Use of gene MRS6 in improvement of tolerance of tobacco to heavy metal cadmium

A heavy metal and gene technology, applied in application, genetic engineering, recombinant DNA technology, etc., can solve problems such as environmental hazards and obvious risks

Active Publication Date: 2018-09-04
DONGHUA UNIV +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, with the improvement of environmental protection requirements, traditional physical and chemical cadmium pollution control methods are subject to many restrictions, and the harm and risk of these methods to the environment are becoming more and more obvious

Method used

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  • Use of gene MRS6 in improvement of tolerance of tobacco to heavy metal cadmium
  • Use of gene MRS6 in improvement of tolerance of tobacco to heavy metal cadmium
  • Use of gene MRS6 in improvement of tolerance of tobacco to heavy metal cadmium

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0045] Genetic transformation of tobacco and PCR identification of transgenic tobacco.

[0046] (1) PCR amplification of MRS6 gene

[0047] Primers were designed according to the yeast genome database information, with Saccharomyces cerevisiae S288C (PLoS Genet, 2012, 8(11): e1003083) and MRS6-GFP strains (according to Nature, 2003 .425：686-691. Methods The genome was constructed on the basis of S288C as a template, and the PCR amplification of MRS6 and MRS6-GFP genes was carried out. The PCR reaction system (25 μL) was: EmeraldAmp MAX PCR Master Mix (2×Premix) 12.5 μL, and the primers (concentration: 1OD ) 1 μL, lower primer (concentration is 1OD) 1 μL, template DNA (concentration is 233ng / μL) 1 μL, ddH 2 O9.5 μL. The PCR amplification program was as follows: pre-denaturation at 95°C for 3 min, denaturation at 95°C for 30 s, annealing at 55°C for 30 s, extension at 72°C for 100 s, 32 cycles, final extension at 72°C for 10 min, and hold at 4°C. A PCR amplification product i...

Embodiment 2

[0070] Effects of MRS6 and MRS6-GFP overexpression on plant growth.

[0071] (1) Sterilize the wild type and the transgenic Sansheng Tobacco seeds obtained in Example 1, the specific steps are:

[0072] i. put the tobacco seeds into the EP tube under aseptic conditions and rinse 3 times with sterile water;

[0073] ii. Soak in 70% ethanol solution for 1min;

[0074] iii. Then soak in 1% sodium hypochlorite solution for 10 minutes, and then rinse the seeds repeatedly with sterile water for 4 to 5 times;

[0075] iv. Finally, place the rinsed seeds on sterile filter paper to absorb excess water.

[0076] (2) Sow 40 sterilized tobacco seeds on MS solid medium, culture in an artificial climate incubator, and culture in dark for 3 days. The temperature was 28°C, and light and dark (16h / 8h) were alternately cultivated for 45-50 days, during which the plant growth was observed and related data were recorded.

[0077] (3) After the tobacco plants were cultivated for about 5 days, ...

Embodiment 3

[0082] Effects of overexpression of MRS6 and MRS6-GFP on cadmium tolerance and cadmium accumulation in plants.

[0083] (1) When the tobacco grows to the 10-leaf stage, the tobacco plants with the same growth potential and the same number are divided into 3 groups and added with different concentrations of CdCl 2 into MS medium for processing. Prepare CdCl with concentrations of 0, 250, and 500 μg / mL 2 Mother liquor, add 3mL CdCl to each group 2 , CdCl in the final medium 2 The actual concentration is 0mg / kg, 15mg / kg and 30mg / kg. After 12 days of treatment, the growth of MRS6 and MRS6-GFP transgenic tobacco plants was significantly better than that of wild-type tobacco plants. Tobacco leaves are divided into four stages from green to yellow, cyan, light yellow, yellow, dark yellow ( Image 6 A). With CdCl 2 With the increase of treatment concentration, the number of mild yellowing of transgenic tobacco leaves is less, the color is lighter (light yellow), and the plants ...

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Abstract

The invention discloses a high cadmium-tolerant tobacco modified by a saccharomyces cerevisiae S288C vesicle transport gene MRS6 and its application. The nucleotide sequence of the gene MRS6 is shownin the formula of SEQ ID NO: 1. The amino acid sequence of the encoded protein is shown in the formula of SEQ ID NO: 2. In the present invention, it is found by the green fluorescent protein trace observation that the MRS6-encoded protein is mainly expressed in the ductal cells of the rhizomes of the transgenic plants. Through the treatment with CdCl2, compared with the wild type tobacco, the cadmium content enriched in the rhizome of the transgenic tobacco is significantly increased, the cadmium content in the leaves is significantly reduced, and the plant grows more rapidly. It is proved that the overexpression of MRS6 can increase the tolerance of tobacco to heavy metal cadmium, enhance the enrichment ability of cadmium, reduce the content of cadmium in leaves, and increase the growth rate of tobacco. Therefore, the tobacco improved by the gene MRS6 has high cadmium-tolerant activity, has broad application prospects in natural environment improvement based on environmental cadmium enriching, and has the great economic benefit potential.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering and relates to an application of MRS6 protein in improving tolerance of tobacco to heavy metal cadmium. Background technique [0002] The "National Soil Pollution Survey Bulletin" jointly issued by the Ministry of Environmental Protection and the Ministry of Land and Resources on April 17, 2014 pointed out that the total excess rate of soil in the country was 16.1%, indicating that we are facing a very serious problem of arable land soil environmental safety. Inorganic pollutants exceeding the standard number accounted for 82.8% of all exceeding the standard points, indicating that it is mainly heavy metal pollution. Heavy metals usually refer to metals with a density above 5.0, such as lead, chromium, copper, cadmium, mercury, etc. Heavy metals, especially the "five poisons" represented by cadmium, chromium, mercury, arsenic, and lead, are heavy metals. Once excessively absorbe...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/82A01H5/00A01H6/82
CPCC12N15/8205C12N15/8271
Inventor 黄志伟沈裕虎程迅王桥刘宝龙王寒冬余圆圆
Owner DONGHUA UNIV
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