band (about 2000bp).Small band is Ubiquitin promoters.
3. build conversion carrier:By the pCAMBIA1301-3 × Flag-Bar built before using Hind III and
BamH I carry out digestion, and the Ago-Gel that digestion products are carried out to 1% carries out electrophoresis, recycles big band.It will be ready to before
Ubiquitin promoters be attached therewith using T4 ligases, by connection product using heat shock method convert Escherichia coli sense
By state Top10, picking monoclonal carries out bacterium solution PCR, positive strain access LB-Kan culture mediums is shaken bacterium, upgrading grain uses BamH
I and Hind III carry out digestion verification again.Spare, the pUb-3 × Flag- that will verify that correct bacterium shake bacterium extraction plasmid
Bar conversion carriers structure is completed.
(2) what the structure of the conversion carrier comprising 2 × CaMV35S promoters and HYG screening-genes was had by oneself based on laboratory
PCAMBIA1301 carriers obtain after being transformed by following steps:
1. using Hind III and BamH I digestions pCAMBIA1301 by digestion products carry out 1% Ago-Gel into
It is spare to recycle small band (877bp) for row electrophoresis.
2. III containing Hind and Pst I restriction endonuclease primers are designed according to CaMV35S promoter sequences, with previous step recycling
Band is template, and PCR amplification is carried out using high-fidelity enzyme, and the Ago-Gel that PCR product is carried out to 1% carries out electrophoresis, recycling
Band.And using Hind III and Pst I restriction endonucleases to recovery product digestion, and glue recycling is carried out to digestion products.
3. using Hind III and Pst I restriction endonucleases to pCAMBIA1301 digestions, and glue recycling is carried out to digestion products.
4. by the recovery product in 2. with 3. in recovery product be attached using T4 ligases.Connection product is used
Heat shock method converts E. coli competent Top10, and picking monoclonal carries out bacterium solution PCR, by positive strain access LB-Kan cultures
Base shakes bacterium, and upgrading grain carries out digestion verification again using BamH I and Hind III.It will verify that correct bacterium carries out shaking bacterium extraction
Plasmid is spare, and p2 × CaMV35S-HYG conversion carriers structure is completed.The nucleotide sequence of 2 × CaMV35S promoters such as SEQ ID
NO:Shown in 7, HYG screening-genes nucleotide sequence such as SEQ ID NO:Shown in 8.
The SpNramp5 gene overexpression binary vectors of 3 heavy metal super-enriched transfer-gen plant of embodiment
Homologous recombination primer, the nucleosides of the homologous recombination primer of design are designed according to the ORF areas both ends of SpNramp5 genes
Acid sequence is:
Upstream sequence (such as SEQ ID NO:Shown in 12):
5‘TTCTGCAGGTCGACTCTAGAGGATCCATGGCATCAACTGTCGGAAACGC3’;
Downstream sequence (such as SEQ ID NO:Shown in 13):
5‘CTTTGTAGTCGGTACCCGGGGATCCCTCTAAGACAGCTCTGCGTTGCGG3’。
Using the 19T carriers containing SpNramp5 as template PCR amplification is carried out using high-fidelity enzyme.PCR product is carried out 1%
Ago-Gel carry out electrophoresis, recycle purpose band, it is spare.PUb-3 × Flag-Bar the conversion carriers that will be built respectively
BamH I single endonuclease digestions are used with p2 × CaMV35S-HYG conversion carriers, digestion products are directly subjected to glue recycling, it is spare.It will be same
Source recombinant fragment carries out homologous recombination with the carrier after digestion.Homologous recombination product is experienced using heat shock method conversion Escherichia coli
State Top10, picking monoclonal carry out bacterium solution PCR, positive strain access LB-Kan culture mediums are shaken bacterium, upgrading grain uses BamH I
Single endonuclease digestion verification is carried out, digestion verification figure is as shown in figure 3, be the enzyme of pUb-SpNramp5-Bar overexpression conversion carriers in figure
Proof diagram is cut, wherein arrow meaning band is purpose band, i.e. plasmid 1-3 is positive plasmid.It will verify that correct plasmid send survey
Sequence company is sequenced.Correct plasmid will be sequenced, Agrobacterium tumefaciems EHA105 is transferred to by electric robin, the bacterium solution after conversion is coated with
On Yep-Kan tablets, when its longer monoclonal bacterium colony, picking monoclonal shakes bacterium, is bacterium solution PCR, positive strain is protected
It deposits spare.Successful SpNramp5 genes overexpression binary vector is built, pUb-SpNramp5-Bar conversion carriers collection of illustrative plates is such as
Shown in Fig. 4, p2 × 35S-SpNramp5-HYG conversion carrier collection of illustrative plates is as shown in Figure 5.
4 transfer-gen plant genetic transformation of embodiment
Choose full, uniformly, the normal Nipponbare rice paddy seed of color and luster using ethyl alcohol, after hypochlorite disinfectant, is placed in more
Hinder on inducing culture, while prepare to infect required Agrobacterium.Callus is grown after 5-7 days, ready before use
EHA105 Agrobacterium tumefaciems is infected, and being placed on co-cultivation culture medium for infecting uses the nothing containing cephalo after 3 days, 3 days
Bacterium water cleans it, and it is (a concentration of to place it in Glufosinate-ammoniumpesticideng later:5mg/L) or hygromycin is (a concentration of:35mg/L)
It is cultivated two weeks on screening and culturing medium, wherein, the screening and culturing medium using Glufosinate-ammoniumpesticideng of pUb-SpNramp5-Bar conversion carriers,
Screening and culturing medium of the use of p2 × 35S-SpNramp5-HYG conversion carriers containing hygromycin.In order to reduce transgenosis false positive
Rate moves on to newly longer resistant calli on new screening and culturing medium, until graininess kanamycin-resistant callus tissue is grown.It will
Resistant calli is transferred on RE-III culture mediums, until it grows seedling.Seedling is transferred on HF culture mediums after growing and lures
It leads and takes root;Wait seedlings grow to bottle cap highly uncap add in sterile water practice seedling.It can be transplanted in Nutrition Soil after young plant grows up.
SpNramp5 transgenic paddy rice genetic transformation stage diagrams as shown in fig. 6, A is evoked callus figure in figure, to obtain resistance be cured by B
Injured tissue figure, C are taken root figure for evoked callus, and D be the transfer-gen plant figure of acquisition.
5 positive transgenic plant of embodiment is identified and adsorption levels measure
Transfer-gen plant is identified, obtains transgenic positive plant, by a transgenic positive plant huge sum of money for acquisition
Belong to cadmium to be handled, detect wherein heavy metal cadmium content, and being compared with control treatment, analyze its adsorption capacity.The positive turns
For gene plant qualification figure as shown in fig. 7, in figure, WT is WT lines, and abscissa is transfer-gen plant, and ordinate is target base
Because of expression, it can be seen from the figure that the gene expression amount of transfer-gen plant F1, F2, F3, F4, F6, F7, F9, F10, F11
Height is positive transgenic plant.Then according to positive plant qualification result, turn that Partial Conversion is horizontal high and expression is stable is selected
Gene strain carries out heavy metal adsorption level identification, and the results are shown in Figure 8.From figure 8, it is seen that transgenic positive plant counterweight
The accumulation of cadmium metal is apparently higher than control Wild plant, wherein, transgenic positive plant F3 to the accumulation of heavy metal cadmium most
Secondly height is transfer-gen plant F2.Integrated data result obtains, the weight provided by the invention for turning S.plumbizincicola SpNramp5 genes
The initiative of metal super enrichment transgenic engineering rice is to solve the effective ways of huge sum of money soil contamination problem.
Finally, it should be noted that the above embodiments are merely illustrative of the technical solutions of the present invention rather than the present invention is protected
The limitation of range is protected, although being explained in detail with reference to preferred embodiment to the present invention, those of ordinary skill in the art should
Understand, technical scheme of the present invention can be modified or replaced equivalently, without departing from the essence of technical solution of the present invention
And range.
Sequence table
<110>Guangdong Kaiyuan Environment Technology Co., Ltd.
<120>Turn the initiative and application of the heavy metal super-enriched transgenic engineering rice of S.plumbizincicola SpNramp5 genes
<160> 13
<170> SIPOSequenceListing 1.0
<210> 1
<211> 1641
<212> DNA
<213>S.plumbizincicola (Sedum plumbizincicola)
<400> 1
atggcatcaa ctgtcggaaa cgccggtggt ggtggttgac ggcagcagtt tatggttggt 60
tcaggaaacg gaagcatatc aaatgctcct ttgattgaag agtctggtga tgaagttaat 120
cagattgttg tacctgataa gacgagttgg aagaactttt ttgcatacct tggtcctggt 180
tttcttgttt caattgcata tattgacccc ggaaactttg agacggattt gcaggccgga 240
gctcagtaca aatatgagtt attgtggatc atattggtgg cctcgtgtgc tgcgcttgtt 300
atacagtctt tggcagccaa cctgggtgtt gtgacaggga agcatctggc ggaacactgt 360
aggaatgagt attctaaggt tcctaacttt attttgtggg ttcttgcgga aattgctgta 420
gttgcgtgtg acatacctga agtgatcggg actgcctttg cattgaacat gctgttccat 480
attccagtct ggtgtggggt ccttttgact gggctcagta cattggttct tcttgcacta 540
cagcagtatg gggtaaggaa attagagtta ttgattgcat tcttggtgtt cactatagca 600
gcagcatttc tagcagagct tggttatgca aatcctgtac cttctgaagt tctaagtggc 660
ttatttgttc ctaaactaaa aggaaatggt gctactggac ttgccatttc acttttcggt 720
gcaatggtca tgccgcataa cttattcttg cactctgctc tggtgctctc aaggaaagta 780
cctagatcag ctcgaggcat aaaagaggcg tgcagattct atatgatgga aagtggattt 840
gctctcatgg tggcgtttct catcaatgta tctgttatat cagtcagtgg tgctgtgtgc 900
agttcatcaa ccttgagtga agaagataaa ttgagttgca gcgacttgga tttgaacaag 960
gcgtcatttt tgttacgaaa tgtcctgggc aaatggagtt caaagctttt cggcatagct 1020
ttgcttgcat ctgggcaaag ttcaaccata gctggaacat atgcagggca gtatgtaatg 1080
cagggatttc taaatctacg cttagaacca tggattcgaa actttctgac aagatgtttg 1140
gcgattgtgc caagtctgat tgttgctctc atcggtggct cagcaggtgc tggacagttg 1200
attatcattg cttcgataat tttatccttc gagcttccgt ttgctctcat cccccttctt 1260
aagttcacaa gttgcaacac aaagatgggt tcacatgcca attccacagc gatatcagtg 1320
attacatgga tcatcggctc tctgatcatg tcaatcaaca tgtactacct tgcatcaagc 1380
ttcataaagc tccttctcca taatgtctcc aatatcggac tgaaagcatt ctatggaata 1440
ctgggctttt ccgggatggc aatctacgtc gctggagtag catacctagt aatcaggaaa 1500
aacaagaaac caacgcatct tttggcattt acaacatcag aaactcagga ggctggcgtg 1560
gttgatacat gtgtaacatc tcttcctaga gaagatattg tcgacatgca attgccgcaa 1620
cgcagagctg tcttagagta g 1641
<210> 2
<211> 21
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 2
ttccggcctt cgactttgac g 21
<210> 3
<211> 23
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 3
aaccgttaac gcgcattcgc aca 23
<210> 4
<211> 2011
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 4
gcatgcctgc agtgcagcgt gacccggtcg tgcccctctc tagagataat gagcattgca 60
tgtctaagtt ataaaaaatt accacatatt ttttttgtca cacttgtttg aagtgcagtt 120
tatctatctt tatacatata tttaaacttt actctacgaa taatataatc tatagtacta 180
caataatatc agtgttttag agaatcatat aaatgaacag ttagacatgg tctaaaggac 240
aattgagtat tttgacaaca ggactctaca gttttatctt tttagtgtgc atgtgttctc 300
cttttttttt gcaaatagct tcacctatat aatacttcat ccattttatt agtacatcca 360
tttagggttt agggttaatg gtttttatag actaattttt ttagtacatc tattttattc 420
tattttagcc tctaaattaa gaaaactaaa actctatttt agttttttta tttaataatt 480
tagatataaa atagaataaa ataaagtgac taaaaattaa acaaataccc tttaagaaat 540
taaaaaaact aaggaaacat ttttcttgtt tcgagtagat aatgccagcc tgttaaacgc 600
cgtcgacgag tctaacggac accaaccagc gaaccagcag cgtcgcgtcg ggccaagcga 660
agcagacggc acggcatctc tgtcgctgcc tctggacccc tctcgagagt tccgctccac 720
cgttggactt gctccgctgt cggcatccag aaattgcgtg gcggagcggc agacgtgagc 780
cggcacggca ggcggcctcc tcctcctctc acggcaccgg cagctacggg ggattccttt 840
cccaccgctc cttcgctttc ccttcctcgc ccgccgtaat aaatagacac cccctccaca 900
ccctctttcc ccaacctcgt gttgttcgga gcgcacacac acacaaccag atctccccca 960
aatccacccg tcggcacctc cgcttcaagg tacgccgctc gtcctccccc cccccccctc 1020
tctaccttct ctagatcggc gttccggtcc atggttaggg cccggtagtt ctacttctgt 1080
tcatgtttgt gttagatccg tgtttgtgtt agatccgtgc tgctagcgtt cgtacacgga 1140
tgcgacctgt acgtcagaca cgttctgatt gctaacttgc cagtgtttct ctttggggaa 1200
tcctgggatg gctctagccg ttccgcagac gggatcgatt tcatgatttt ttttgtttcg 1260
ttgcataggg tttggtttgc ccttttcctt tatttcaata tatgccgtgc acttgtttgt 1320
cgggtcatct tttcatgctt ttttttgtct tggttgtgat gatgtggtct ggttgggcgg 1380
tcgttctaga tcggagtaga attctgtttc aaactacctg gtggatttat taattttgga 1440
tctgtatgtg tgtgccatac atattcatag ttacgaattg aagatgatgg atggaaatat 1500
cgatctagga taggtataca tgttgatgcg ggttttactg atgcatatac agagatgctt 1560
tttgttcgct tggttgtgat gatgtggtgt ggttgggcgg tcgttcattc gttctagatc 1620
ggagtagaat actgtttcaa actacctggt gtatttatta attttggaac tgtatgtgtg 1680
tgtcatacat cttcatagtt acgagtttaa gatggatgga aatatcgatc taggataggt 1740
atacatgttg atgtgggttt tactgatgca tatacatgat ggcatatgca gcatctattc 1800
atatgctcta accttgagta cctatctatt ataataaaca agtatgtttt ataattattt 1860
tgatcttgat atacttggat gatggcatat gcagcagcta tatgtggatt tttttagccc 1920
tgccttcata cgctatttat ttgcttggta ctgtttcttt tgtcgatgct caccctgttg 1980
tttggtgtta cttctgcagg tcgactctag a 2011
<210> 5
<211> 552
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 5
atgagcccag aacgacgccc ggccgacatc cgccgtgcca ccgaggcgga catgccggcg 60
gtctgcacca tcgtcaacca ctacatcgag acaagcacgg tcaacttccg taccgagccg 120
caggaaccgc aggagtggac ggacgacctc gtccgtctgc gggagcgcta tccctggctc 180
gtcgccgagg tggacggcga ggtcgccggc atcgcctacg cgggcccctg gaaggcacgc 240
aacgcctacg actggacggc cgagtcgacc gtgtacgtct ccccccgcca ccagcggacg 300
ggactgggct ccacgctcta cacccacctg ctgaagtccc tggaggcaca gggcttcaag 360
agcgtggtcg ctgtcatcgg gctgcccaac gacccgagcg tgcgcatgca cgaggcgctc 420
ggatatgccc cccgcggcat gctgcgggcg gccggcttca agcacgggaa ctggcatgac 480
gtgggtttct ggcagctgga cttcagcctg ccggtaccgc cccgtccggt cctgcccgtc 540
accgagattt ga 552
<210> 6
<211> 66
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 6
gactacaaag accatgacgg tgattataaa gatcatgaca tcgactacaa ggatgacgat 60
gacaag 66
<210> 7
<211> 1700
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 7
gtccccagat tagccttttc aatttcagaa agaatgctaa cccacagatg gttagagagg 60
cttacgcagc aggtctcatc aagacgatct acccgagcaa taatctccag gaaatcaaat 120
accttcccaa gaaggttaaa gatgcagtca aaagattcag gactaactgc atcaagaaca 180
cagagaaaga tatatttctc aagatcagaa gtactattcc agtatggacg attcaaggct 240
tgcttcacaa accaaggcaa gtaatagaga ttggagtctc taaaaaggta gttcccactg 300
aatcaaaggc catggagtca aagattcaaa tagaggacct aacagaactc gccgtaaaga 360
ctggcgaaca gttcatacag agtctcttac gactcaatga caagaagaaa atcttcgtca 420
acatggtgga gcacgacaca cttgtctact ccaaaaatat caaagataca gtctcagaag 480
accaaagggc aattgagact tttcaacaaa gggtaatatc cggaaacctc ctcggattcc 540
attgcccagc tatctgtcac tttattgtga agatagtgga aaaggaaggt ggctcctaca 600
aatgccatca ttgcgataaa ggaaaggcca tcgttgaaga tgcctctgcc gacagtggtc 660
ccaaagatgg acccccaccc acgaggagca tcgtggaaaa agaagacgtt ccaaccacgt 720
cttcaaagca agtggattga tgtgatatct ccactgacgt aagggatgac gcacaatccc 780
actatccttc gcaagaccct tcctctatat aaggaagttc atttcatttg gagagaacac 840
gggggacctg caggtcccca gattagcctt ttcaatttca gaaagaatgc taacccacag 900
atggttagag aggcttacgc agcaggtctc atcaagacga tctacccgag caataatctc 960
caggaaatca aataccttcc caagaaggtt aaagatgcag tcaaaagatt caggactaac 1020
tgcatcaaga acacagagaa agatatattt ctcaagatca gaagtactat tccagtatgg 1080
acgattcaag gcttgcttca caaaccaagg caagtaatag agattggagt ctctaaaaag 1140
gtagttccca ctgaatcaaa ggccatggag tcaaagattc aaatagagga cctaacagaa 1200
ctcgccgtaa agactggcga acagttcata cagagtctct tacgactcaa tgacaagaag 1260
aaaatcttcg tcaacatggt ggagcacgac acacttgtct actccaaaaa tatcaaagat 1320
acagtctcag aagaccaaag ggcaattgag acttttcaac aaagggtaat atccggaaac 1380
ctcctcggat tccattgccc agctatctgt cactttattg tgaagatagt ggaaaaggaa 1440
ggtggctcct acaaatgcca tcattgcgat aaaggaaagg ccatcgttga agatgcctct 1500
gccgacagtg gtcccaaaga tggaccccca cccacgagga gcatcgtgga aaaagaagac 1560
gttccaacca cgtcttcaaa gcaagtggat tgatgtgata tctccactga cgtaagggat 1620
gacgcacaat cccactatcc ttcgcaagac ccttcctcta tataaggaag ttcatttcat 1680
ttggagagaa cacgggggac 1700
<210> 8
<211> 1026
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 8
atgaaaaagc ctgaactcac cgcgacgtct gtcgagaagt ttctgatcga aaagttcgac 60
agcgtctccg acctgatgca gctctcggag ggcgaagaat ctcgtgcttt cagcttcgat 120
gtaggagggc gtggatatgt cctgcgggta aatagctgcg ccgatggttt ctacaaagat 180
cgttatgttt atcggcactt tgcatcggcc gcgctcccga ttccggaagt gcttgacatt 240
ggggagttta gcgagagcct gacctattgc atctcccgcc gtgcacaggg tgtcacgttg 300
caagacctgc ctgaaaccga actgcccgct gttctacaac cggtcgcgga ggctatggat 360
gcgatcgctg cggccgatct tagccagacg agcgggttcg gcccattcgg accgcaagga 420
atcggtcaat acactacatg gcgtgatttc atatgcgcga ttgctgatcc ccatgtgtat 480
cactggcaaa ctgtgatgga cgacaccgtc agtgcgtccg tcgcgcaggc tctcgatgag 540
ctgatgcttt gggccgagga ctgccccgaa gtccggcacc tcgtgcacgc ggatttcggc 600
tccaacaatg tcctgacgga caatggccgc ataacagcgg tcattgactg gagcgaggcg 660
atgttcgggg attcccaata cgaggtcgcc aacatcttct tctggaggcc gtggttggct 720
tgtatggagc agcagacgcg ctacttcgag cggaggcatc cggagcttgc aggatcgcca 780
cgactccggg cgtatatgct ccgcattggt cttgaccaac tctatcagag cttggttgac 840
ggcaatttcg atgatgcagc ttgggcgcag ggtcgatgcg acgcaatcgt ccgatccgga 900
gccgggactg tcgggcgtac acaaatcgcc cgcagaagcg cggccgtctg gaccgatggc 960
tgtgtagaag tactcgccga tagtggaaac cgacgcccca gcactcgtcc gagggcaaag 1020
aaatag 1026
<210> 9
<211> 781
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 9
agagatagat ttgtagagag agactggtga tttcagcgtg tcctctccaa atgaaatgaa 60
cttccttata tagaggaagg tcttgcgaag gatagtggga ttgtgcgtca tcccttacgt 120
cagtggagat atcacatcaa tccacttgct ttgaagacgt ggttggaacg tcttcttttt 180
ccacgatgct cctcgtgggt gggggtccat ctttgggacc actgtcggca gaggcatctt 240
gaacgatagc ctttccttta tcgcaatgat ggcatttgta ggtgccacct tccttttcta 300
ctgtcctttt gatgaagtga cagatagctg ggcaatggaa tccgaggagg tttcccgata 360
ttaccctttg ttgaaaagtc tcaatagccc tttggtcttc tgagactgta tctttgatat 420
tcttggagta gacgagagtg tcgtgctcca ccatgttatc acatcaatcc acttgctttg 480
aagacgtggt tggaacgtct tctttttcca cgatgctcct cgtgggtggg ggtccatctt 540
tgggaccact gtcggcagag gcatcttgaa cgatagcctt tcctttatcg caatgatggc 600
atttgtaggt gccaccttcc ttttctactg tccttttgat gaagtgacag atagctgggc 660
aatggaatcc gaggaggttt cccgatatta ccctttgttg aaaagtctca atagcccttt 720
ggtcttctga gactgtatct ttgatattct tggagtagac gagagtgtcg tgctccacca 780
t 781
<210> 10
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 10
acaaatctat ctctctcgag tctaccatga gcccagaacg 40
<210> 11
<211> 40
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 11
ttattatgga gaaactcgag tcaaatctcg gtgacgggca 40
<210> 12
<211> 49
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 12
ttctgcaggt cgactctaga ggatccatgg catcaactgt cggaaacgc 49
<210> 13
<211> 49
<212> DNA
<213>Artificial sequence (Artificial Sequence)
<400> 13
ctttgtagtc ggtacccggg gatccctcta agacagctct gcgttgcgg 49
Claims (15)
1. a kind of initiative for the heavy metal super-enriched transgenic engineering rice for turning S.plumbizincicola SpNramp5 genes, feature exist
In, including:
(1) clone of heavy metal super-enriched gene
Clone SpNramp5 genes in S.plumbizincicola;
(2) structure of overexpression binary vector
Based on plant expression vector, conversion carrier of the structure comprising strong promoter, screening-gene clones step (1)
To SpNramp5 genes the conversion carrier is inserted by methods of homologous recombination, obtain overexpression binary vector;
(3) heavy metal super-enriched transfer-gen plant structure
Overexpression binary vector obtained by step (2) is transferred to by Agrobacterium tumefaciems in Rice Callus, is trained with screening
It supports base and carries out screening and culturing, the transgenic resistance Rice Callus of acquisition is by inducing, breaking up, taking root, the forging of transgenic seedling
Refining and transplanting, screen heavy metal super-enriched transgenic positive plant;
Wherein, the SpNramp5 gene nucleotide series such as SEQ ID NO:Shown in 1, the rice varieties are Nipponbare.
2. the wound of the heavy metal super-enriched transgenic engineering rice as described in claim 1 for turning S.plumbizincicola SpNramp5 genes
System, which is characterized in that in the step (1), the method for cloning SpNramp5 genes in S.plumbizincicola is:
Using the cDNA of S.plumbizincicola as template, with such as SEQ ID NO:2 and SEQ ID NO:Sequence shown in 3 is carried out for primer pair
PCR amplification obtains SpNramp5 genes.
3. the wound of the heavy metal super-enriched transgenic engineering rice as described in claim 1 for turning S.plumbizincicola SpNramp5 genes
System, which is characterized in that the plant expression vector is pCAMBIA1301 carriers, and the strong promoter starts for Ubiquitin
Son, the screening-gene are Bar screening-genes, and the conversion carrier further includes 3 × Flag labels.
4. the wound of the heavy metal super-enriched transgenic engineering rice as claimed in claim 3 for turning S.plumbizincicola SpNramp5 genes
System, which is characterized in that the construction method of the conversion carrier includes the following steps:
(a) 3 × Flag labels are added between pCAMBIA1301 vector multiple cloning sites Kpn I and the Sac I;
(b) the Bar screening-genes on the pCAMBIA3301 carriers are cloned using pcr amplification reaction;
(c) using the carrier obtained in restriction endonuclease Xho I digestion steps (a), and by the load after the Bar screening-genes and digestion
Body carries out homologous recombination;
(d) using the Ubiquitin promoters on Hind III and BamH I digestion pUN1301 carriers;
(e) using the recombinant vector obtained in restriction endonuclease Hind III and BamH I digestion steps (c), and by described in
Ubiquitin promoters are connect with the recombinant vector after digestion.
5. the wound of the heavy metal super-enriched transgenic engineering rice as claimed in claim 4 for turning S.plumbizincicola SpNramp5 genes
System, which is characterized in that the nucleotide sequence of the Ubiquitin promoters such as SEQ ID NO:Shown in 4, the Bar screens base
Because of nucleotide sequence such as SEQ ID NO:Shown in 5, the nucleotide sequence such as SEQ ID NO of 3 × Flag labels:Shown in 6.
6. the wound of the heavy metal super-enriched transgenic engineering rice as claimed in claim 4 for turning S.plumbizincicola SpNramp5 genes
System, which is characterized in that in step (b), the PCR primer sequence of the Bar screening-genes on clone's pCAMBIA3301 carriers
Row such as SEQ ID NO:10 and SEQ ID NO:Shown in 11.
7. the wound of the heavy metal super-enriched transgenic engineering rice as claimed in claim 3 for turning S.plumbizincicola SpNramp5 genes
System, which is characterized in that the screening and culturing medium includes glufosinate-ammonium.
8. the wound of the heavy metal super-enriched transgenic engineering rice as described in claim 1 for turning S.plumbizincicola SpNramp5 genes
System, which is characterized in that the plant expression vector is pCAMBIA1301 carriers, and the strong promoter starts for 2 × CaMV35S
Son, the screening-gene are HYG screening-genes.
9. the wound of the heavy metal super-enriched transgenic engineering rice as claimed in claim 8 for turning S.plumbizincicola SpNramp5 genes
System, which is characterized in that the screening-gene that the HYG screening-genes are carried for the pCAMBIA1301 carriers, the conversion carrier
Construction method include the following steps:
(a) using pCAMBIA1301 carriers described in restriction endonuclease Hind III and Pst I digestions;
(b) using pCAMBIA1301 carriers described in restriction endonuclease Hind III and BamH I digestions, CaMV35S promoters are obtained;
(c) drawing containing restriction endonuclease Hind III and Pst I restriction enzyme sites is designed according to the nucleotide sequence of CaMV35S promoters
Object, and pcr amplification reaction is carried out by template of CaMV35S promoters, it obtains containing Hind III and Pst I restriction enzyme sites
CaMV35S promoters, and digestion is carried out to the CaMV35S promoters that PCR is obtained using restriction endonuclease Hind III and Pst I;
(d) digestion products obtained in step (c) are recycled, and the digestion carrier with being obtained in step (a) is connect.
10. the heavy metal super-enriched transgenic engineering rice as claimed in claim 8 for turning S.plumbizincicola SpNramp5 genes
Initiative, which is characterized in that the nucleotide sequence of 2 × CaMV35S promoters such as SEQ ID NO:Shown in 7, the HYG screenings
The nucleotide sequence of gene such as SEQ ID NO:Shown in 8.
11. the heavy metal super-enriched transgenic engineering rice as claimed in claim 8 for turning S.plumbizincicola SpNramp5 genes
Initiative, which is characterized in that the screening and culturing medium contains hygromycin.
12. the heavy metal super-enriched transgenic engineering rice for turning S.plumbizincicola SpNramp5 genes as described in claim 3 or 8
Initiative, which is characterized in that in step (2), the structure of the overexpression binary vector includes the following steps:
SpNramp5 genes are cloned using pcr amplification reaction, the homologous recombination PCR primer of clone's SpNramp5 genes includes
BamH I restriction enzyme sites.
13. the heavy metal super-enriched transgenic engineering rice as claimed in claim 12 for turning S.plumbizincicola SpNramp5 genes
Initiative, which is characterized in that the homologous recombination PCR primer sequence such as SEQ ID NO of clone's SpNramp5 genes:12 and SEQ
ID NO:Shown in 13.
14. the heavy metal super-enriched transgenic engineering rice as described in claim 1 for turning S.plumbizincicola SpNramp5 genes
Initiative, which is characterized in that the Agrobacterium tumefaciems is EHA105.
15. as claim 1~14 any one of them turns the heavy metal super-enriched transgenosis of S.plumbizincicola SpNramp5 genes
Application of the heavy metal super-enriched transfer-gen plant of gained in heavy metal-polluted soil reparation in the initiative of engineering rice.
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