CN105274121A - Gene capable of promoting cadmium accumulation and application of gene - Google Patents
Gene capable of promoting cadmium accumulation and application of gene Download PDFInfo
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Abstract
The invention discloses a gene capable of promoting cadmium accumulation and application of the gene. The gene capable of promoting cadmium accumulation is separated from super accumulation type sedum alfredii hance, the polynucleotide sequence is shown by (a) or (b), (a) is a polynucleotide sequence shown by SEQ ID No.1; (b) is a polynucleotide sequence which is complementary with the polynucleotide sequence in the (a) according to a base complementary pairing rule. The gene is derived from a super accumulation type sedum alfredii hance plant, an expression vector of the gene is constructed, agrobacterium is converted with an expression vector electric shock method, arabidopsis thaliana is soaked with a mature agrobacterium transformation method, and finally, a transgenic arabidopsis thaliana plant is obtained. The content of cadmium in the blade of the transgenic arabidopsis thaliana plant can be remarkably increased by over expression of the gene on the blade of the transgenic arabidopsis thaliana plant, compared with an ordinary arabidopsis thaliana plant, the content of cadmium in the transgenic arabidopsis thaliana subjected to cadmium treatment is improved by 50%-64%, and the absorption rate of the cadmium in the transgenic arabidopsis thaliana plant under heavy metal cadmium stress is remarkably higher than that of the cadmium in the ordinary arabidopsis thaliana plant.
Description
Technical field
The present invention relates to gene engineering technology field, particularly a kind of gene and application thereof promoting Cd accumulation.
Background technology
In recent years along with China's modernization construction and industrialized develop rapidly, the various environmental problems caused by heavy metals exceeding standard are day by day serious, and attract attention gradually.It is reported, current China is by nearly 2,000 ten thousand hm of cultivated area of the heavy metal contaminations such as cadmium, arsenic, chromium, lead
2, account for 1/5 of total area under cultivation, the whole nation is annual, and because of heavy metal contamination, underproduction grain reaches more than 1,000 ten thousand tons, and the financial loss caused thus is difficult to estimate.Point out in " the national Soil Pollution Investigation publication " announced in April, 2014, on the whole, the edatope situation of China also exists larger problem, the soil contamination problem in some regions is comparatively serious, and the quality of ploughing is more troubling, the soil contamination problem on some the industry and mining ground fallen into disuse is also comparatively outstanding.Due to nondegradation and the difficult scavenging of heavy metal element, how low cost, high-level efficiency, carry out improvement safely and become study hotspot.
Phytoremediation is a kind of cleaning method relying on plant, fixed by plant extraction, plant degradation, plant, phytovolatilization, the effect such as rhizosphere filtration, remove the pollutent in environment, it be with low cost, technology is simple, sustainable, environmentally friendly, be subject to people and welcome.At present in developed country, phytoremediation is widely used in the reparation of contaminated soil, surface water, underground water, throw out, refuse landfill etc., reaches commercial level.Under heavy metal stress, although the upgrowth situation of most plant can be subject to significantly suppressing and showing poisoning symptom, but still can find at occurring in nature the plant that a class is special, its can not only containing high density heavy metal environment in normal growth, also ultraproduct can also be absorbed to a certain extent and tire out heavy metal ion, be therefore defined as hyperaccumulative plant.Super accumulation type Sedum alfredii Hance has certain super accumulation ability to Cd, Zn, Pb, and its over-ground part can accumulate 8000 μ gg
-1above cadmium and without obvious poisoning symptom.Compared with other hyperaccumulative plants, super accumulation type Sedum alfredii Hance ultraproduct can tire out Cd, Zn, Pb tri-heavy metal species, biomass is comparatively considerable, vegetative propagation is utilized to be conducive to carrying out amount reproduction, and there is perennial, fast growth and the easy feature such as harvesting, in phytoremediation field, there is larger potential value.In the face of day by day serious soil pollution by heavy metal problem, find the phytoremediation gene of heavy metal hyperaccumulative and illustrate its function for the improvement of China's heavy metal contamination with repair significant.
Summary of the invention
An object of the present invention is to provide a kind of gene promoting Cd accumulation, and it is obtained by being separated in the leaf cDNA from super accumulation type Sedum alfredii Hance;
Two of object of the present invention is to provide expression vector and the transgenic Arabidopsis plants of the gene of being correlated with containing above-mentioned promotion Cd accumulation.
Three of object of the present invention is to provide the gene of being correlated with containing above-mentioned promotion Cd accumulation and is promoting the application in Arabidopis thaliana transfer-gen plant Cd accumulation.
The invention discloses a kind of gene promoting Cd accumulation, it is obtained by being separated in the leaf cDNA from super accumulation type Sedum alfredii Hance, it is characterized in that, its polynucleotide sequence is for shown in (a) or (b):
Polynucleotide sequence shown in (a) SEQIDNo.1;
B () and polynucleotide sequence in (a) are according to the polynucleotide sequence of base pair complementarity principle complementation.
Preferably, wherein, its polynucleotide sequence is also for shown in (c):
The sequence of the cDNA of the polynucleotide sequence (a) shown in (c) SEQIDNO.1 or (b) middle polynucleotide sequence: be the polynucleotide sequence shown in SEQIDNO.2.
By an albumen for the coded by said gene of promotion Cd accumulation, it is characterized in that, its aminoacid sequence is the aminoacid sequence shown in SEQIDNO.3.
A kind of expression vector of the gene containing promotion Cd accumulation.
Preferably, wherein, the construction process of described expression vector specifically comprises the following steps:
5.1) from super accumulation type Sedum alfredii Hance transcript profile sequence, analysis obtains Nramp6 gene fragment, with the design of this fragment forward primer Nramp6-3F and universal primer B26, reverse primer Nramp6-5R and universal primer APL, carry out respectively 3 '-RACE and 5 '-RACE obtain Nramp6 gene fragment 3 ' end and 5 ' end group because of PCR fragment, and direct Sequencing splicing acquisition DNA sequence dna;
5.2) according to described cDNA sequence, pair of primers Nramp6-F1 and Nramp6-R1 is redesigned at its 5 ' end and 3 ' end non-coding region, extract Sedum alfredii Hance blade total serum IgE reverse transcription becomes cDNA, with this cDNA for template and with Nramp6-F1 and Nramp6-R1 for primer carries out Standard PCR reaction, obtain the cDNA full length sequence of Nramp6, pair of primers Nramp6-F2 and Nramp6-R2 is redesigned at 5 ' end and 3 ' end with the open reading frame sequence of Nramp6 full length sequence, and with described cDNA for template, pcr amplification goes out the encoding sequence of Nramp6, afterwards encoding sequence is connected on pMD19-T cloning vector.
Preferably, wherein, the polynucleotide sequence of described forward Nramp6-3F and reverse primer Nramp6-5R is the polynucleotide sequence shown in SEQIDNo.4 and SEQIDNo.5; The polynucleotide sequence of universal primer B26 and universal primer APL is the polynucleotide sequence shown in SEQIDNo.6 and SEQIDNo.7; Primer Nramp6-F1 and Nramp6-R1 polynucleotide sequence are the polynucleotide sequence shown in SEQIDNo.8 and SEQIDNo.9; Primer Nramp6-F2 and Nramp6-R2 polynucleotide sequence are the polynucleotide sequence shown in SEQIDNo.10 and SEQIDNo.11.
A kind of Arabidopis thaliana transfer-gen plant of expression vector of the gene containing promotion Cd accumulation.
Promote that the gene of Cd accumulation is promoting the application in Arabidopis thaliana transfer-gen plant Cd accumulation.
The invention has the beneficial effects as follows:
The invention provides a kind of plant code gene with increase plant cadmium absorption and accumulation, this gene stems from super accumulation type Sedum alfredii Hance plant, build the expression vector of this gene, utilize this expression vector electric shocking method transformation Agrobacterium, the conversion method for agrobacterium of application maturation infects Arabidopis thaliana, final acquisition transgenic Arabidopsis plants, the overexpression of this gene on the blade of transgenic Arabidopsis plants obviously can increase the content of cadmium in transgenic arabidopsis blade, after Cadmium treated, the cadmium content of transgenic arabidopsis improves 50% ~ 64% than with common Arabidopsis plant for contrasting, the absorption rate of cadmium in lower transgenic Arabidopsis plants is coerced apparently higher than common Arabidopsis plant at heavy metal cadmium.
The term definition arrived involved in the present invention
Unless otherwise defined, otherwise all technology used herein and scientific terminology all have with those skilled in the art usually understand identical implication.Although any method, device and the material similar or equivalent with person described herein can be used in practice of the present invention or test, preferred method, device and material are described now.
Term " base pair complementarity principle " means in DNA molecular structure, the distance had between fixing number and DNA two chains due to the hydrogen bond between base remains unchanged, make base pairing must follow certain rule, certain and the Thymine (T, thymus pyrimidine) of Here it is Adenine (A, VITAMIN B4) matches, Guanine (G, guanine) certain and Cytosine (C, cytosine(Cyt)) matches, and vice versa.
Term " expression vector " means on the basis of cloning vector basic framework, increase Expression element (as promotor, RBS, terminator etc.), enables the carrier that goal gene is expressed.If expression vector pKK223-3 is a coli expression carrier with typical expression structure.Its basic framework is Plasmid replication origins from pBR322 and pUC and ampicillin resistance gene.In Expression element, have a heterozygosis tac strong promoter and terminator, have RBS site (if utilize this site, requiring interval 5-13bp between ATG) in promotor downstream, multiple clone site thereafter can load the target gene that will express.Goal gene in the present invention is the cDNA of polynucleotide sequence in the polynucleotide sequence (a) shown in SEQIDNO.1 or (b).
Term " polynucleotide " means the deoxyribonucleotide of sub-thread or bifilar form, dezyribonucleoside (DNA), ribonucleoside or ribonucleotide (RNA) and polymkeric substance thereof.As described in the present invention disulfide isomerase gene " DNA " or " cDNA " and etc.Except nonspecific restriction, otherwise the nucleic acid of the known analogue containing natural nucleotide contained in described term, and described analogue has the binding characteristic that is similar to reference nucleic acid and carries out metabolism in the mode of the Nucleotide being similar to natural generation.Unless other specific restriction, otherwise described term also means oligonucleotide analogs, and it comprises PNA (peptide nucleic acid(PNA)), DNA analogue used in antisense technology (thiophosphatephosphorothioate, phosphamide acid esters etc.).Unless otherwise, otherwise the specific nucleic acid sequence sequence that also impliedly contains its conservative varient (including, but is not limited to degenerate codon replace) of modifying and complementary sequence and clearly specify.Particularly, the 3rd sequence replaced through mixing base and/or deoxyinosine residue by producing one of them or more than one selected (or all) codon replaces to realize degenerate codon (people such as Batzer, NucleicAcidRes.19:5081 (1991); The people such as Ohtsuka, J.Biol.Chem.260:2605-2608 (1985); With people such as Cassol, (1992); The people such as Rossolini, MolCell.Probes8:91-98 (1994)).Wherein, the cDNA of the disulfide isomerase gene mentioned in the present invention is the RNA reverse transcription obtained after transcribing by the DNA of disulfide isomerase gene.
Term " amino acid " means the fundamental unit forming protein, gives protein specific molecular morphosis, makes his molecule have biochemical activity.Protein is bioactive molecule important in organism, comprises the metabolic ferment of catalysis and enzyme.Different amino acid dehydrating condensations forms peptide (original segments of protein), is proteinogenous precursor.
Term " intrinsic protein ", " foreign protein " and " albumen " mean the polymkeric substance of amino-acid residue.It is the aminoacid polymers of non-naturally encoded amino acids that described term is applicable to natural generation aminoacid polymers and one of them or more than one amino-acid residue.
Accompanying drawing explanation
Fig. 1 is the ORF amplification electrophorogram of the gene of promotion Cd accumulation of the present invention, and wherein, M is DNAMarker2000; 1-3:Nramp6PCR amplified band;
Fig. 2 is the cadmium content figure in the transgenic line blade of Cadmium treated 1 week and 2 weeks in the present invention, wherein WT: be the contrast of non-transgenic Arabidopis thaliana, N1 ~ N3: be transgenic arabidopsis strain;
Fig. 3 is the cadmium ion flow velocity figure in the present invention in transgenic line root system, wherein WT: be the contrast of non-transgenic Arabidopis thaliana, N1 ~ N3: be transgenic arabidopsis strain.
Embodiment
Below in conjunction with accompanying drawing, the present invention is described in further detail, can implement according to this with reference to specification sheets word to make those skilled in the art.
Should be appreciated that used hereinly such as " to have ", other element one or more do not allotted in " comprising " and " comprising " term or the existence of its combination or interpolation.
The invention provides a kind of gene promoting Cd accumulation, it is obtained by being separated in the gene from super accumulation type Sedum alfredii Hance, it is characterized in that, its polynucleotide sequence is for shown in (a) or (b):
Polynucleotide sequence shown in (a) SEQIDNo.1;
B () and polynucleotide sequence in (a) are according to the polynucleotide sequence of base pair complementarity principle complementation.
Preferred version is, its polynucleotide sequence is also for shown in (c):
The sequence of the cDNA of the polynucleotide sequence (a) shown in (c) SEQIDNO.1 or (b) middle polynucleotide sequence: be the polynucleotide sequence shown in SEQIDNO.2.
By an albumen for the coded by said gene of promotion Cd accumulation, it is characterized in that, its aminoacid sequence is the aminoacid sequence shown in SEQIDNO.3.
A kind of expression vector of the gene containing promotion Cd accumulation.
Preferred version is, the construction process of described cloning vector specifically comprises the following steps:
5.1) from super accumulation type Sedum alfredii Hance transcript profile sequence, analysis obtains Nramp6 gene fragment, with the design of this fragment forward primer Nramp6-3F and universal primer B26, reverse primer Nramp6-5R and universal primer APL, carry out respectively 3 '-RACE and 5 '-RACE obtain this gene 3 ' end and 5 ' end group because of PCR fragment, and direct Sequencing splicing acquisition cDNA sequence;
5.2) cDNA sequence is obtained according to gene sequencing splicing, pair of primers Nramp6-F1 and Nramp6-R1 is redesigned at its 5 ' end and 3 ' end non-coding region, extract Sedum alfredii Hance blade mRNA total serum IgE reverse transcription becomes cDNA, with this cDNA for template and with Nramp6-F1 and Nramp6-R1 for primer carries out Standard PCR reaction, obtain the cDNA complete genome sequence full length sequence of Nramp6, open reading frame sequence according to Nramp6 full length sequence redesigns pair of primers Nramp6-F2 and Nramp6-R2 at 5 ' end and 3 ' end, and with described cDNA for template, pcr amplification goes out the encoding sequence of Nramp6, afterwards encoding sequence is connected on pMD19-T cloning vector.
Preferred version is, the polynucleotide sequence of described forward Nramp6-3F and reverse primer Nramp6-5R is the polynucleotide sequence shown in SEQIDNo.4 and SEQIDNo.5; The polynucleotide sequence of universal primer B26 and universal primer APL is the polynucleotide sequence shown in SEQIDNo.6 and SEQIDNo.7; The polynucleotide sequence of primer Nramp6-F1 and Nramp6-R1 is the polynucleotide sequence shown in SEQIDNo.8 and SEQIDNo.9; Primer Nramp6-F2 and Nramp6-R2 polynucleotide sequence are the polynucleotide sequence shown in SEQIDNo.10 and SEQIDNo.11.
A kind of Arabidopis thaliana transfer-gen plant of expression vector of the gene containing promotion Cd accumulation.
Promote that the gene of Cd accumulation is promoting the application in Arabidopis thaliana transfer-gen plant Cd accumulation.
Below, by conjunction with concrete testing sequence, from the gene of super accumulation type Sedum alfredii Hance, obtain the gene promoting Cd accumulation with sake of clarity the application, this gene is checked order, and apply this gene constructed expression carrier acquisition transgenic Arabidopsis plants.
In simple terms, the present invention analyzes and obtains Nramp6 gene fragment from super accumulation type Sedum alfredii Hance transcript profile sequence, with this fragment design forward Nramp6-3F and reverse primer Nramp6-5R, carry out 3 ' end and 5 ' end PCR fragment that 3 '-RACE and 5 '-RACE obtains this gene respectively, and direct Sequencing splicing.
CDNA sequence is obtained according to gene sequencing splicing, pair of primers Nramp6-F1 and Nramp6-R1 is redesigned at 5 ' end and 3 ' end non-coding region, extract Sedum alfredii Hance blade total serum IgE reverse transcription becomes cDNA, with this cDNA for template and with Nramp6-F1 and Nramp6-R1 for primer carries out Standard PCR reaction, obtain the cDNA full length sequence of Nramp6 and be connected to pMD19-T cloning vector sequence verification.
By obtaining the Arabidopis thaliana transgenic line containing Sedum alfredii Hance Nramp6 gene, by determination and analysis, demonstrate the gene that Nramp6 is an important Cd uptake accumulation.To the separation of this gene and the analysis of clone and biological function, important promoter action will be played for Phytoremediation of Soils Contaminated by Heavy Metals.
Realize concrete technological step of the present invention as follows:
1. the separation of Sedum alfredii Hance Nramp6 gene and sequential analysis
Analyze from super accumulation type Sedum alfredii Hance transcript profile sequence and obtain Nramp6 gene fragment, analyze and find that this clone does not cover the complete coding region of this gene.
In order to obtain the complete coding region sequence of this fragment, we to increase 3 ' end of this gene and 5 ' terminal fragment by 3 '-RACE and 5 '-RACE technology.Extracting the Sedum alfredii Hance blade total serum IgE under Cd stress according to NorgenRNA test kit, is templated synthesis Invitrogensuperscript Ш firststrandsynthesisc test kit synthesis cDNA with it.Match with this fragment forward primer Nramp6-3F and 3 '-RACE universal primer B26, reverse primer Nramp6-5R and 5 '-RACE universal primer APL matches and makes Standard PCR and obtain 5 ' terminal fragment and 3 ' terminal fragment respectively, PCR fragment is after reclaiming kits, be connected to pMD19-T cloning vector, order-checking obtains 5 ' end and 3 ' the end sequence information of Nramp6.
Pair of primers Nramp6-F2 and Nramp6-R2 is redesigned at 5 ' end and 3 ' end with the ORF sequence information of Nramp6, and with the cDNA synthesized above for template, connect into pMD19-T cloning vector after pcr amplification, picking 8 clones serve the order-checking of marine life engineering corporation, and each survey is respectively led to 2 times.Sequencing result information biology ceases professional DNA and splices software Contig3.1 splicing.Obtain the gene coded sequence of Nramp6.According to the open reading frame (ORF) of this sequence, extrapolate the aminoacid sequence of this gene coded protein.
2. build the plant expression vector containing Sedum alfredii Hance Nramp6 gene.The expression vector built, with electric shocking method transformation Agrobacterium, obtains the engineering Agrobacterium containing expression vector, with the Agrobacterium transgenic method titbit infestation method arabidopsis thaliana transformation that this experiment is ripe, obtains containing Nramp6 gene transgenic Arabidopis thaliana.By showing the test of transgenic arabidopsis determination and analysis, Nramp6 gene can improve the cadmium content of transgenic arabidopsis blade, and the cadmium content plant of transgenic arabidopsis improves 50 ~ 64% than contrast.Under heavy metal cadmium is coerced, significantly improve cadmium ion flow velocity in transgenic Arabidopsis plants at short notice, in transgenic Arabidopsis plants, the absorption rate of cadmium is apparently higher than the common Arabidopsis plant of control group.
The acquisition of embodiment 1, Nramp6cDNA coding region
1) extraction of Sedum alfredii Hance blade RNA, quality examination and total cDNA first chain synthesis;
2) from total cDNA first chain, Nramp6cDNA fragment upstream primer Nramp6-F2:5 '-ATGGCATCAACTGTCGGAAACGC-3 ' is synthesized with polymerase chain reaction (PCR)
Downstream primer Nramp6-R2:5 '-CTACTCTAAGACAGCTCTGCGTTGCGG-3 '
Pcr amplification condition: 94 DEG C × 5min → (94 DEG C × 30sec → 58 DEG C × 30sec → 72 DEG C × 2min) × 32 circulations → 72 DEG C × 10min, obtain specific PCR amplification product and see accompanying drawing 1.
3) pMD19-Nramp6 cloning vector builds and the parsing of gene base
PCR primer is cloned into Takara company pMD19-T carrier by T and obtains pMD19-Nramp6 carrier, and proceeds to DH5 α intestinal bacteria by thermal shock method.DH5 α bacterium liquid containing pMD19-Nramp6 carrier is served the order-checking of marine life Engineering Co., Ltd.Carry out splicing the sequence SEQIDNo.2 obtained in sequence table to order-checking with software Chromas and Contig.
The functional study of embodiment 2, Nramp6 gene accumulation cadmium
1) design forward primer Nramp6-F2 (5 ' end of primer adds CACC joint) and reverse primer Nramp6-R2, pcr amplification goes out cDNA fragment Nramp6 gene whole coding sequence;
2) utilize gateway method that Nramp6 gene is inserted into plant expression vector pH2GW7.0, obtain the expression vector pH2GW7.0-Nramp6 containing Nramp6 gene;
3) pH2GW7.0-Nramp6 is proceeded to Agrobacterium EHA105 with electric-shocking method, obtain Agrobacterium engineering cell system.Titbit infestation method is adopted to carry out transformation of Arabidopsis thaliana, being lain low by Arabidopis thaliana after infecting puts in the plastic tub of lucifuge, spray preservative film on a small amount of water bonnet and divide evaporation to prevent water, on preservative film upper cover, newspaper is to keep dark surrounds, after cultivating 16-24h, throw off preservative film, and take out Arabidopis thaliana, continue to be placed in Arabidopis thaliana room and cultivate; After Arabidopis thaliana maturation, collect seed, save backup in 4 DEG C after drying;
4) by dried T0 for seed at the ethanol disinfection through 75% on Bechtop, be sprinkling upon 1/2MS equably (containing Hyg20mgL
-1) on solid medium; Light culture 2d under 4 DEG C of environment, is transferred to culture dish in Arabidopis thaliana room subsequently and cultivates, and cultivates about one week; The Arabidopsis plant with Hyg resistance of normal growth can be transferred in Nutrition Soil subsequently, and cultivate in Arabidopis thaliana room; After PCR and RT-qPCR detects, choose the high positive plant of expression amount as transfer-gen plant strain, by culture until results T3 is for homozygote seed and in-4 DEG C of preservations, with biological function analysis later;
5) transgenic line and non-transgenic strain all carry out Cd stress test, compare the indexs such as Cd accumulation ability and uptake rate, analyze the effect of Nramp6 gene in Cd stress process.As shown in Figures 2 and 3, by showing the test of transgenic arabidopsis determination and analysis, under heavy metal cadmium is coerced, significantly improve cadmium ion flow velocity in transgenic Arabidopsis plants at short notice, in transgenic Arabidopsis plants, the absorption rate of cadmium is apparently higher than the common Arabidopsis plant of control group, and the cadmium content plant of transgenic arabidopsis improves 50 ~ 64% than contrast.
The present invention provides a kind of brand-new hyperaccumulative plant for nature, its can not only containing high density heavy metal environment in normal growth, can also absorb to a certain extent and ultraproduct tire out heavy metal ion, for China's heavy metal contamination improvement and repair significant.
Although embodiment of the present invention are open as above, but it is not restricted to listed in specification sheets and embodiment utilization, it can be applied to various applicable the field of the invention completely, for those skilled in the art, can easily realize other amendment, therefore do not deviating under the universal that claim and equivalency range limit, the present invention is not limited to specific details and illustrates here and the legend described.
Claims (8)
1. promote a gene for Cd accumulation, it is obtain from the leaf cDNA of super accumulation type Sedum alfredii Hance, it is characterized in that, promotes that the polynucleotide sequence of the gene of Cd accumulation is for shown in (a) or (b):
Polynucleotide sequence shown in (a) SEQIDNo.1;
B () and polynucleotide sequence in (a) are according to the polynucleotide sequence of base pair complementarity principle complementation.
2. the gene promoting Cd accumulation as claimed in claim 1, it is characterized in that, its polynucleotide sequence is also for shown in (c):
The sequence of the cDNA of the polynucleotide sequence (a) shown in (c) SEQIDNO.1 or (b) middle polynucleotide sequence: be the polynucleotide sequence shown in SEQIDNO.2.
3., by the albumen of coded by said gene promoting Cd accumulation as claimed in claim 1, it is characterized in that, its aminoacid sequence is the aminoacid sequence shown in SEQIDNO.3.
4. the cloning vector containing, for example the gene of promotion Cd accumulation according to claim 1.
5. the cloning vector of the gene containing promotion Cd accumulation as claimed in claim 4, it is characterized in that, the construction process of described cloning vector specifically comprises the following steps:
5.1) from super accumulation type Sedum alfredii Hance transcript profile sequence, analysis obtains Nramp6 gene fragment, with this fragment design forward primer Nramp6-3F and universal primer B26, reverse primer Nramp6-5 and universal primer APL, carry out respectively 3 '-RACE and 5 '-RACE obtain Nramp6 gene fragment 3 ' end and 5 ' end group because of PCR fragment, and direct Sequencing splicing acquisition cDNA sequence;
5.2) according to described cDNA sequence, pair of primers Nramp6-F1 and Nramp6-R1 is redesigned at its 5 ' end and 3 ' end non-coding region, extract Sedum alfredii Hance blade total serum IgE reverse transcription becomes cDNA, with this cDNA for template and with Nramp6-F1 and Nramp6-R1 for primer carries out Standard PCR reaction, obtain the cDNA full length sequence of Nramp6, pair of primers Nramp6-F2 and Nramp6-R2 is redesigned at 5 ' end and 3 ' end with the open reading frame sequence of Nramp6 full length sequence, and with described cDNA for template, pcr amplification goes out the encoding sequence of Nramp6, afterwards encoding sequence is connected on pMD19-T cloning vector.
6. the cloning vector of the gene containing promotion Cd accumulation as claimed in claim 5, it is characterized in that, the polynucleotide sequence of described forward Nramp6-3F and reverse primer Nramp6-5R is the polynucleotide sequence shown in SEQIDNo.4 and SEQIDNo.5; The polynucleotide sequence of universal primer B26 and universal primer APL is the polynucleotide sequence shown in SEQIDNo.6 and SEQIDNo.7; The polynucleotide sequence of primer Nramp6-F1 and Nramp6-R1 is the polynucleotide sequence shown in SEQIDNo.8 and SEQIDNo.9; Primer Nramp6-F2 and Nramp6-R2 polynucleotide sequence are the polynucleotide sequence shown in SEQIDNo.10 and SEQIDNo.11.
7. the Arabidopis thaliana transfer-gen plant of the expression vector of the gene containing promotion Cd accumulation according to claim 1.
8. gene as claimed in claim 1 is promoting the application in Arabidopis thaliana transfer-gen plant Cd accumulation.
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| CN108192916A (en) * | 2018-01-30 | 2018-06-22 | 广东开源环境科技有限公司 | Turn the initiative and application of the heavy metal super-enriched transgenic engineering rice of S.plumbizincicola SpNramp5 genes |
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| CN108866081A (en) * | 2018-07-25 | 2018-11-23 | 中国林业科学研究院亚热带林业研究所 | A kind of raising cadmium resistance and the gene of cadmium content and application thereof |
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