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Application of AtPrx64 gene in improving aluminum tolerance of plants

A 1. atprx64, tolerance technology, applied in the fields of application, plant products, genetic engineering, etc., to achieve the effect of improving tolerance and easy promotion of planting

Active Publication Date: 2016-08-17
KUNMING UNIV OF SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the role of the AtPrx64 gene in the process of aluminum stress has not been reported yet. We transferred the gene into tobacco, tested the ability of the transgenic AtPrx64 gene tobacco to resist aluminum, and discussed its possible mechanism

Method used

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  • Application of AtPrx64 gene in improving aluminum tolerance of plants
  • Application of AtPrx64 gene in improving aluminum tolerance of plants
  • Application of AtPrx64 gene in improving aluminum tolerance of plants

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1: Obtaining of AtPrx64 gene cDNA fragment

[0027] Arabidopsis total RNA was extracted with TRIzol Reagent according to the instructions. The specific operation steps are as follows: take about 0.1 g of young plant leaves and put them into a mortar with liquid nitrogen, and grind them into powder; add 1 mL of TRIzoL reagent in the mortar and continue to grind them into a solution, and put them into 2 mL centrifuge tube; then add 200 µL chloroform, shake for 15 s to mix well, centrifuge at 12000 rpm at 4°C for 15 min; transfer the supernatant to a new 2 mL EP tube, add 500 µL isopropanol, mix well, store at -20°C Place for 10 min; centrifuge at 4°C, 12,000 rpm for 10 min; discard the supernatant, and wash the precipitate with 0.8-1 mL of 75% ethanol; centrifuge at 4°C, 12,000 rpm for 1 min, discard 75% ethanol; repeat washing once to completely remove the salt Impurities: Vacuum-dried the precipitate or air-dried, treated with 20 µL of diethylpyrocarbonate (...

Embodiment 2

[0030] Example 2: Construction and Transformation of AtPrx64 Gene Plant Expression Vector pK-35S-AtPrx64

[0031] The cDNA fragment of the AtPrx64 gene was connected to the pMD-18T vector, transformed into DH5α by heat shock, and a single colony with resistance was obtained by screening with ampicillin. After the PCR and double-enzyme digestion detection were correct, the sequence was performed, and the sequencing work was entrusted to BGI. The single colonies with correct sequencing were expanded and cultured to extract the plasmid, and after double digestion, they were connected to the entry cloning vector pENTR-2B, transformed into DH5α by heat shock, and screened with kanamycin (Km) to obtain resistant single colonies, double digestion After detection and sequencing, the culture was expanded, and the plasmid was extracted to obtain the entry cloning vector pENTR-2B-AtPrx64. Under the action of LR Mix Enzyme, the destination vector pK2GW7 and the entry cloning vector pENTR-...

Embodiment 3

[0033] Example 3: Detection of genome, mRNA level, protein expression level and POD activity of transgenic plants

[0034] (1) Detection of the integration of AtPrx64 gene in the tobacco genome: Genomic DNA was extracted from tobacco leaves by CTAB method as a template for PCR amplification to detect the integration of AtPrx64 in the transgenic tobacco genome. Take 0.1 g of tobacco leaves and freeze them quickly in liquid nitrogen, quickly grind them into powder in a mortar, and add 900 µL of 2×CTAB buffer (20 mM EDTA, 2% CTAB and 1.4 M NaCl) preheated to 65 °C. ), after grinding and mixing, place in a water bath at 65 °C for 15 min, add 500 µL of chloroform-isoamyl alcohol (24:1) mixture after cooling, shake up and down, and centrifuge at room temperature. Take the supernatant into EP tubes, add an equal volume of isopropanol and 1 / 10 sodium acetate, mix well, place at -20 °C for 20 min, and centrifuge at 4 °C for 20 min. After discarding the supernatant, freshen it twice wi...

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Abstract

The invention discloses application of an AtPrx64 gene in improving aluminum tolerance of plants. According to the application, the AtPrx64 gene is recombined into a plant expression vector, wild tobacco is transformed, and transgenic AtPrx64 tobacco can be screened; the experimental result shows that under the condition of aluminum stress, the relative growth rate and the soluble protein content of roots of the transgenic tobacco are gradually reduced along with increase of aluminum concentration, and the reduction rate of the transgenic tobacco is lower than that of wild tobacco; the contents of H2O2 and MDA of the transgenic tobacco are gradually increased along with increase of the aluminum concentration, and the increase rate of the transgenic tobacco is lower than that of the wild tobacco; no matter whether aluminum stress acts or not, the activity of plasmalemmas H<+>-ATPase and the secretion of citric acid of the transgenic tobacco are both higher than those of the wild tobacco; the test on the aluminum content in the roots under aluminum stress of different concentrations shows that the aluminum content in the roots of the transgenic tobacco is remarkably lower than that of the wild tobacco; the tolerance of the transgenic AtPrx64 tobacco to aluminum stress can be remarkably improved.

Description

technical field [0001] The invention belongs to the field of plant genetic engineering, and in particular relates to the application of AtPrx64 gene in improving plant aluminum tolerance. Background technique [0002] Various metabolic reactions in different tissues and organs of plants will produce reactive oxygen species (reactive oxygen species, ROS). Under normal growth conditions, the ROS content in plants is low and will not cause damage to plants, but under environmental stress such as soil acidification or salinization, drought, low temperature or insufficient light, and pathogen infection and damage, plants will There will be many changes in morphology, physiology, biochemistry and other aspects, which will lead to intracellular metabolic disorder and produce a large amount of active oxygen. These ROS have two effects on plants. On the one hand, reactive oxygen species have a strong toxic effect on plant cells, which can trigger the oxidation of plant cell componen...

Claims

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Application Information

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IPC IPC(8): C12N9/08A01H5/00
CPCC12N9/0065C12N15/8271
Inventor 李昆志吴远双杨志丽陈丽梅徐慧妮
Owner KUNMING UNIV OF SCI & TECH
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