Method for enhancing and carrying out suppression expression on activity of miRNA in embryogenic callus of longan

A technology of embryogenic callus and callus, which is applied in the field of enhancing and inhibiting the expression of miRNA activity in embryogenic callus of longan, can solve the problems that have not been reported in the research, achieve wide application range, high test efficiency, and test short cycle effect

Inactive Publication Date: 2016-09-28
FUJIAN AGRI & FORESTRY UNIV
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Problems solved by technology

However, due to the particularity of plant embryogenic callus, there are often many difficulties in miRNA transformation. For example, longan is a recalcitrant plant, and its embryogenic callus is endogenous, which cannot be achieved by traditional genetic transformation methods.
Therefore, finding new ways to solve this problem has become the key to further in-depth research, but no relevant research has been reported so far.

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  • Method for enhancing and carrying out suppression expression on activity of miRNA in embryogenic callus of longan
  • Method for enhancing and carrying out suppression expression on activity of miRNA in embryogenic callus of longan
  • Method for enhancing and carrying out suppression expression on activity of miRNA in embryogenic callus of longan

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Embodiment Construction

[0021] The present invention will be further described in detail with reference to specific embodiments and drawings, but the specific embodiments do not limit the protection scope of the present invention in any sense.

[0022] The specific implementation steps are as follows:

[0023] Design and synthesis of mature miRNA mimetics and efficient miRNA blockers

[0024] Find and obtain the target miRNA mature body sequence from miRbase, and send it to Shanghai Gemma Pharmaceutical Technology Co., Ltd. to synthesize the corresponding oligonucleotide (miRNA-agomir) and antisense oligonucleotide (miRNA-antagomir), and carry out methylation respectively modification, thiolation, and cholesterol modification.

[0025] Proliferation Culture and Screening of Embryogenic Callus of Longan

[0026] The longan embryogenic callus was inoculated into the liquid suspension medium and cultured for 1-2 generations, and the suspension culture cell lines with loose texture, bright color and go...

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Abstract

The invention provides a method for enhancing and carrying out suppression expression on miRNA in the embryogenic callus of longan. An miRNA mimic agomir and efficient blocker antagomir, which undergo chemical modification and special mark design according to mature miRNA, are guided into the embryogenic callus cells of longan by adopting a cell transfection agent Lipofectamine 2000 reagent, thus achieving specific enhancement or suppression of the activity of target miRNA in the longan living body, and meanwhile, miRNA expression system optimization and effect duration identification are carried out. The method has the advantages of simplicity and rapidness in operation, obvious interference effects, stability, durability, and the like, avoids the defects that depending on the transgenic technology, existing methods are long in experimental periods and waste time and energy, provides an efficient and convenient method for research of miRNA in the callus of longan and has important significance to miRNA expression regulation and function identification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a method for enhancing and inhibiting the activity of miRNA expressed in longan embryogenic callus. Background technique [0002] MicroRNA (miRNA) is an important non-coding small molecule RNA, usually 18-25nt. The mode of action of miRNA is mainly through mediating target gene methylation at the transcriptional level, or performing target mRNA splicing or inhibiting translation at the post-transcriptional level, so as to realize the body's regulatory function. At present, a large number of research reports have shown that miRNAs are involved in the regulation of plant growth and development, such as flowering, aging, stress resistance, and disease resistance. The traditional miRNA research method is mainly through the isolation of related miRNA precursors to construct expression vectors for transformation. In recent years, the enhancement and inhibition of target miRNA expression th...

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N15/88
CPCC12N15/88
Inventor 林玉玲赖瑞联赖钟雄程春振刘生财陈裕坤张梓浩
Owner FUJIAN AGRI & FORESTRY UNIV
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