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A HRM-based method for detecting mutations in the tobacco cadmium transport gene nthma4

A technology for transferring genes and tobacco, applied in the field of agricultural biology, to achieve the effect of improving the detection throughput

Active Publication Date: 2021-11-16
GUIZHOU TOBACCO SCI RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, there is no report on the use of HRM technology to screen mutant genes in tobacco

Method used

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  • A HRM-based method for detecting mutations in the tobacco cadmium transport gene nthma4
  • A HRM-based method for detecting mutations in the tobacco cadmium transport gene nthma4
  • A HRM-based method for detecting mutations in the tobacco cadmium transport gene nthma4

Examples

Experimental program
Comparison scheme
Effect test

Embodiment Construction

[0013] (1) Sample collection and tobacco genomic DNA extraction

[0014] Young leaves were collected at the tobacco cluster stage, and 6 plants were sampled from each line. After stacking every 3 leaves, an equal amount of leaves were obtained by using a puncher to construct a gene pool. The tobacco genomic DNA was extracted by the improved CTAB method, and finally dissolved in An appropriate amount of TE buffer was used to measure the DNA concentration of each sample with a Nanodrop 2000 ultraviolet spectrophotometer, and to detect the DNA quality by 1% agarose gel electrophoresis. Qualified DNA samples were uniformly diluted to 100ng / μl and stored in a 96-well PCR plate at -20°C.

[0015] (2) PCR amplification

[0016] Using the extracted genomic DNA as a template, PCR amplification was performed using primer pairs HZ1, HZ2, HZ3, HZ4, HZ5 and HZ6. 10 μl PCR system includes 5 μl of 2×Gene Solution mastermix (Shanghai Shuomeng Biotechnology Co., Ltd.), 0.5 μl of 20×EvaGreen ...

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Abstract

The invention discloses a method for detecting the mutation of tobacco cadmium transport gene NtHMA4 by HRM, which is characterized in that it comprises the following steps: (1) using CTAB method to extract genomic DNA of leaves of tobacco samples at seedling stage; (2) using the genomic DNA as a template , utilize primer pair to carry out PCR amplification; (3) PCR amplification product utilizes HRM detection instrument LightScanner96 to carry out mutation site scanning; (4) according to HRM curve result, judge the genotype of tobacco sample to be tested; When the HRM of sample to be tested When the curve is consistent with the wild-type control Guiyan No. 1, it is a wild-type material; if other curve types appear, and the peak value of the HRM fluorescence curve ΔF ≥ 0.05, it is a suspected gene mutation material; (5) DNA sequencing to verify the HRM screening results; (6) Cadmium phenotype determination of NtHMA4 material.

Description

technical field [0001] The invention belongs to the field of agricultural biotechnology, and in particular relates to a method for screening tobacco low-cadmium mutant materials. Background technique [0002] Cadmium (Cd) is one of the most toxic and biologically mobile heavy metal elements in soil and the environment, and cadmium pollution ranks first in soil heavy metal pollution in my country. Global approx. 2.35x10 12 square meters of arable land is polluted by heavy metals, about 2.79x10 in China 9 square meters of cadmium-contaminated farmland. In soil, even extremely low concentrations of cadmium can be absorbed and enriched by plants, and enter the human body through the food chain, thereby damaging the functions of the kidneys, lungs, cardiovascular and musculoskeletal systems, and seriously endangering human health. [0003] Tobacco is an economic crop with strong cadmium enrichment ability. And the cadmium content in cigarettes has a high correlation with the ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/156C12Q2527/107
Inventor 张吉顺任学良赵海军张超王仁刚
Owner GUIZHOU TOBACCO SCI RES INST
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