Chloroplast DNA extracting method of sonneratia plant

A technology for chloroplasts and plant leaves is applied in the field of chloroplast DNA extraction of sea mulberry plants, and achieves the effects of short time consumption, avoiding RNA pollution and high purity

Inactive Publication Date: 2019-09-27
LINGNAN NORMAL UNIV
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0005] However, there is currently no method for extracting chloroplast DNA from plants of the genus Sonsanthesia

Method used

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  • Chloroplast DNA extracting method of sonneratia plant
  • Chloroplast DNA extracting method of sonneratia plant
  • Chloroplast DNA extracting method of sonneratia plant

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Experimental program
Comparison scheme
Effect test

Embodiment 1

[0054] The chloroplast separation and DNA extraction of embodiment 1 Sonneratia plant

[0055] The extraction and separation method of chloroplasts of the genus Sonsangia plants refers to the article published by Palmer et al. (Palmer J D, Stein DB. Conservation of chloroplast genome structure among vascular plants. CurrGenet, 1986, 10:823-833.), and improvements are made. All operating steps were carried out at 4°C unless otherwise specified, and all reagents and instruments needed to be pre-cooled in advance. The specific experimental methods and experimental results are as follows:

[0056] (1) Isolation and purification of chloroplasts of Sonangula genus

[0057] Collect 30 g of young, healthy and free of diseases and insect pests of Sonneratia sinensis young leaves that have not yet unfolded, and place them in a 4°C refrigerator for dark cultivation for 36 hours, so as to reduce the starch content in the leaves. Remove the veins of the leaves, weigh them, and cut them i...

Embodiment 2

[0060] Embodiment 2 Fluorescence microscope observation of the chloroplasts of the genus Sonangula

[0061] 1. Experimental method

[0062] The filtrate of the chloroplasts of the genus Sonanga chloroplast prepared in step (1) in Example 1 was centrifuged for 3 min under the centrifugal conditions of 100 × g, 200 × g, and 300 × g respectively, and the supernatant was transferred to a new 10 mL tube. Take 5uL of the precipitate and examine it under a fluorescent microscope (10× eyepiece, 40× objective lens).

[0063] 2. Experimental results

[0064] The results of fluorescent microscope examination of the chloroplasts of the genus Sonnensis under different centrifugation conditions are as follows: figure 1 As shown, it can be seen that under the fluorescence microscope, spherical and complete red chloroplast particles can be clearly seen, and their sizes are concentrated at about 200 μm. The number increased successively, among them, the number of chloroplasts of the genus S...

Embodiment 3

[0065] Example 3 Detection of the Integrity of Chloroplasts of Sangria genus Plants

[0066] 1. Experimental method

[0067] According to the results of fluorescence microscope observation in Example 2, take the chloroplasts of the genus Nutonia genus obtained under the centrifugation condition of 100 × g in Example 2, centrifuge the supernatant at 5000 × g for 5 min, discard the supernatant, leave the precipitate, and take 5uL of the precipitate in the fluorescence microscope. Microscopic examination was carried out under a microscope (10× eyepiece, 40× objective lens).

[0068] 2. Experimental results

[0069] The integrity detection results of the chloroplasts of the sangria genus obtained under the condition of 100×g centrifugation are as follows: figure 2 As shown, it can be seen that no nuclei were found, and a large number of high-purity chloroplasts of the Sonneratia plant were obtained, and the chloroplasts were all kept intact.

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Abstract

The invention discloses a chloroplast DNA extracting method of a sonneratia plant. The method comprises the following steps: performing dark treatment on leaves of the sonneratia plant first, performing homogenate, centrifugal treatment and filtration to obtain filtrate, namely sonneratia plant chloroplast, then adding cracking buffering liquid into the obtained chloroplast for fully cracking, firstly extracting the obtained cracking mixture solution by using phenol, then extracting by using phenol-chloroform, centrifuging to obtain a precipitate, dissolving the obtained precipitate, and performing RNase digestion to obtain the chloroplast DNA of the sonneratia plant, wherein the concentration of lauryl sodium sulfate in the cracking buffering liquid is 3.5 to 4.5 percent. By utilizing the method, high-quality and high-purity chloroplast DNA of the sonneratia plant is extracted, separated and obtained successfully, and the method is of great significance to analyze evolutionary relationship of the sonneratia plant, design an SSR primer to analyze the genetic diversity of the existing sonneratia plant population, analyze the expansion and evolution of exotic sonneratia plant at home and the like, and has a broad promotion and application prospect.

Description

technical field [0001] The invention belongs to the technical field of plant genetic engineering. More specifically, it relates to a method for extracting chloroplast DNA of Sonsangia plants. Background technique [0002] The mangrove ecosystem is located at the dynamic interface between sea and land, and is periodically flooded by seawater. As a unique sea-land marginal ecosystem, it has extremely high ecological, social, and economic values, especially in the areas of shore embankment, The development of offshore fisheries, maintenance of biodiversity, regulation of microclimate, environmental purification, development of ecotourism, and maintenance of ecological balance in coastal zones are particularly prominent, and are currently important contents of global marine biodiversity protection and global climate change research. Mangrove plants of the genus Sonneratia belong to the family Sonneratiaceae, and are an important group in tropical and subtropical coastal mangrov...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/1003
Inventor 张颖左欢欢钟军弟
Owner LINGNAN NORMAL UNIV
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