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Metallothionein DaMT3a and application of coding gene thereof

A metallothionein, gene technology, applied in metallothionein, application, genetic engineering, etc.

Active Publication Date: 2021-05-25
LIUPANSHUI NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, the function of metallothionein gene in ginseng has not been reported yet.

Method used

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  • Metallothionein DaMT3a and application of coding gene thereof
  • Metallothionein DaMT3a and application of coding gene thereof
  • Metallothionein DaMT3a and application of coding gene thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Example 1. Acquisition of metallothionein and its coding gene

[0028] Analyze the nucleotide sequences of metallothionein from Arabidopsis thaliana, cassava and sweet potato that have been registered in NCBI, and screen and assemble a 400bp metallothionein by searching the EST sequence database of tubers and leaves of ginseng that we have established. After gene assembly sequence (contig), design a pair of specific primers to amplify the full-length cDNA sequence including the complete reading frame.

[0029] The specific method is as follows:

[0030] Specific primers were designed as follows:

[0031] F (5' end): 5'-ACAACACAAGCCATTCAATCTAT-3';

[0032] R (3' end): 5'-TTCATTAAAATAAAAGAGAGGCAG-3'.

[0033] The leaf cDNA of Dioscorea alata L. (Dioscorea alata L.; Hainan University Danzhou Campus Dioscorea Germplasm Resource Garden, No. Da56; hereinafter referred to as Dioscorea Da56) was used as a template (obtained by reverse transcription with random primers), and ...

Embodiment 2

[0036] Example 2. Analysis of gene expression pattern of ginseng potato DaMT3a

[0037] Tissue-specific expression of DaMT3a gene in ginseng potato

[0038] Using the random reverse-transcribed cDNA of Da56 tuber, Lingyuzi, leaf, root, stem and male flower RNA as a template, the DaMT3a gene-specific primers (F: 5'-ATGGGATGGTGATTGTTGAGG-3'; R: 5'-CCCATCATTCTCAGCAGCAGT-3' ) for real-time fluorescent quantitative PCR. The total reaction system is 20 μL, including 2 μL template, 10 μL 2×SYBR Premix and 10 μmol·L -1 The upstream and downstream primers were 0.3 μL each; the amplification program was pre-denaturation at 95°C for 30s; 45 cycles at 94°C for 5s, 60°C for 20s, and 72°C for 20s. The results showed that the expression abundance of the gene varied greatly among tissues, and it was mainly expressed in stems, followed by leaves, roots and male flowers, and basically not expressed in tubers and subtilis ( figure 1 ).

[0039] The effect of low temperature on the expressio...

Embodiment 3

[0047] Example 3. Prokaryotic expression and functional verification of DaMT3a gene

[0048] Utilize the pGEX-4T-1 expression vector (the carrier is purchased from TransGen Biotech company) to construct the prokaryotic expression vector of DaMT3a gene (the expression vector in this embodiment is only an example, and the present invention can also adopt other expression plasmids and virus vectors, etc.), while Escherichia coli expression strain E.coli BL21(DE3) (competent state purchased from Tiangen Biochemical Technology Co., Ltd.) was used to induce the recombinant protein, and the effect of the recombinant protein on the growth of the BL21 strain was determined. The specific method is as follows:

[0049] Acquisition of recombinant vector containing DaMT3a gene coding region

[0050] Design primers for DaMT3a gene coding region

[0051] F:5'-CGC GAATTC (EcoR I restriction site) ATGTCTAGCTGTGGCAACTGTG-3',

[0052] R:5'-CCC CTCGAG (Xho I restriction site) TCACTTGCCGCAGT...

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Abstract

The invention belongs to the technical field of biology, and discloses metallothionein DaMT3a and an application of a coding gene of the metallothionein DaMT3a. The full-length cDNA of the Dioscorea alata L. metallothionein DaMT3a gene is cloned for the first time, and gene expression analysis shows that the gene has specific expression in leaves, up-regulated expression under the induction of ethephon and abscisic acid, up-regulated expression under low temperature and high stress and down-regulated expression under mechanical damage stress, so that the gene is possibly closely related to stress resistance of potatoes, and the gene can be used as an important target gene for transgenic breeding of Dioscorea alata L.. Further, the gene is transferred into a prokaryotic expression strain, and the growth of the transgenic strain under normal conditions, heavy metal ions (Cd<2+>), NaCl stress and hydrogen peroxide stress is obviously improved, so that the gene can be used as an important gene resource and can be applied to stress-resistant and heavy metal stress-resistant gene engineering of other plants or microorganisms except for the ginseng and the sweet potatoes.

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to the application of metallothionein DaMT3a and its coding gene. Background technique [0002] Metallothioneins (MTs) are a class of low-molecular (6-7kDa) polypeptides rich in cysteine ​​(Cys) and capable of binding heavy metals. They are ubiquitous in microorganisms, higher animals, plants and humans. According to the distribution and arrangement of metallothionein gene Cys residues in plants that have been cloned so far, they can be divided into four types: Type1, Type2, Type3 and Type4. The four types of metallothionein genes have the characteristics of tissue-specific expression, among which Type1 is mainly expressed in roots, Type2 is mainly expressed in leaves, Type3 is mainly expressed in mature fruits and leaves, and Type4 is mainly expressed in seeds. The functional research of the sulfur protein gene shows that its functions are mainly reflected in: (1) it can bind he...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/70C12N1/21C12N15/11C07K14/825C12R1/19
CPCC07K14/825C12N15/70Y02A50/30
Inventor 黄亚成刘林娅苏龙兴黄东益王才全曾林
Owner LIUPANSHUI NORMAL UNIV
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