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Method for extracting cotton mitochondria and ribose nucleic acid (RNA) of cotton mitochondria

A mitochondria and cotton technology, applied in the field of plant biology, can solve the problems of cotton mitochondrial DNA and mitochondrial RNA extraction difficulties and other problems

Active Publication Date: 2013-02-27
CHINA AGRI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, compared with some other crops, the extraction of cotton mtRNA and mtRNA is more difficult, and there is no report on the extraction method of cotton mtRNA

Method used

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  • Method for extracting cotton mitochondria and ribose nucleic acid (RNA) of cotton mitochondria
  • Method for extracting cotton mitochondria and ribose nucleic acid (RNA) of cotton mitochondria
  • Method for extracting cotton mitochondria and ribose nucleic acid (RNA) of cotton mitochondria

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0067] Embodiment 1, the extraction of cotton mitochondrial RNA

[0068] 1. Solution preparation

[0069] Solution A: NaCl, Tris-HCl, EDTA-Na 2 Mix with DEPC water, sterilize at 121°C for 40 minutes, add PVP-40 and BSA after cooling, and add β-mercaptoethanol before use, which is solution A; in solution A, the concentration of NaCl is 1.2mol / L, and the concentration of Tris-HCl The concentration is 0.05mol / L, EDTA-Na 2 The concentration of PVP-40 is 2mmol / L, the concentration of PVP-40 is 2.5% (volume percentage), the concentration of BSA is 0.1% (volume percentage), and the concentration of β-mercaptoethanol is 5mmol / L.

[0070] Solution B: Tris-HCl, EDTA-Na 2 , CTAB, NaCl, spermidine, PVP-40 and DEPC water were mixed, sterilized at 121°C for 40 minutes after 2 days at room temperature, and β-mercaptoethanol was added before use, which was solution B; in solution B, the concentration of Tris-HCl was 100mmol / L, EDTA-Na 2 The concentration of CTAB is 25mmol / L, the concent...

Embodiment 2

[0114] Embodiment 2, the extraction of cotton mitochondrial RNA

[0115] 1. Solution preparation

[0116] Solution A: NaCl, Tris-HCl, EDTA-Na 2 Mix with DEPC water, sterilize at 121°C for 40 minutes, add PVP-40 and BSA after cooling, and add β-mercaptoethanol before use, which is solution A; in solution A, the concentration of NaCl is 0.5mol / L, and the concentration of Tris-HCl The concentration is 0.01mol / L, EDTA-Na 2 The concentration of PVP-40 is 0.5mmol / L, the concentration of PVP-40 is 0.5%) (volume percentage), the concentration of BSA is 0.05% (volume percentage), and the concentration of β-mercaptoethanol is 1mmol / L.

[0117] Solution B: Tris-HCl, EDTA-Na 2 , CTAB, NaCl, spermidine, PVP-40 and DEPC water were mixed, sterilized at 121°C for 40 minutes after 2 days at room temperature, and β-mercaptoethanol was added before use, which was solution B; in solution B, the concentration of Tris-HCl was 20mmol / L, EDTA-Na 2 The concentration of CTAB is 5mmol / L, the conce...

Embodiment 3

[0153] Embodiment 3, the extraction of cotton mitochondrial RNA

[0154] 1. Solution preparation

[0155] Solution A: NaCl, Tris-HCl, EDTA-Na 2 Mix with DEPC water, sterilize at 121°C for 40 minutes, add PVP-40 and BSA after cooling, and add β-mercaptoethanol before use, which is solution A; in solution A, the concentration of NaCl is 2.0mol / L, and the concentration of Tris-HCl The concentration is 1.00mol / L, EDTA-Na 2 The concentration of BSA is 4mmol / L, the concentration of PVP-40 is 5% (volume percentage), the concentration of BSA is 0.5% (volume percentage), and the concentration of β-mercaptoethanol is 10mmol / L.

[0156] Solution B: Tris-HCl, EDTA-Na 2 , CTAB, NaCl, spermidine, PVP-40 and DEPC water, sterilized at 121°C for 40 minutes after 2 days at room temperature, and added β-mercaptoethanol before use, which was solution B; in solution B, the concentration of Tris-HCl was 200mmol / L, EDTA-Na 2 The concentration of CTAB is 50mmol / L, the concentration of CTAB is 1...

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Abstract

The invention discloses a method for extracting cotton mitochondria and ribose nucleic acid (RNA) of the cotton mitochondria. The method comprises the following steps that: (1) roots of cotton chlorisis seedlings are ground by liquid nitrogen, 4-DEG C solution 1 is added and is uniformly mixed through oscillation, and the still standing is carried out for 2 to 30 min on ice; the solution 1 consists of solution A and prolease K, and the concentration of the prolease K is 0.01 to 1 mg / mL; (2) the 4-DEG C (500 to 2000)*g centrifugation is carried out for 5 to 40min, and supernate is taken; the 4-DEG C (1000 to 4000)*g centrifugation is carried out for 5 to 40min, and supernate is taken; and the 4-DEG C (8000 to 30000)*g centrifugation is carried out for 15 to 120min, and precipitates are taken; and (3) the solution A is used for washing resuspension precipitates, the (8000 to 30000)*g centrifugation is carried out for 15 to 120min, and precipitates are collected, and the mitochondria is obtained. The method can solve the technical difficult problem of the cotton mitochondria study in the prior art, and high-quality mitochondrial RNA (mtRNA) can be effectively obtained from the cotton mitochondria. The extracted mtRNA is applicable to relevant molecular biology experiments.

Description

technical field [0001] The invention relates to plant biotechnology, in particular to a method for extracting cotton mitochondria and RNA thereof. Background technique [0002] Mitochondria is the most important organelle in higher organisms and one of the semi-autonomous genetic systems of the cytoplasm. Mitochondria are important organelles for biological respiration. They have relatively independent genetic material and are regulated by nuclear genes. In plant cells, mitochondrial RNA accounts for only about 1% of total RNA. Therefore, the isolation of high-purity mitochondrial RNA (mtRNA) is necessary for the construction of mitochondrial cDNA library and the study of mitochondrial transcripts. [0003] Cotton tissue cells are rich in secondary metabolites such as gossypol and tannin, which are easily oxidized; when the tissue cells are broken, these secondary metabolites can combine with DNA or RNA to form a stable sticky brown complex. Therefore, compared with some ...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/04C12N15/10
Inventor 华金平李双双苏爱国王玉美熊敏雷彬彬
Owner CHINA AGRI UNIV