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Preparation method of intact chloroplasts of Salix matsudana leaves

A chloroplast and complete technology, applied in the field of agricultural biology, can solve the problems of no preparation method of dry willow leaves, public reports, etc.

Inactive Publication Date: 2018-06-19
INST OF FORESTRY CHINESE ACAD OF FORESTRY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, there is no public report on the preparation method of complete chloroplasts from the leaves of willow

Method used

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  • Preparation method of intact chloroplasts of Salix matsudana leaves
  • Preparation method of intact chloroplasts of Salix matsudana leaves
  • Preparation method of intact chloroplasts of Salix matsudana leaves

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0021] Example 1 Preparation of Whole Willow Leaf Chloroplasts by the Method of the Invention

[0022] 1) Sampling and homogenization: Take the fully expanded functional leaves of the dry willow branches in the early morning of the growing season, wash with deionized water, blot the filter paper, and cut the leaves into 5mm 2 Of small pieces. Take 10g leaves and transfer them to WaringBlender homogenizer, add 200mL separation solution (see Table 1), strictly control the number and time of homogenization, first low speed 2 times and then high speed 2 times, 5 seconds each time, 1 minute interval.

[0023] 2) Filtration: filter the homogenate through 4 layers of gauze, collect the filtrate in an ice bath beaker, wrap the residue with gauze after filtration, gently squeeze, and combine the filtrate.

[0024] 3) Preparation of crude chloroplast precipitate: divide the filtrate into 50 mL centrifuge tubes, centrifuge at 200g for 3 minutes in a corner rotor, transfer the supernatant to a n...

Embodiment 2

[0031] Example 2 Chloroplast fluorescence microscope examination

[0032] The chloroplast suspension obtained by the invention is dropped on the center of the slide glass, and a cover glass is added to prepare a temporary mount. Use Zeiss Imager.A1 fluorescence microscope microscopy. Observe the chloroplast image in the bright field to determine whether there is contamination of other organelles in the field of view; observe the chloroplast fluorescence image of the chloroplast under excitation light to determine the activity of the chloroplast. The results showed that the chloroplasts were evenly distributed in the field of vision, and all chlorophyll fluorescence was emitted, and no contamination of other organelles was found.

Embodiment 3

[0033] Example 3 Integrity inspection of chloroplast envelope

[0034] The potassium ferricyanide method is used to test the integrity of the chloroplast envelope obtained by the invention. Take two equal-volume chloroplast suspensions, one to burst in deionized water and the other without bursting treatment, and use an oxygen electrode to detect the oxygen release rate of the two chloroplasts depending on potassium ferricyanide, according to formula 1. By calculating the integrity rate of the chloroplast envelope, it can be determined that the integrity rate of the chloroplast prepared by the method of the present invention is above 80%.

[0035] Formula 1:

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Abstract

The invention belongs to the technical field of agrobiology and discloses a preparation method of intact chloroplasts of Salix matsudana leaves. The method comprises the following steps: the Salix matsudana leaves are collected and subjected to the following processes in sequence: 1) homogenization; 2) filtration; 3) preparation of coarse chloroplast precipitates; 4) suspension of the coarse chloroplast precipitates; 5) gradient preforming; 6) isodensity centrifugation; 7) recovery of the intact chloroplasts, and finally, suspension of the intact chloroplasts is obtained. With adoption of themethod, purity of the obtained chloroplasts is high, and envelope integrity can reach 80% or higher. The foundation is laid for conducting research of photosynthetic physiology and molecular biology of Salix matsudana at the chloroplast level, and one novel idea is provided for separation of organelles of other wood plants.

Description

Technical field [0001] The invention belongs to the field of agricultural biotechnology, and relates to a preparation method of complete chloroplasts of Salix matsudana leaves. Background technique [0002] Salix matsudana is a Salix plant of the Salicaceae family. It is an important species for farmland protection, plain afforestation and landscaping in northern my country. It has a wide range of ecological adaptability and high production and application value. Dry willow leaves show early, deciduous late, and leaves fixed CO 2 It has a long annual accumulation time, rich photosynthetic pigment content, strong light energy absorption and transformation ability, and high photosynthetic rate. It is a representative tree species for conducting research on photosynthetic physiology of woody plants. Chloroplast is the basic structural unit of photosynthesis. Obtaining a complete chloroplast is a necessary prerequisite for conducting chloroplast photosynthetic electron transport, ca...

Claims

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Application Information

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IPC IPC(8): C12N5/04
CPCC12N5/04
Inventor 刘俊祥郎蓬蓬孙振元李振坚彭向永张少伟于永畅
Owner INST OF FORESTRY CHINESE ACAD OF FORESTRY
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