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Gene for improving cadmium resistance and cadmium content and reducing cadmium stress DNA damage and application of gene

A DNA damage and cadmium resistance technology, applied in the field of genetic engineering, can solve problems such as unresearched, and achieve the effects of increasing the root length of seedlings, improving soil heavy metal pollution, and increasing cadmium resistance and cadmium content

Active Publication Date: 2021-06-29
ZHEJIANG CHINESE MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, how the plant alleviates the cytotoxicity of Cd in the presence of high concentrations of Cd and maintains the mechanism of genome stability has not yet been studied.

Method used

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  • Gene for improving cadmium resistance and cadmium content and reducing cadmium stress DNA damage and application of gene
  • Gene for improving cadmium resistance and cadmium content and reducing cadmium stress DNA damage and application of gene
  • Gene for improving cadmium resistance and cadmium content and reducing cadmium stress DNA damage and application of gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0039] Construction of cDNA library of cadmium hyperaccumulating mine type Sedum sedum

[0040] (1) Using the screened cadmium hyperaccumulating mine type Southeast Sedum (collected from an ancient lead-zinc mining area in Quzhou City, Zhejiang Province, and then transplanted to our laboratory) hydroponic seedlings as the material, treated with 400 μM·L -1 Cadmium chloride (CdCl 2 ) After the stress treatment, the total RNA of Sedum sedum under cadmium stress was extracted with a plant tissue total RNA extraction kit (Total RNA Purification Kit), and purified mRNA was obtained through oligo(dT)-fiber column filtration;

[0041] (2) Obtaining full-length cDNA

[0042] cDNA strand synthesis using SMART TM cDNA library construction kit completed;

[0043] Synthesis of the first strand of cDNA: using the purified mRNA obtained in the above step (1) as a template, SMART TM IVoligonucleotide (10μM) (sequence shown in SEQ.ID.No.3: 5'-AAGCAGTGGTATCAACGCAGAGTGGCCATTACGGCCGGG-3') ...

Embodiment 2

[0050] Screening and identification of cadmium-resistant transgenic Arabidopsis

[0051] (1) Extract the mixed plasmid of the cadmium hyperaccumulation mine type Sedum sedum cDNA library obtained in Example 1, and electro-transform Agrobacterium EHA105:

[0052] Use a plasmid DNA extraction kit to extract the mixed plasmids of the cDNA library, and electro-transform the extracted mixed plasmids into the EHA105 strain. The voltage used for electro-transformation is 2.5 V; the transformed products are cultured in 5 mL of LB liquid for 3 hours and then transferred to 100 mL of liquid LB (containing Fuping 50mg·L -1 +Kan 20mg·L -1 ) at 28°C for 3 days to obtain an Agrobacterium engineering cell line.

[0053] (2) Heterologous expression of Sedum sedum cDNA library in Arabidopsis thaliana

[0054] Arabidopsis thaliana transformation was carried out by the tidbit infection method, and seeds were harvested and dried after the Arabidopsis thaliana matured. The dried TO generation ...

Embodiment 3

[0061] (1) Construction of yeast expression vector

[0062] Using the screened cadmium hyperaccumulating mine type Southeast Sedum (collected from an ancient lead-zinc mining area in Quzhou City, Zhejiang Province, and then transplanted to our laboratory) hydroponic seedlings as materials, total RNA from plant tissues was extracted with Total RNA Prification Kit. The kit (Norgen Company) extracted the total RNA of Sedum sedum under cadmium stress, obtained cDNA after reverse transcription, used it as a template, and designed the upstream and downstream specific primer SaCRRP-F1 according to the ORF of SaCRRP (such as SEQ.ID.No. 10: ATGGAGCATGTTAATTCAG) and SaCRRP-R1 (as shown in SEQ.ID.No.11: TTAGATTTTATCAGAGAG) to obtain the ORF of SaCRRP by PCR, and construct it into the yeast expression vector pYES2-SaCRRP after correct sequencing to obtain a positive recombinant plasmid.

[0063] (2) Resistance identification of yeast transformants under cadmium stress

[0064] The pYES2-...

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Abstract

The invention discloses a gene for improving cadmium resistance and cadmium content and reducing cadmium stress DNA damage and application of the gene. By constructing a sedum alfredii cDNA library, by means of model plant arabidopsis thaliana, the gene SaCRRP capable of improving cadmium resistance and cadmium content and reducing the genome DNA damage degree of transgenic arabidopsis thaliana under cadmium stress is found from the sedum alfredii cDNA library for the first time. It is found that the gene SaCRRP (Cd stress related protein) can improve the cadmium resistance of yeast; when the gene is transferred into the arabidopsis thaliana, the cadmium resistance and cadmium content of the transgenic arabidopsis thaliana are both improved; and the damage degree of the genome DNA of the transgenic arabidopsis thaliana under the cadmium stress condition is lower than that of a wild type. The gene SaCRRP provides a gene resource for cultivating a novel plant high-cadmium-resistance germplasm for plant bioremediation of heavy metal contaminated soil.

Description

technical field [0001] The invention belongs to the technical field of genetic engineering, and in particular relates to a gene for improving cadmium resistance and cadmium content and reducing DNA damage under cadmium stress and use thereof. Background technique [0002] With the rapid development of my country's industrialization construction, the problem of heavy metal pollution in soil is becoming more and more serious. How to repair heavy metal contaminated soil has become a hot spot in environmental protection work. The traditional methods of treating heavy metal-contaminated soils mainly involve technologies such as physical remediation, chemical remediation and biological remediation. However, these traditional soil heavy metal remediation techniques have disadvantages such as long processing time, high processing cost, large workload, and small application range, and cannot fundamentally remove heavy metals in soil. Phytoremediation technology is an emerging enviro...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/82C07K14/415A01H5/00A01H6/20
CPCC07K14/415C12N15/8271
Inventor 刘明英
Owner ZHEJIANG CHINESE MEDICAL UNIVERSITY
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