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Method for detecting tobacco cadmium transport gene NtHMA4 mutation by HRM

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: 2018-07-06
GUIZHOU TOBACCO SCI RES INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

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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  • Method for detecting tobacco cadmium transport gene NtHMA4 mutation by HRM
  • Method for detecting tobacco cadmium transport gene NtHMA4 mutation by HRM
  • Method for detecting tobacco cadmium transport gene NtHMA4 mutation by HRM

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Experimental program
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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 Nanodrop2000 ultraviolet spectrophotometer, and the DNA quality was detected 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×GeneSolution mastermix (Shanghai Shuomeng Biotechnology Co., Ltd.), 0.5 μl of 20×EvaGreen...

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Abstract

The invention discloses a method for detecting tobacco cadmium transporter gene NtHMA4 mutation by HRM. The method comprises the following steps: (1) extracting genomic DNA of seedling-stage leaves ofa tobacco sample by a CTAB method; (2) using the genomic DNA as a template, and using a primer pair for PCR amplification; (3) scanning mutation sites of a PCR amplification product by HRM detectioninstrument LightScanner96; (4) judging the genotype of the tobacco sample to be tested based on HRM curve results; when the HRM curve of the sample to be tested is consistent with that of wild-type control Guiyan No. 1, determining the sample to be tested is a wild-type material; if the HRM curve of the sample to be tested is other curve type, and HRM fluorescence curve peak delta F is greater than or equal to 0.05, the sample to be tested is a suspected gene mutation material; (5) verifying HRM screening results by DNA sequencing; and (6) determining cadmium phenotypes of NtHMA4 materials.

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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IPC IPC(8): C12Q1/6895C12Q1/686C12N15/11
CPCC12Q1/686C12Q1/6895C12Q2600/156C12Q2527/107
Inventor 赵海军任学良张吉顺张超王仁刚
Owner GUIZHOU TOBACCO SCI RES INST