Gene capable of promoting cadmium accumulation and application of gene
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A gene and hyper-accumulation technology, applied in the field of genetic engineering, can solve problems such as inestimable economic losses
Inactive Publication Date: 2016-01-27
RES INST OF SUBTROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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According to reports, the area of cultivated land polluted by heavy metals such as cadmium, arsenic, chromium, and lead in my country is nearly 20 million hm 2 , accounting for about 1/5 ...
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Embodiment 1
[0057] Embodiment 1, the acquisition of Nramp6 cDNA coding region
[0058] 1) Extraction, quality inspection and first-strand synthesis of total cDNA of Sedum sedum leaves RNA;
[0059] 2) Synthesize the Nramp6 cDNA fragment upstream primer Nramp6-F2 from the first strand of the total cDNA by polymerase chain reaction (PCR): 5'-ATGGCATCAACTGTCGGAAACGC-3'
[0060] Downstream primer Nramp6-R2: 5'-CTACTCTAAGACAGCTCTGCGTTGCGG-3'
[0061] PCR amplification conditions: 94°C×5min→(94°C×30sec→58°C×30sec→72°C×2min)×32 cycles→72°C×10min, see the attached figure 1 .
[0062] 3) Construction of pMD19-Nramp6 cloning vector and gene base analysis
[0063] The PCR product was cloned into the pMD19-T vector of Takara Company by T to obtain the pMD19-Nramp6 vector, and transformed into DH5α Escherichia coli by heat shock method. The DH5α bacterial liquid containing the pMD19-Nramp6 vector was sent to Shanghai Bioengineering Co., Ltd. for sequencing. The sequencing was spliced with the s...
Embodiment 2
[0064] Example 2, Functional Research of Nramp6 Gene Accumulation of Cadmium
[0065] 1) Design the forward primer Nramp6-F2 (the 5' end of the primer plus a CACC adapter) and the reverse primer Nramp6-R2, and PCR amplify the full coding sequence of the cDNA fragment Nramp6 gene;
[0066] 2) using the gateway method to insert the Nramp6 gene into the plant expression vector pH2GW7.0 to obtain the expression vector pH2GW7.0-Nramp6 containing the Nramp6 gene;
[0067] 3) Transform pH2GW7.0-Nramp6 into Agrobacterium EHA105 by electric shock method to obtain the Agrobacterium engineered cell line. Transformation of Arabidopsis thaliana was carried out by floc infection method. Lay the infected Arabidopsis flat in a light-proof plastic pot, spray a small amount of water and cover with plastic wrap to prevent water from evaporating. Newspaper to keep a dark environment, after 16-24 hours of cultivation, remove the plastic wrap, take out the Arabidopsis, and continue to cultivate in...
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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 [0001] The invention relates to the technical field of genetic engineering, in particular to a gene for promoting cadmium accumulation and its application. Background technique [0002] In recent years, with the rapid development of my country's modernization and industrialization, various environmental problems caused by excessive heavy metals have become increasingly serious, and have gradually attracted people's attention. According to reports, the area of cultivated land polluted by heavy metals such as cadmium, arsenic, chromium, and lead in my country is nearly 20 million hm 2 , Accounting for about 1 / 5 of the total arable land area, the country's annual production of grain due to heavy metal pollution is reduced by more than 10 million tons, and the economic losses caused by this are incalculable. In the "National Soil Pollution Survey Bulletin" published in April 2014, it was pointed out that in general, my country's soil environment has serious pr...
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IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00
Inventor 韩小娇方洁卓仁英刘明英乔桂荣
Owner RES INST OF SUBTROPICAL FORESTRY CHINESE ACAD OF FORESTRY
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