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Method for efficiently extracting high-quality nucleic acid substances from olive leaves

An olive and nucleic acid technology, applied in the field of biology, can solve the problems of accelerating the oxidation and browning of tissue samples, affecting the purity of nucleic acid extraction products, and the failure of olive samples to be used normally, so as to inhibit the oxidation process, solve the difficulty of extraction, and prevent oxidation. Effect

Pending Publication Date: 2019-09-13
POMOLOGY RES INST FUJIAN ACAD OF AGRI SCI
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, olives are rich in polysaccharides, polyphenols, proteins, and secondary metabolites, which often have strong interference effects in the extraction process of their nucleic acid substances, making it difficult to efficiently obtain high-quality olive nucleic acid substances
For example, a large amount of polysaccharides in olives can easily lead to RNA degradation and destroy its integrity. At the same time, polysaccharides can also wrap DNA, making it impossible to resuspend and dissolve during extraction; rich polyphenols in olives can easily accelerate tissue sample oxidation and browning. Extraction fails, and polyphenols are easy to combine with proteins or nucleic acids to form macromolecules or polymers, which affects the extraction effect and quality
In addition, proteins and other secondary metabolites in olives are often difficult to remove and cannot be effectively purified, affecting the purity of nucleic acid extraction products and interfering with subsequent application processes
Through our extensive research, we also found that the traditional unimproved nucleic acid extraction method cannot be used normally in olive samples

Method used

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  • Method for efficiently extracting high-quality nucleic acid substances from olive leaves
  • Method for efficiently extracting high-quality nucleic acid substances from olive leaves
  • Method for efficiently extracting high-quality nucleic acid substances from olive leaves

Examples

Experimental program
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Effect test

Embodiment 1

[0026] In this example, the old leaves of 3 germplasms of Fulan No. 1, Sanlengola and Hejiang medicinal green fruit respectively originating from Fujian, Guangdong, and Sichuan were preserved in the Olive Germplasm Resource Garden of the Fujian Academy of Agricultural Sciences Institute of Fruit Trees. and young leaves as materials to extract its genomic DNA, the specific implementation steps include:

[0027] (1) Pick up 3 parts of the old and young leaves of olive germplasm, wash them, absorb the water on the surface of the leaves with filter paper, let them cool for a short time, and remove the veins quickly.

[0028] (2) Grinding. Weigh 0.5 g of the sample, add 10 mg of PVP to the mortar, grind it into a homogenate under liquid nitrogen, and transfer it to a new centrifuge tube.

[0029] (3) Cracking. Add 10 mL of CTAB lysate with a mass volume ratio of 3% preheated at 65 °C, add 100 μL of β-mercaptoethanol, place in a warm bath at 65 °C for 30 min, and mix by inverting ...

Embodiment 2

[0038] In this example, the young leaves of 3 germplasms of Chi No. 2, Niulan No. 2 and Ruian No. 3 in Fujian, Guangxi, and Zhejiang provinces were used in the olive germplasm resource nursery of the Ministry of Agriculture and Rural Affairs of the Fujian Academy of Agricultural Sciences. As the material, the total RNA was extracted. In this example, all reagents and consumables are treated with DEPC water or high pressure to inactivate RNA hydrolase. The specific implementation steps include:

[0039] (1) Pick up 3 parts of the old and young leaves of olive germplasm, wash them, absorb the water on the surface of the leaves with filter paper, let them cool for a short time, and remove the veins quickly.

[0040] (2) Grinding. Weigh 0.5 g of the sample, add 10 mg of PVP to the mortar, grind it into a homogenate under liquid nitrogen, and transfer it to a new centrifuge tube.

[0041] (3) Cracking. Add 10 mL of CTAB lysate with a mass volume ratio of 3% preheated at 65 °C, a...

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Abstract

The invention discloses a method for efficiently extracting high-quality nucleic acid substances from olive leaves. The operation steps mainly include sampling, grinding, pyrolysis, impurity removal,precipitation, purification, dissolution and the like. Aiming at the characteristics that the olive is rich in impurities such as polyphenols, polysaccharides and proteins, on the basis of a traditional CTAB method, deep optimization is carried out on the characteristics of olive leaves, systematic improvement is carried out on sample dosage, reagent types, reagent concentration, reaction time, reaction volume, operation steps and the like, and a method suitable for high-efficiency and high-quality extraction of nucleic acid substances of olive tender leaves, mature leaves or old leaves is developed. The method can select and extract olive leaf genome DNA or total RNA according to actual requirements, has wide applicability and strong practicability, and has good application value.

Description

technical field [0001] The invention relates to the field of biology, in particular to a method for efficiently extracting nucleic acid substances from olive leaves. Background technique [0002] Olive is a famous and precious fruit tree in tropical and subtropical regions of my country, and its fruit is a homologous raw material for medicine and food announced by the Ministry of Health. my country is the origin of olive and the distribution center of genetic diversity, and its germplasm resources are extremely rich. However, due to the lack of unified olive naming standards among producing areas, and the frequent exchange of germplasm resources, homonyms and synonyms are very common. Therefore, the identification of olive germplasm resources based on modern molecular marker technology is of great importance to olive germplasm resources. It is of great significance to excavate and identify new varieties. In addition, since olive belongs to the small family of fruit trees, ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/101C12Q2527/125C12Q2523/32
Inventor 赖瑞联吴如健陈瑾冯新杨俊陈义挺
Owner POMOLOGY RES INST FUJIAN ACAD OF AGRI SCI
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