Transgenic plant plasmid p3301-121-ZnT1 for repairing polluted water bodies and application thereof

A technology of transgenic plants and plasmids, applied in the field of bioremediation of pollution, can solve the problems of long plant growth cycle, high living environment requirements, and small biomass.

Inactive Publication Date: 2019-10-15
INST OF GEOGRAPHICAL SCI & NATURAL RESOURCE RES CAS
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0022] In order to solve the problems of multi-metal complex pollution in polluted water, hyperaccumulator plants have high requirements for the living environment of water bodies, and the plants themselves have a long growth cycle and small biomass. It is planned to use transgenic ZNT1 water peanuts to carry out bioremediation of polluted water bodies.

Method used

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  • Transgenic plant plasmid p3301-121-ZnT1 for repairing polluted water bodies and application thereof
  • Transgenic plant plasmid p3301-121-ZnT1 for repairing polluted water bodies and application thereof
  • Transgenic plant plasmid p3301-121-ZnT1 for repairing polluted water bodies and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Establishment of plant tissue culture system

[0075] Take wild water peanut plants, wash them with clean water for 10 minutes, and cut off the shorter stems with germs and the shorter stems with axillary buds. Sterilize with 75% ethanol for 1 min and 1% NaClO for 8 min on a sterile operating table, wash the sterilized stems with sterile water 6-7 times, remove residual water on sterile filter paper, and then place them on the callus Tissue induction medium (MS+2mg / L2,4D+1mg / L6BA+20mg / L sucrose) and shoot induction medium (1 / 2 MS+2mg / L6BA+0.1mg / LNAA +30mg / L sucrose) culture medium , until it grows into callus and subculture seedlings (such as Figure 12-13 ). The stem segment with axillary buds was asexually propagated in a sterile environment as the original material for tissue culture.

Embodiment 2

[0077] Construct targeting vector and transform into plants:

[0078] (1) Construction of the target carrier:

[0079] Retrieve the ZNT1 gene sequence (AF133267.1) in Genebank, entrust Nanjing Yidao Biotechnology Co., Ltd. to chemically synthesize the ZnT1 target gene fragment, and make the ZNT1 gene (the applicant retrieved the ZNT1 cds sequence on GenBank, and chemically synthesize the ZNT1 target gene by the biological company) fragment, and then amplified by PCR), digested with restriction endonucleases SmaI and XbaI, connected to the plasmid expression vector p3301-121 after double digestion with the same enzymes, and named p3301 -121-Znt1 plasmid (for its structure see figure 1 ), into Agrobacterium strain EHA105 by electroporation;

[0080] (2) Agrobacterium leaf disk transformation method to transfer the target gene

[0081] Receptor material selection: Cut the leaves of the sterile plants cultivated in experiment (4) into small pieces of 1cm×1cm, cut the stem lengt...

Embodiment 3

[0086] Resistance of transgenic plants to heavy metals zinc and cadmium

[0087] Reserve Zn ion solutions of 25mg / L, 75mg / L, 125mg / L, and 175mg / L, and take 5 parallel samples. Use atomic absorption spectrophotometry to detect Cd in the sample, take 5 parallel samples of transgenic plants and sterile seedling plants under the Cd concentrations of 0 mg / L, 20 mg / L, and 40 mg / L to measure, and calculate the standard for the results. difference and take the mean value, and measure the Cd in the root of the plant, the heavy metal content in the stem and leaf is as attached Figure 4 - attached Figure 7 As shown, it can be seen from the figure that with the planting of transgenic plants, the concentration of Zn and Cd in the solution decreased significantly, while the concentration of heavy metals in the plant also increased significantly with the growth of time. When the heavy metal concentration in the medium was 175 mg / L, the adsorption capacity of transgenic plants could reach...

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Abstract

The invention relates to a technology for applying transgenic plants to repair polluted water bodies. A target plasmid p3301-121-ZnT1 is obtained by chemical synthesis of the published ZnT1 gene sequence related to heavy metal tolerance, the agrobacterium containing the target plasmid is transferred to water peanuts by using a leaf disc method to increase the tolerance and accumulation of plants on Zn, Cd and Mn, and transgenic plants with high accumulation of heavy metal elements are screened and cultivated. The transgenic water peanuts can repair the wastewater polluted by metal elements such as Zn, Cd and Mn. The technology can be widely applied to the field of biological repair of heavy metal polluted soil and water bodies, and has good repair effects and broad application prospects.

Description

technical field [0001] The invention relates to the technical field of bioremediation pollution, in particular to the remediation of water bodies polluted by various heavy metals by using transgenic plants. Specifically, it is a transgenic plant plasmid p3301-121-ZnT1 for repairing polluted water bodies and its application. Background technique [0002] With the development of genetic engineering technology, the research on the molecular mechanism of metal-resistant genes is also deepening. Using genetic engineering to remediate heavy metal-contaminated soil, the existing research results include the following categories. [0003] 1. Metal binding protein gene [0004] (1) Metallothionein genes (metallothioneins, MTs) [0005] Metallothionein (MT) is a complex gene family that often exists in eukaryotic organisms. MT genes have the characteristics of good heat resistance and strong conformation. It is a class of low molecular weight polypeptides rich in cysteine ​​and co...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/02A01H6/10A01H6/50C02F3/32C02F101/20
CPCC02F3/32C02F2101/20C07K14/415C12N15/8243
Inventor 卢宏玮卢静昭王伟鹏姚天次康瑜何理
Owner INST OF GEOGRAPHICAL SCI & NATURAL RESOURCE RES CAS
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