New application of tobacco Snakin2 gene

A gene, tobacco technology, applied in the new application field of the tobacco Snakin2 gene and its encoded protein, can solve problems such as affecting the accumulation of heavy metal cadmium

Active Publication Date: 2016-05-25
SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is no report that this kind of protein can participate in the detoxification of heavy metal cadmium, and the report that affects the accumulation of heavy metal cadmium in organisms

Method used

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  • New application of tobacco Snakin2 gene
  • New application of tobacco Snakin2 gene
  • New application of tobacco Snakin2 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: The expression of NtSnakin2 gene is regulated by the induction of heavy metal cadmium and other stress factors and hormones

[0045] The tobacco variety used in the present invention is Safflower Nicotiana SR1 (Nicotianatabacum L.cv.SR1), and the seeds are preserved and provided by the South China Botanical Garden of the Chinese Academy of Sciences. Tobacco seeds were germinated using 1 / 2 liquid MS medium, and after two weeks they grew into seedlings, and the tobacco seedlings were transferred to 1 / 2 MS liquid medium to continue culturing for 2 weeks. Then use CdCl containing 2 (50μM), GA (100μM), JA (90μM) and mannitol (300mM) 1 / 2MS liquid medium to process tobacco seedlings, collect stress treatment 0.5g each of tobacco young leaves and young roots after 0h, 6h and 24h, Used to extract total RNA. The extraction of RNA was carried out according to the specification of HiPurePlantRNAKits (R4151) of Magen Company. cDNA was reverse transcribed using a two-st...

Embodiment 2

[0050] Example 2: Cloning of NtSnakin2 gene and construction of yeast recombinant expression vector NtSN2-pYES260

[0051] Tobacco was taken as the seedling leaves of Nicotiana tabacum SR1 (NicotianatabacumL.cv.SR1, preserved in the South China Botanical Garden), and the RNA of the young leaves was extracted and reverse-transcribed into cDNA. Using the cDNA as a template, the primer NtSN2YEF was designed: 5'-TTTCAGGGCGCCATGGCCATTTTCAAACTCTCTTTG-3' and NtSN2YER: 5'-CGTTACTAGTGGATCTTAAGGGCATTTGCGTCTGTTG-3, (SEQ ID NO.3 and SEQ ID NO.4), using high-fidelity Taq enzyme PCR to amplify the full length of the cDNA reading frame of the NtSnakin2 gene. For the PCR system used, refer to the instruction manual of PrimeSTARHSDNAPolymerase from TaKaRa Company. The amplified DNA fragment was in accordance with the instructions of HiPureGelPureDNAKits of Magen Company. The recovered fragment was used for insertion into the yeast expression vector pYES60. Saccharomyces cerevisiae expression...

Embodiment 3

[0052] Example 3: Expression of NtSnakin2 gene in Saccharomyces cerevisiae improves tolerance of yeast to heavy metal cadmium

[0053] Saccharomyces cerevisiae strains WT and Δycf1 (purchased from Euroscarf, the European Yeast Research Center, http: / / web.uni-frankfurt.de / fb15 / mikro / euroscarf / , strain numbers Y00000 and Y04069), were cultured with pYES260 plasmid and recombinant NtSN2- The above yeasts were transformed with pYES260. Since the NtSnakin2 gene is placed under the regulation of the yeast galactose-induced promoter PGAL1 (see figure 2 shown), NtSN2-pYES260 was transformed into Saccharomyces cerevisiae and placed on the growth selection synthetic medium (Selective Growth Synthetic Medium, SDmedium) supplemented with galactose to induce heterologous overexpression of NtSnakin2 in yeast.

[0054] The method used for yeast transformation is the lithium acetate conversion method, and the specific steps are as follows:

[0055] 1) Inoculate a single colony of the yeast...

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Abstract

The invention discloses an application of a tobacco NtSnakin2 protein and/or NtSnakin2 gene in improvement of tolerance or accumulation of plants on heavy metal cadmium. By constructing a saccharomyces cerevisiae transgene over-expression vector of the NtSnakin2 gene, the NtSnakin2 gene is over expressed in yeasts, tolerance of the yeasts on the heavy metal cadmium can be improved, cadmium content in the yeasts is reduced, discharge of cadmium in the yeasts is promoted, and tolerance of the yeasts on hydrogen peroxide also can be improved as the gene is expressed in the yeasts. The NtSnakin2 gene can be applied to the fields such as engineering bacteria, genetic engineering breeding of plants aiming at change of cadmium content in the plants and microorganism and phytoremediation for environmental heavy metal cadmium pollution.

Description

technical field [0001] The invention belongs to the field of biogenetic engineering, in particular to the new application of tobacco Snakin2 gene and its coded protein. Background technique [0002] Heavy metal cadmium is a non-essential toxic element for plant growth. As environmental pollution becomes more and more serious, more and more toxic active cadmium ions are released from the earth's crust into water, atmosphere and soil. Cadmium is easily absorbed by plants in the soil and can be enriched in the food chain to harm the human body. It has become one of the heavy metals that seriously harm the ecological environment. Tobacco is a crop that is easy to accumulate cadmium (enrichment coefficient 5-10), and the absorbed cadmium is mainly distributed and accumulated in the leaves. The cadmium in tobacco leaves can enter the human body through smoke, increasing the risk of health threats. Therefore, the safety of heavy metals in tobacco has become a hot topic in tobacco...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/29C07K14/415C12N15/81C12N1/19C12R1/865
CPCC07K14/415C12N15/81C12N2800/102
Inventor 张美郭艳莫辉
Owner SOUTH CHINA BOTANICAL GARDEN CHINESE ACADEMY OF SCI
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