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Protein with cadmium combination performance, and encoding gene and applications thereof

A coding gene, coding technology, applied in application, genetic engineering, plant genetic improvement, etc.

Inactive Publication Date: 2017-03-29
INST OF BOTANY CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no report on site-directed mutation of CusF gene to promote high cadmium accumulation and cadmium resistance in transgenic plants

Method used

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  • Protein with cadmium combination performance, and encoding gene and applications thereof
  • Protein with cadmium combination performance, and encoding gene and applications thereof
  • Protein with cadmium combination performance, and encoding gene and applications thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0074] Example 1. Acquisition and Functional Research of Cell Wall-localized mCusF Transgenic Plants

[0075] 1. Cloning of CusF gene

[0076] (1) Primers CusF-F and CusF-R were designed according to the sequence of the CusF gene (SEQ ID NO: 9), and the mature sequence of CusF was amplified using the genome of Escherichia coli DH5α as a template. The reaction conditions are: 94°C, 5min; 94°C, 30s; 58°C, 30s; 72°C, 25s; 72°C, 5min; the number of cycles is 30 cycles.

[0077] CusF-F: 5'-5'- GGATCC GCTAACGAACATCATCATGAAACCA-3' (the underlined part is the recognition sequence of BamHI, and the sequence thereafter is the 1st-25th position of sequence 9 in the sequence listing);

[0078] CusF-R: 5'- CTGCAG TTACTGGCTGACTTTAATATCCTGT-3' (the underlined part is the recognition sequence of Pst I, and the following sequence is the reverse complementary sequence of the 246-270th position of sequence 9 in the sequence listing).

[0079] (2) PCR amplified products were subjected to 1%...

Embodiment 2

[0185]Example 2, Acquisition and Functional Research of Cytoplasmic Localized mCusF Transgenic Plants

[0186] 1. Cloning of mCusF gene

[0187] Design primers mCusF-F and mCusF-R2, the specific sequences are as follows:

[0188] mCusF-F: 5'- GGATCC GCTAACGAACATCATCATGAAACCA-3' (the underlined part is the recognition sequence of BamH I, and the sequence thereafter is the 724-748th position of sequence 7);

[0189] mCusF-R2: 5'- CTGCAG TTACTGGCTGACTTTAATATCCTGT-3' (the underlined part is the recognition sequence of Pst I, and the following sequence is the reverse complementary sequence of No. 969-993 of Sequence 7).

[0190] Using the mCusF mature protein coding gene (position 64-333 of Sequence 5) obtained in Example 1 as a template, PCR amplification was performed using primers mCusF-F and mCusF-R2. The reaction conditions of PCR were consistent with those of the first round of PCR reaction in Example 1.

[0191] PCR amplified products were subjected to 1% agarose gel ...

Embodiment 3

[0232] Example 3, Acquisition and functional research of vacuolar-localized mCusF transgenic plants

[0233] 1. Vacuole-localized recombinant vector mCusF vac - Construction of GFP

[0234] Vacuole-localized mCusF recombinant expression vector mCusF vac - The construction of GFP is a recombinant expression vector mCusF located on the cell wall cw -GFP was performed on the basis of. details as follows:

[0235] ① To introduce the vacuolar sorting sequence AFVY at the C-terminus of GFP, design primers GFP-F and V-GFP-R.

[0236] GFP-F: 5'-GA GGTACC ATGGTGAGCAAGGGCGAGGAGCT-3' (the underlined part is the recognition sequence of Kpn I, and the sequence thereafter is the 346-368th position of sequence 8);

[0237] V-GFP-R:

[0238] 5'-GC GAGCTC TCAGTACACAAAAGGCCTTGTACAGCTCGTCCATGC-3' (the underlined part is the recognition sequence of Sac I, and the following sequence is the reverse complementary sequence of the 1043-1077th position of sequence 8).

[0239] mCusF cw The ...

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Abstract

The invention discloses a protein with cadmium combination performance, and the encoding gene and applications thereof. The protein is one randomly selected from following proteins: I, a protein with a sequence represented by the 22th to the 110th sits of sequence 1 or sequence 2 in a sequence table; II, a protein with a sequence represented by the 1th to the 113th sits of sequence 1 or sequence 2 in the sequence table; III, a protein with a sequence represented by sequence 3 in the sequence table; and IV, a protein with a sequence represented by sequence 4 in the sequence table, or a protein with a sequence represented by sequence 4 after base deletion of the 116th to the 354th sites. The encoding gene of the protein is transferred arabidopsis thaliana via genetic engineering technology, so that accumulation ability of the overground part of arabidopsis thaliana on heavy metal Cd is increased obviously, and Cd tolerance of obtained transgenic plants is increased obviously. The above gene transferring method is high in practicability, can be used for obtaining plants which possess heavy metal tolerance and heavy metal ion accumulation ability, and can be planted in heavy metal polluted soil, and obtaining novel plant varieties used for soil restoration.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and relates to a protein with cadmium binding performance, its coding gene and its application, especially the application in cultivating transgenic plants with high cadmium accumulation and cadmium resistance. Background technique [0002] With the rapid development of modern industry, the increase in the types and quantities of agricultural chemicals and the aggravation of urban pollution, heavy metal pollution has become increasingly serious. Unlike other organic pollution, heavy metal pollution is an irreversible process, and pollutants will not be degraded by plants and microorganisms over time. Heavy metal pollution in soil, water and atmosphere not only seriously endangers the natural environment, but also poses a huge threat to human health. Heavy metals can enter the human body through the food chain and affect the normal metabolism of the human body. The treatment of heavy met...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/415C12N15/29C12N15/82A01H5/00
CPCC07K14/415C12N15/8205C12N15/8271
Inventor 张海燕余朋丽袁金红
Owner INST OF BOTANY CHINESE ACAD OF SCI
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