Hyperaccumulator solanum nigrum metallothionein gene (MT-L3) sequence and cloning method thereof

A technique for metallothionein and hyperaccumulation plants, which is applied in the field of hyperaccumulation plant nightshade metallothionein gene (MT-L3) sequence and its cloning, and can solve the problem that the mechanism is not very clear.

Inactive Publication Date: 2014-07-23
SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The expression of plant MT-L gene is tissue-organ-specific and developmental-stage-specific. At present, the most studied is the effect of metal ions on the expression of plant MT-L gene. Studies have reported the relationship between plant MT-L gene and the tolerance of heavy metal Cd. There is a certain correlation, but the mechanism of metal ions affecting the expression of MT-L gene is still not very clear, especially what role MT-L gene plays in heavy metal hyperaccumulation plants is currently a research hotspot in this field

Method used

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  • Hyperaccumulator solanum nigrum metallothionein gene (MT-L3) sequence and cloning method thereof
  • Hyperaccumulator solanum nigrum metallothionein gene (MT-L3) sequence and cloning method thereof
  • Hyperaccumulator solanum nigrum metallothionein gene (MT-L3) sequence and cloning method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Solanum nigrum MT-L3 gene is shown in SEQ ID NO.1:

[0019] atgacggaca agtgtagcaa ttgcgattgc gctgatgtca gccaatgcgt gaggaaggagagccaatatg atgtcgtcat cgtcgaaaag agctacatcg agacggtggt gatgatggacgttggagcgg aagaacatga tggaaaatgc aaatgcggca gcagctgcgc ctgtgttaactgcacttagtg ctcatta

[0020] (a) Sequence features:

[0021] ●Length: 200bp 1-198 nucleotides

[0022] ●Type: Nucleotide sequence

[0023] ●Chain type: single chain

[0024] ●Topology: Linear

[0025] (b) Molecular type: double-stranded DNA

[0026] (c) Assumption: No

[0027] (d) Antonym: No

[0028] (e) Original source: Solanum nigrum

Embodiment 2

[0029] The cloning method of embodiment 2 Solanum nigrum MT-L3 gene

[0030] The method for cloning the above-mentioned MT-L3 gene from Solanum nigrum is:

[0031] (1) Extracting total RNA from the leaves of the fresh hyperaccumulated plant Solanum nigrum;

[0032] (2) Using total RNA as a template, the first-strand cDNA was synthesized using reverse transcriptase AMV and random primers (Random Primers, Invitrogen, USA);

[0033] (3) Using the synthesized first-strand cDNA as a template, use degenerate primers MT-L3-F and MT-L3-R for PCR amplification, and the amplified fragment size is about 200bp (see figure 1 );

[0034] (4) After the PCR product was cloned into the pMD-18T vector, the positive clones were taken and sequenced to obtain the gene sequence of the hyper-enriched plant Solanum nigrum type 3 metallothionein protein.

[0035] Wherein the primers include:

[0036] MT-L3-F: 5'-ATGTCGGACAAGTGYRGYARTTG-3'

[0037] MT-L3-R: 5'-TTAATGAGCACAAGTGCAGTTAAC-3'

[0038]...

Embodiment 3

[0075] Example 3 Expression of hyper-enriched plant Solanum nigrum type 3 metallothionein under different concentrations of Cd stress

[0076] The Solanum nigrum seedlings shown in the above-mentioned sequence table SEQ ID NO.1 are planted in the soil with different concentrations of Cd (2.5mg / kg, 5mg / kg and 40mg / kg) and no Cd added to the maturity stage, take different The total RNA of Solanum nigrum leaves of the treatment group (2.5mg / kg, 5mg / kg and 40mg / kg) and the control group (no Cd treatment), the total RNA extraction method refers to the above example, using a fluorescence quantitative PCR instrument (ABIStep-One Plus Real Time PCR System, USA) for differential expression analysis, quantitative PCR primers were MT-L3-F and MT-L3-R. The results showed that Cd stress treatment could significantly induce the expression of MT-L3, which was 2.4 times, 3.7 times, and 31.3 times that of the control, respectively (see figure 2 ), which is closely related to the ability of S...

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Abstract

The invention belongs to the field of gene clone in molecular biology, and specifically relates to a hyperaccumulator solanum nigrum metallothionein gene (MT-L3) sequence and a cloning method thereof. The hyperaccumulator solanum nigrum metallothionein gene sequence is type 3 metallothionein gene, and the gene sequence is shown by a nucleotide sequence in SEQ ID NO. 1. The successive cloning and sequencing of the hyperaccumulator solanum nigrum type 3 metallothionein gene helps to establish a base for further analyzing the biological stress resistant capability under the influence of MT-L3, and screening and applying other functions of MT-L3.

Description

technical field [0001] The invention belongs to the field of gene cloning in molecular biology, in particular to a super-enriched plant nightshade metallothionein gene (MT-L3) sequence and a cloning method thereof. Background technique [0002] Metallothioneins (MTs) are a class of low molecular weight, high Cys content and metal-binding polypeptides, which were first discovered in cadmium-accumulating horse kidney tissue. Known as plant metallothionein (Metallothionein Like, MT-L). It was first discovered from soybean roots by Casterline and Barnett in 1977. According to the position and arrangement of Cys, plant metallothionein proteins are divided into three categories. MT-L1 has 6 Cys-X-Cys units, each of which contains 3 C-terminal and N-terminal domains. Between the two domains is a Cys-free spacer composed of about 40 amino acid residues. A large number of amino acid residues is a common feature of most plant MTs; MT-L2 is similar to MT-L1, but the first and second...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C12N15/10C07K14/825
Inventor 张倩茹魏树和焦洪静
Owner SHENYANG INST OF APPLIED ECOLOGY - CHINESE ACAD OF SCI
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