Application of metallothionein DaMT2a and coding gene thereof

A technology of metallothionein and gene, applied in the direction of metallothionein, application, genetic engineering, etc., to achieve the effect of promoting growth and development

Active Publication Date: 2021-03-09
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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  • Application of metallothionein DaMT2a and coding gene thereof
  • Application of metallothionein DaMT2a and coding gene thereof
  • Application of metallothionein DaMT2a and 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 600bp metallothionein by searching our established tuber and leaf EST sequence database 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'-GATTTGTGATTAGGGTTGAGAG-3';

[0032] R (3' end): 5'-AAACAGCAAAGAAAGGCCTA-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 F and R were used as primers. The f...

Embodiment 2

[0036] Example 2. Analysis of the expression pattern of ginseng potato DaMT2a gene

[0037] Tissue-specific expression of DaMT2a 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 DaMT2a gene-specific primers (F: 5'-TAGTGTTTGTGTGTTGTGTTTGTGTT-3'; R: 5'-CCAAGGATAAGACCAGAGAGAGG-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 level of this gene in male flower and stem was higher than that in other 4 tissues ( figure 1 ).

[0039] The effect of low temperature on the expression of DaMT2a gene

[0040] Select ginseng Da56 tissue-cultured seedlings with 6-8 fully exp...

Embodiment 3

[0047] Example 3. Prokaryotic expression and functional verification of DaMT2a gene

[0048] The prokaryotic expression vector of DaMT2a gene was constructed by using the pGEX-4T-1 expression vector (the vector was purchased from TransGen Biotech Company), and the E. coli expression strain E.coli BL21(DE3) was used (the competent state was purchased from Tiangen Biochemical Technology Co., Ltd.) Induce the recombinant protein and measure the effect of the recombinant protein on the growth of the BL21 strain, the specific method is as follows:

[0049] Obtaining the recombinant vector containing DaMT2a gene coding region

[0050] Design primers for DaMT2a gene coding region

[0051] F:5'-CGC GAATTC (EcoRI restriction site) ATGTCTTGCTGCGGAGGA-3',

[0052] R:5'-CCC CTCGAG (XhoI restriction site) TCATTTGCAGTTGCAGGGAT-3',

[0053] Using pMD18-DaMT2a as a template, PCR amplification was carried out. The amplification program was: pre-denaturation at 95°C for 4 minutes; denat...

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Abstract

The invention belongs to the technical field of biology, and discloses application of metallothionein DaMT2a and a coding gene thereof. Full-length cDNA of the metallothionein DaMT2a gene of Dioscoreaalata L. is cloned for the first time; gene expression analysis shows that the DaMT2a gene is highly expressed in male flowers and stems, expression is up-regulated under the induction of ethephon and abscisic acid, meanwhile expression of the gene is up-regulated under the induction of low temperature and mechanical injury stress, and expression is down-regulated under high temperature stress, so that the gene is possibly closely related to stress resistance of the Dioscorea alata L., and the gene can be used as an important target gene for transgenic breeding of the Dioscorea alata L.. Thegene is further transferred into a prokaryotic expression strain, and the growth of a transgenic strain under normal conditions, heavy metal ions (Cu<2+> and Cd<2+>), NaCl stress and hydrogen peroxidestress is obviously improved, so that the gene can be used as an important gene resource, and the gene can be applied to stress resistance and heavy metal stress resistance gene engineering of otherplants or microorganisms except for the Dioscorea alata L..

Description

technical field [0001] The invention belongs to the field of biology, and in particular relates to the application of metallothionein DaMT2a 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): C12N15/29C07K14/825C12N15/70C12N1/21C12N15/11C12R1/19
CPCC07K14/825C12N15/70Y02A50/30
Inventor 黄亚成刘林娅李淳妤黄东益袁小素彭欣
Owner LIUPANSHUI NORMAL UNIV
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