RNA in-situ hybridization optimization method for cucumber seedling gene localization research

Abstract

The invention relates to an RNA in-situ hybridization optimization method for cucumber seedling gene localization research. The method comprises the following steps: firstly, improving a plant culture, sampling and fixing method according to the characteristics of cucumber seedling plant materials so as to obtain a complete plant structure containing target tissues; secondly, optimizing operatingtechniques such as slice preparation, hybridization pretreatment, hybridization incubation slice sealing and chromogenic reaction microscopic examination in a targeted manner so that the reliability,accuracy and specificity of a gene localization result are improved; finally, designing a treatment scheme suitable for cucumber seedling mature tissue after the chromogenic reaction is ended in a targeted mode so that interference of cell inclusions can be eliminated, and directly observing the distribution of hybridization signals. The method is not only used for improving the sensitivity and accuracy of experimental results, but also reduces the economic cost by improving the utilization rate of drugs, reducing the consumption of experimental articles, increasing the yield of reaction products, saving the labor cost and the like, so that the method is a scientific research method considering scientificity, innovativeness and practicability.

Description

technical field [0001] The invention relates to the field of plant biotechnology, in particular to an RNA in situ hybridization optimization method used for the research on gene positioning of cucumber seedlings. Background technique [0002] Cucumber (Cucumis sativus L.) is the main vegetable crop in my country and also an important economic crop. According to the statistics of the World Food and Agriculture Organization (FAO), China’s annual cucumber production accounted for 48.3% of the world’s annual cucumber production in 1961, and this proportion has reached 77.5% in 2017. China is the world’s most important cucumber cultivation area. . Therefore, it is of great significance to study the physiological cultivation mechanism and gene function characteristics of cucumber for the construction of modern agriculture, the improvement of cultivation management mode and the selection of high-quality new varieties. With the advancement of genomics and bioinformatics, the whole...

AI Technical Summary

Problems solved by technology

[0006] (1) Cultivation of plant materials: Cucumber seedling root system cultivated with traditional peat vermiculite and perlite matrix will adhere to many matrix impurities that are difficult to remove. However, violent washing not only cannot completely remove impurities, but also destroys the organizational structure of cucumber root system;

[0007] (2) Gene probe preparation: the traditional system connects the cDNA fragment of the target gene to pGEMT or pGEMTeasy, and then obtains the in vitro transcription template by recovering the enzyme-cut linear vector product. However, this method not only increases the economic cost, but also directly affects the yield of the template. The effect of ligation efficiency and enzyme digestion efficiency;

The traditional sample cutting mode, fixative formula, gradient ethanol dehydration system and paraffin embedding methods are not applicable, resulting in tissue shrinkage, damage, and structural incompleteness (such as figure 1 shown);

[0009] (4) Preparation of hybrid slices: the traditional system is not optimized for this step, and this step has an important impact on the results of in situ hybridization of mature tissues of cucumber seedlings in the later stage;

[0010] (5) Hybridization pretreatment: The traditional system has not optimized the preservation of samples treated in this step, and at this stage, various reagent solutions are used multiple times and consume a lot;

[0011] (6) Sealing by hybridization incubation: The traditional system has not been optimized for this step, and the technical method of this step has a direct impact on the accuracy of the gene mapping results of mature tissues of cucumber seedlings;

[0012] (7) Microscopic examination of chromogenic reaction: the traditional system is not optimized for this step, and this step has a direct impact on the specificity of gene localization in mature tissues of cucumber seedlings;

[0014] This requires a new set of in situ hybridization optimization schemes suitable for mature tissues of cucumber seedlings to solve the steps of cucumber seedling material source, sampling, fixation, probe preparation, hybridization process and post-processing of hybridization chromogenic termination when the existing system is applied. technical deficiencies in

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