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Creation and application of heavy metal super-enrichment genetically modified engineering rape for transferring sedum plumbizincicola SpHMA2 and SpNramp5

A technology of transgenic engineering and sedum with ore, which is applied in the field of transgenic plants and heavy metal pollution control, can solve the problems of limited tolerance, special growth environment, heavy metal return of wild heavy metal-enriched plants, and achieve the improvement of heavy metal enrichment ability, genetic Good conversion effect and high expression effect

Inactive Publication Date: 2019-11-19
深圳市沃地污染修复技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] The purpose of the present invention is to provide a method and application of a heavy metal hyper-enrichment transgenic engineering rapeseed that has been transformed with Sedum sedum SpHMA2 and SpNramp5 genes, so as to solve the problem of limited tolerance, slow growth, small biomass, and poor growth in wild heavy metal-enriched plants. The special environment and the return of heavy metals

Method used

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  • Creation and application of heavy metal super-enrichment genetically modified engineering rape for transferring sedum plumbizincicola SpHMA2 and SpNramp5
  • Creation and application of heavy metal super-enrichment genetically modified engineering rape for transferring sedum plumbizincicola SpHMA2 and SpNramp5
  • Creation and application of heavy metal super-enrichment genetically modified engineering rape for transferring sedum plumbizincicola SpHMA2 and SpNramp5

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Example 1 Double gene cloning of SpHMA2 and SpNramp5

[0046] The seedlings of Sedum sedatus were ground into powder with liquid nitrogen, the RNA was extracted by Trizol method, reverse-transcribed into cDNA using TOYOBO reverse transcription kit, and Primer5 was used to design the cDNA sequence of the SpHMA2 gene provided by NCBI. Downstream primers, and synthetic primers, the nucleotide sequence of the designed primers is:

[0047] Upstream sequence: 5'GAACACGGGGGACTCTAGAGGATCC 3' (as shown in SEQ ID NO: 3);

[0048] Downstream sequence: 5'CATTTATTTCAACCGGTCCGACC 3' (shown in SEQ ID NO:4).

[0049] According to the cDNA sequence of the SpNramp5 gene provided by NCBI, use Primer5 to design its upstream and downstream primers, and synthesize the primers. The nucleotide sequence of the designed primers is:

[0050] Upstream sequence: 5'TTCCGCACTCGACTTTGACG 3' (as shown in SEQ ID NO:5);

[0051] Downstream sequence: 5'AACTTGATCCGATTCGCACA 3' (shown in SEQ ID NO: 6). ...

Embodiment 2

[0053] The construction of the transformation vector of embodiment 2 heavy metal hyper-enrichment transgenic plant

[0054] The construction of the transformation vector containing 2×CaMV 35S promoter and Hyg selection gene is based on the laboratory’s own pCAMBIA1301 vector, which is obtained after transformation through the following steps:

[0055] Use Primer5 to design the PCR upstream and downstream primers of the CaMV 35S promoter gene, and synthesize the primers. The nucleotide sequence of the designed primers is:

[0056] Upstream sequence: 5'AAGCTTTGAGACTTTTCAACAAAGGGT 3' (as shown in SEQ ID NO: 9);

[0057] Downstream sequence: 5'GGATCCTCAGCGTGTCCTTCCAAATG 3' (shown in SEQ ID NO: 10).

[0058] ① Obtain 2×CaMV 35S promoter: insert the strain containing pCAMBIA1301 vector into LB-Kan medium, and shake the bacteria to extract the plasmid. pCAMBIA1301 was digested with HindⅢ and BamH I, the digested product was electrophoresed on 1% agarose gel, and a small band (about...

Embodiment 3

[0060] Example 3 Overexpression of SpHMA2 and SpNramp5 double genes in transgenic plants with heavy metal hyperaccumulation Binary vector

[0061] Homologous recombination primers were designed according to the two ends of the ORF region of the SpHMA2 gene, and the nucleotide sequence of the designed homologous recombination primers was:

[0062] Upstream sequence (shown as SEQ ID NO: 11):

[0063] 5'AGAGAACACGGGGGACTCTAGAATGGCCTTAGGCGACGAAAAG 3';

[0064] Downstream sequence (shown as SEQ ID NO: 12):

[0065] 5'ACGATCGGGGAAATTCGAGCTCCTACTCCACAACAATCTCGGACAATC 3'.

[0066] Design PCR primers according to the two ends of the ORF region of SpNramp5 gene, the nucleotide sequence of the designed PCR primers is:

[0067] Upstream sequence (shown as SEQ ID NO:13):

[0068] 5'GAAGATCTATGGCATCAACTGTCGGAAACGC3';

[0069] Downstream sequence (shown as SEQ ID NO: 14):

[0070] 5'GGGTAACCCCTACTCTAAGACAGCTCTGCGTT 3'.

[0071] The 19T vector containing SpHMA2 and the 19T vector conta...

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Abstract

The invention discloses a creation method of heavy metal super-enrichment genetically modified engineering rape for transferring sedum plumbizincicola SpHMA2 and SpNramp5. The method comprises the following steps of cloning SpHMA2 and SpNramp5 dual genes of sedum plumbizincicola; using a plant expression vector as a foundation, constructing a transformation vector containing a strong promoter andthe screening genes, connecting the heavy metal super-enrichment SpNramp5 gene to the restriction enzyme cutting site of the transformation vector, and inserting the transformation vector into the SpHMA2 gene to obtain a dual-gene overexpression binary vector; and transferring the obtained dual-gene overexpression binary vector into the hypocotyl cutting stock of the rape through root knot agrobacterium tumefaciens, breeding genetically modified seedlings and screening genetically modified positive plants expressed with dual-gene pyramiding. The breeding method provided by the invention can solve the problems that wild heavy metal enrichment plants are limited in tolerance, slow in growth, small in biomass, special in growing environment, and the like, and heavy metals return. In addition,the invention further provides an application of the heavy metal super-enrichment transfer dual-gene plant obtained by the creation method to repair of heavy metals in soil.

Description

technical field [0001] The invention belongs to the field of transgenic plants and heavy metal pollution control, and in particular relates to a creation method and application of heavy metal hyper-enrichment transgenic engineering rapeseed that has been transformed with Sedum sedum SpHMA2 and SpNramp5 double genes. Background technique [0002] Heavy metal pollution has become a major environmental problem worldwide. Heavy metal pollution not only threatens the production and development of animals, plants and microorganisms, but also threatens human health and even life through the food chain. Heavy metals that cause soil pollution mainly include As, Cd, Co, Cr, Cu, Hg, etc., which are generally compound pollution of several heavy metals. Heavy metal pollution in heavy metal mining areas and high-speed economic development areas is particularly serious, resulting in many agricultural products in our country being refused to enter the international market due to excessive h...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/84A01H5/00A01H6/20B09C1/10
CPCB09C1/105B09C2101/00C12N15/8259
Inventor 白帆陈坤明袁博刘佳兰李永辉魏伟刘捷
Owner 深圳市沃地污染修复技术有限公司
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