In-situ hybridization method for dianthus chinensis

A Chinese carnation and in situ hybridization technology, applied in the field of molecular biology of horticultural plants, can solve the problems of low signal sensitivity, gene expression detection, unclear coloring, etc., achieve low hybridization background, clear coloring, improve flexibility and reliability operational effect

Inactive Publication Date: 2019-04-19
HUAZHONG AGRICULTURAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the signal sensitivity of in situ hybridization in Chinese carnation (Dianthuschinensis) is not high, the background signal is too strong, and the hybridization efficiency is low, the hybridization time is long, and the coloring is not clear. expression for good detection
At present, there is no method for in situ hybridization of Chinese Dianthus flower buds

Method used

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  • In-situ hybridization method for dianthus chinensis
  • In-situ hybridization method for dianthus chinensis
  • In-situ hybridization method for dianthus chinensis

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0071] 1. Fixation and embedding of Chinese Dianthus organs and tissues

[0072] Step A1: Fixation of Chinese Dianthus Buds

[0073] Take 0.3-0.5cm long Chinese Dianthus flower buds, place them in 10ml centrifuge tubes, fix them with EAF fixative respectively, label them (indicating date, time, sample) for 30min, open the lid, and vacuum at 0.08MPa for 30min.

[0074] Refer to Table 1 to prepare gradients to prepare ethanol (alcohol) solutions.

[0075] Table 1 Configuration of gradient concentration ethanol solution

[0076]

Absolute ethanol

Ultra-pure water

30% ethanol solution

30ml

Dilute to 100ml

50% ethanol solution

50ml

Dilute to 100ml

75% ethanol solution

75ml

Dilute to 100ml

85% ethanol solution

85ml

Dilute to 100ml

95% ethanol solution

95ml

Dilute to 100ml

100% ethanol

100ml

Dilute to 100ml

[0077] Step A2: Dehydration of Chinese Dianthus Buds

[0078] From th...

Embodiment 2

[0116] Step C1: Deparaffinization and rehydration of samples:

[0117] According to the method described in the above-mentioned Example 1, the embedded Dianthus flower bud sample was obtained using a German Lycra RM2265 microtome, and the wax block was sliced, with a thickness of 9 μm, and a drop of DEPC water was dropped on the adhesive slide, and the slice was placed on it. Spread the slices on a heating plate at 37°C and dry them, and bake the stretched slices in an oven at 37°C for more than 12 hours. Take out the slides, wash them twice with xylene, 10 minutes each time, then rehydrate with 100%, 95%, 70%, 50%, 30% ethanol aqueous solution, each treatment for 2 minutes, wash twice with DEPC, each time 2min.

[0118] Step C2: Protein Digestion of Samples

[0119] Specifically, this embodiment is to move the slide with the sample into 1×PBS buffer, let it stand for 2min, then incubate the slide in 0.2M HCl for 15min, then put it in DEPC water for 3min, and rinse Excess H...

Embodiment 3

[0131] Step C1: Deparaffinization and rehydration of samples

[0132] According to the method described in the above-mentioned Example 1, the embedded Dianthus flower buds were obtained. Use a Leica RM2265 microtome to slice the wax block with a thickness of 10 μm. Drop a drop of DEPC water on the adhesive slide, place the slice on it, and incubate at 37°C. Spread the slices on a hot plate and dry them, and bake the stretched slices in an oven at 37°C for more than 12 hours. Take out the slides and wash them twice with xylene for 10 minutes each time, then rehydrate with ethanol solutions with gradient concentrations of 100%, 95%, 70%, 50% and 30% for 1 minute each time, and wash with DEPC water Twice, 2 minutes each time.

[0133] Step C2: Protein Digestion of Samples

[0134] Specifically, this embodiment is to move the slide with the sample into 1×PBS buffer, let it stand for 2min, then incubate the slide in 0.2M HCl for 15min, then put it in DEPC water for 3min, and rins...

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Abstract

The invention belongs to the field of gardening plant molecular biology methods, and particularly relates to an in-situ hybridization method for dianthus chinensis flower buds. The in-situ hybridization method includes the following steps that a sample is prepared, an RNA probe is prepared, pretreatment is conducted before hybridization, hybridization liquor is prepared, pre-hybridization is conducted, hybridization is conducted, and background detection is conducted. Firstly, the in-situ hybridization method for dianthus chinensis is provided and can effectively eliminate background interference, improve detection sensitivity and accuracy and be well applied to genetic transformation of dianthus chinensis. The established in-situ hybridization method for dianthus chinensis is stable, accurate and beneficial to research on a gene expression mode during flower growing of dianthus chinensis.

Description

technical field [0001] The invention belongs to the technical field of molecular biology of horticultural plants, and in particular relates to an in-situ hybridization method of Chinese dianthus. The invention is mainly used for the in-situ hybridization detection of target gene expression in carnation flower buds. Background technique [0002] In situ hybridization (in situ hybridization) refers to the process of using specific labeled known nucleic acid sequences as probes to hybridize with nucleic acids in cells or tissue sections, so as to accurately locate specific nucleic acid sequences. In situ hybridization can be performed on cell or tissue samples. This method can intuitively reflect the spatiotemporal expression and developmental pattern of the target gene in the development of tissues and organs, and can be used to analyze the role of the target gene in the formation of corresponding tissues or organs. [0003] At present, good progress has been made in in situ ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6841
CPCC12Q1/6841C12Q2565/518
Inventor 傅小鹏张晓妮包满珠
Owner HUAZHONG AGRICULTURAL UNIVERSITY
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