Method for extracting RNA in early development stage of cotton fiber cells

A cotton fiber and cell technology, applied in the field of RNA extraction in the early stage of cotton fiber cell development, can solve the problems of systematic errors, reduced specificity, fiber cell differentiation, unclear initial research, etc., and achieves accurate transcriptome data and high RNA quality. Effect

Active Publication Date: 2020-09-01
HENAN UNIVERSITY
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Problems solved by technology

[0008] (1) Previous studies have obtained RNA samples of early cotton fiber development cells, which are mainly composed of fiber cells and other parts of the ovule except fiber cells. Fiber cells only account for less than 1% of the total cells, which severely limits fiber development. The study of transcription level at the initial stage
[0009] (2) Although the specific early cells of cotton fiber development can be obtained by using laser microdissection technology, the total amount obtained rarely requires two amplifications to meet the needs of high-throughput library construction and sequencing. Continuous amplification The process introduces systematic errors that reduce the accuracy of transcript-level findings
[0010] (3) Although the ultra-low temperature mechanical oscillation method can separate relatively specific early cells of cotton fiber development, due to the increased fragility of the ovule tissue under low temperature conditions, under the condition of mechanical oscillation with glass beads, except for the shedding of fiber cells, other A large amount of non-fibrous tissue will also fall off, resulting in a significant reduction in the specificity of obtaining RNA samples
The differentiation and initiation of fiber cells are crucial to fiber development, but the research on the differentiation and initiation of fiber cells is still unclear. Isolating early specific cotton fiber cells and obtaining high-quality early transcriptome data of cotton fiber cell development will help reveal The process of fiber differentiation and initiation is also conducive to the excavation of genes related to fiber initiation, laying the foundation for the cultivation of high-quality cotton fiber varieties

Method used

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  • Method for extracting RNA in early development stage of cotton fiber cells
  • Method for extracting RNA in early development stage of cotton fiber cells
  • Method for extracting RNA in early development stage of cotton fiber cells

Examples

Experimental program
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Effect test

Embodiment 1

[0044] Example 1: Cotton variety TM-1 (from Cotton Research Institute, Chinese Academy of Agricultural Sciences) Extraction and quality detection of fiber RNA at different developmental stages - 1DPA to 5DPA.

[0045] 1) Take materials. From the TM-1 plant, 40 cotton ovaries of -1DPA, 0DPA, 1DPA, 2DPA, 3DPA, 4DPA and 5DPA were respectively taken.

[0046] 2) cracking. Take seven 15mL centrifuge tubes, add 8mL of prepared lysate respectively, and mark them. For the flowers in step 1, carefully cut open the ovary wall with a blade in the ultra-clean workbench, gently clamp the fungus handle with tweezers, take out the complete ovules and put them into the corresponding centrifuge tubes.

[0047] 3) Vacuum treatment. Put the centrifuge tube in step 2 into the desiccator, vacuumize to -0.9Mpa, 10min-20min (-1DPA sample vacuumizes 10min, 0DPA sample vacuumizes 12min, 1DPA sample vacuumizes 14min, 2DPA sample vacuumizes 16min , 3DPA samples were vacuumed for 18 minutes, 4DPA and...

example 2

[0061] Example 2: Imaging observation of ovule tissue by scanning electron microscope environment

[0062] Take 1 ovary of TM-1 0DPA cotton, carefully cut open the ovary wall with a razor blade, gently clamp the fungus with tweezers, take out 6 complete ovules with the same development status, and 3 ovules are directly subjected to electron microscope environment without any treatment scanning. The other 3 ovules were lysed, put the removed ovules into a 1.5mL centrifuge tube containing 800μL lysate, vacuumed to -0.9Mpa, 12min, vortexed for 5min, ddH 2 O was washed three times, and the electron microscope environment was scanned at a magnification of 1000 times.

[0063] The results of electron microscope imaging showed that the epidermal cells of the ovules without lysis treatment had normal protrusions, and the fiber cells were evenly and plumply distributed in the epidermis ( figure 2 ). The ovule epidermis tissue after vacuum lysis treatment, because the ovule epiderma...

example 3

[0064] Example 3: Ovule semi-thin section observation

[0065] Take 1 ovary of TM-1 0DPA cotton, carefully cut open the ovary wall with a blade, gently clamp the funicular handle with tweezers, take out 6 complete ovules with the same development status, and fix the 3 ovules directly without any treatment . The other 3 ovules were lysed, put the removed ovules into a 1.5mL centrifuge tube containing 800μL lysate, vacuumed to -0.9Mpa, 12min, vortexed for 5min, fixed with fixative, sliced, and observed.

[0066] The process of semi-thin sectioning is as follows:

[0067] 1) Sample fixation. Put the ovules into a 2mL centrifuge tube filled with 1mL of fixative solution (the components of the fixative solution are 4% paraformaldehyde and 5% glutaraldehyde), and vacuumize to -0.9MPa until the ovules are completely immersed in the fixative solution.

[0068] 2) Wash. Wash 3 times with 0.1M Pbs (phosphate buffered saline), 15 min each time.

[0069] 3) Dehydration. Use 30% etha...

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Abstract

The invention belongs to the technical field of biology, and discloses a method for extracting cotton fiber cell early development RNA, which comprises the following steps: taking a plurality of ovaryin different development stages, and taking complete ovules; putting the ovules into a lysis solution, vacuumizing, centrifuging and taking a supernate; filtratioffing the supernatant, adding absolute ethyl alcohol into the filtered supernatant to precipitate nucleic acid, collecting the obtained solution, adding a deproteinization solution RW1 to wash protein, adding a DNaseI working solution todigest DNA, adding the deproteinization solution RW1 and a rinsing solution RW to wash other impurities except the nucleic acid, and collecting the eluted RNA solution; and finally, performing RNA concentration and purity detection. The method is suitable for any cotton variety. The method can be used for rapidly extracting the cotton fiber cell development early-stage specific RNA which is difficult to separate in -1DPA, 0DPA, 1DPA, 2DPA, 3DPA, 4DPA and 5DPA periods, and meets the requirements of transcriptional library building.

Description

technical field [0001] The technology belongs to the field of biotechnology, and in particular relates to a method for extracting early RNA of cotton fiber cell development. Background technique [0002] Cotton is an important economic crop and the main source of natural fibers. It has considerable economic significance in textile production and also has important biological significance. Cotton fiber is a single cell formed by special differentiated protrusions from the outer epidermis of the ovule, and is a model system for studying cell elongation and cell wall synthesis. Cotton fiber development includes four stages: initiation of fiber cell differentiation, fiber cell elongation, secondary wall thickening, and dehydration maturation. Fiber initiation generally occurs from 3 days before flowering to 5 days after flowering, that is, about -3DPA to 5DPA (days post anthesis). The initiation of fiber cell differentiation is an extremely complex and crucial process. It not ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/10
CPCC12N15/101C12N15/1017C12Q2523/308
Inventor 邹长松李志芳郑炬瑞胡佳敏王鹏宇王朋宝程祥飞李成迈克尔·斯科特·杜博
Owner HENAN UNIVERSITY
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