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Gene engineering application for elsholtziasplendens metallothionin gene EhMT1

A metallothionein and genetic engineering technology, applied in the field of metal binding proteins in response to heavy metal stress in plants, to achieve the effect of improving tolerance

Inactive Publication Date: 2008-01-02
NANJING AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

So far, there is no functional description of the metallothionein gene in C. haizhouensis

Method used

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  • Gene engineering application for elsholtziasplendens metallothionin gene EhMT1

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] Seeds of Cystis haizhou were collected from the copper mining area. When the seedlings of Pansia haizhou grew to the 7-8 leaf stage, they were treated with 50 μM CuSO 4 After 48 hours of treatment, the leaves were taken immediately and placed in liquid nitrogen for cryopreservation. Part of the leaves were taken, crushed with a mortar, added to a 1.5ml centrifuge tube filled with lysate (purchased from Beijing Tiangen Company), shaken fully, and then transferred into a glass homogenizer. After homogenization, transfer to a 1.5ml centrifuge tube to extract total RNA. The quality of total RNA was identified by formaldehyde denaturing gel electrophoresis, and then the RNA content was determined on a spectrophotometer. The three fragments amplified by RT-PCR and RACE were spliced ​​to obtain the full-length sequence, and primers at both ends were designed according to the sequence

[0029] Primer P1: 5′-ACGTCGATCACTAAGAAAAG-3′

[0030] Primer P2: 5′-CTTAGACGCATAAACACGAA-...

Embodiment 2

[0035] The primers at both ends of EhMT1 designed in Example 1 were used to conduct semi-quantitative RT-PCR to analyze the expression of the aboveground and underground parts of the A. haizhou seedlings, and the expression of the 18SrRNA gene (Sancenon et al., 2003) was used as the internal reference. The results showed that the expression of EhMT1 in the seedlings of A. haizhouensis in 50μM CuSO 4 It reaches a maximum at 48 hours under treatment.

Embodiment 3

[0037] According to the full-length sequence of EhMT1 obtained in Example 1, primers P3 and P4 for amplifying the complete coding frame were designed, and the sequences were as follows:

[0038] Primer P3: 5'-G TCTAGA AAGAAAATGTCGAGTGGA-3′

[0039] Primer P4: 5′- CCCGGG AGGCAGTATATCTGACAG-3′

[0040] Restriction endonuclease sites were introduced in order to construct expression vectors. Using the amplified product obtained in Example 1 as a template, after PCR amplification, the cDNA of EhMT1 was cloned into the intermediate vector pGEM-T, and further cloned into the binary expression vector pBI121, and the expression was identified under the premise of ensuring the correct reading frame. vector, which is then transformed into Agrobacterium, and then into tobacco. After the obtained transgenic plants are verified by PCR and RT-PCR, the heavy metal resistance evaluation of the plants is carried out. T of the transgenic plant 0 Generation and control plants were treated wi...

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Abstract

The invention discloses a gene engineering applying method of elsholtzia metal sulphur protein gene EhMT1 in Haizhou, wherein the GenBank entry number of cDNA sequence of EhMT1 is DQ059081 and the coded amino acid sequence is AAY57923; EhMT1 participates the accumulation of Haizhou elsholtzia root; the analysis of mRNA expression is that the Haizhou elsholtzia is induced by excessive copper; EhMT1 can be goal gene leading-in plant, which improves the copper resistance of plant and the accumulation of copper.

Description

technical field [0001] The invention relates to the genetic engineering application of the metallothionein gene EhMT1 of A. haizhouensis, which belongs to the field of genetic engineering, and specifically relates to a metal-binding protein for plant heavy metal stress response. technical background [0002] Metallothionein is a low-molecular-weight, cysteine-rich protein with metal-binding ability ubiquitous in the biological world (Cobbett et al., 2003). At present, more than 50 metallothionein genes have been cloned from at least 38 plant species. The research results show that there are some organic compounds in the cytoplasm, which can chelate with heavy metal ions, reduce the free ion activity of heavy metals in the cytoplasm, and thus protect the cell protoplasm (Clemens, 2001; Hall, 2002), and metallothionein is one of them. Peptides that reduce the activity of free ions. Metallothionein is also involved in abiotic stress responses such as oxidative stress, high sa...

Claims

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Application Information

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IPC IPC(8): C12N15/29C07K14/415C12N15/82
Inventor 沈振国夏妍王桂萍黄骥鲍永美
Owner NANJING AGRICULTURAL UNIVERSITY
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