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Method for exploring key regulatory factors for regulating plant development and application of key regulatory factors

A technology for plant development and regulatory factors, applied in the field of molecular biology, can solve the problems of little value, regulatory factors or genes that cannot truly reflect the process of plant development, high probability of false positives for miRNA and target genes, etc.

Pending Publication Date: 2022-03-01
INT CENT FOR BAMBOO & RATTAN
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, in the existing technology, only batches of candidate miRNAs and target genes are discovered through omics methods, and then miRNA-target gene pairs are predicted by software according to the interaction characteristics between miRNAs and target genes, so the nodes in the regulatory network obtained The false positive probability of miRNA and target genes is very high, resulting in the discovery of regulatory factors or genes that cannot truly reflect the plant development process, and are of little value in guiding breeding

Method used

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  • Method for exploring key regulatory factors for regulating plant development and application of key regulatory factors
  • Method for exploring key regulatory factors for regulating plant development and application of key regulatory factors
  • Method for exploring key regulatory factors for regulating plant development and application of key regulatory factors

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Experimental program
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Embodiment 1

[0048] In this embodiment, the regulatory factors of plant tissues have been excavated, including the following steps:

[0049] (1) Plant tissues or organs are collected, and three replicates are set for each sample.

[0050] (2) cDNA library construction

[0051] 1. Use the kit purchased by Tiangen Biochemical Technology (Beijing) Co., Ltd. to extract the RNA of the plant tissue in step (1), and perform the specific operation according to the kit instructions.

[0052] 2. Agilent Bioanalyzer 2100 system (Agilent Technologies, CA, USA) was used to detect the purity, concentration and integrity of RNA samples.

[0053] 3. After the sample is qualified, construct the library according to the following process:

[0054] a. Enrichment of eukaryotic mRNA with magnetic beads containing Oligo(dT);

[0055] b. Add FragmentationBuffer to randomly interrupt the mRNA;

[0056] c. Using mRNA as a template, use six base random primers (random hexamers) to synthesize the first cDNA stra...

Embodiment 2

[0242] Utilize the method described in embodiment 1 to excavate the regulatory factor of the shoot shoot procambium in the middle stage of Phyllostachys pubescens development, the results are as follows:

[0243] After testing, 137 miRNAs were identified in the early shoots of Phyllostachys pubescens, including 120 known and 17 new miRNAs; 38652 genes were identified. However, 130 miRNAs were identified in mid-developmental shoots, including 115 known and 15 new miRNAs; 39584 genes were identified. Expression analysis identified 30 differentially expressed miRNAs and 61 differentially expressed genes in the two groups of samples; degradome sequencing analysis identified 101 sets of miRNA-target gene pairs. The dual-luciferase reporter assay verified that 65 groups of miRNA-target gene pairs could interact in cells. Fluorescence in situ hybridization Fish experiments identified ped-miR160a-5p, etc., which can play a role in the procambium in the middle stage of shoot shoot dev...

Embodiment 3

[0245] Utilize the method described in embodiment 1 to excavate the regulatory factors of the shoot shoot top meristem in the early stage of Phyllostachys pubescens development, the results are as follows:

[0246] After detection, 129 miRNAs, including 117 known and 12 new miRNAs; 38462 genes were identified in the bud samples of Phyllostachys pubescens. However, 137 miRNAs were identified in early shoot shoot samples, including 120 known and 17 new miRNAs; 38652 genes were identified. Expression analysis identified 26 differentially expressed miRNAs and 75 differentially expressed genes in the two groups of samples; degradome sequencing analysis identified 98 sets of miRNA-target gene pairs. The dual-luciferase reporter assay verified that 56 groups of miRNA-target gene pairs could interact in cells. Fluorescence in situ hybridization (FISH) assay identified ped-miR166a-3p that can play a role in the apical meristem in the early shoot development ( Figure 7 ).

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Abstract

The invention discloses a method for exploring key regulatory factors for regulating plant development, and belongs to the technical field of molecular biology. By means of transcriptome sequencing, small RNA sequencing and degradation group sequencing, a differential expression miRNAs-target gene pair related to a plant development molecular mechanism is obtained through screening; the action relationship of the differentially expressed miRNAs-target gene pair is verified by using a dual-luciferase report detection technology; the action position of the differentially expressed miRNAs-target gene pair in plant tissue cells is positioned by using a fluorescence in-situ hybridization technology, so that a key regulation factor for regulating plant development can be obtained. According to the invention, the positioning of the node gene and miRNA in the developmental tissue is accurately realized, and the miRNAs-target gene pair which plays a key regulation role in specific plant tissues or cells in a specific developmental stage can be accurately obtained.

Description

technical field [0001] The invention relates to the technical field of molecular biology, in particular to a method for discovering and regulating key regulatory factors of plant development and an application thereof. Background technique [0002] miRNA is a kind of non-coding small molecule RNA with a length of about 21nt, which is a key regulator of almost all biological processes in eukaryotes. In plants, miRNA can bind to the complementary sequence of the target mRNA molecule to degrade it or inhibit its translation, and regulate the expression of the target gene at the post-transcriptional level. . miRNA can combine upstream regulatory genes and downstream target genes to play an important role in key developmental processes such as embryonic development, meristem and organogenesis in plants. [0003] Existing research results show that almost all miRNAs are regulated by multiple transcription factors, and miRNAs also act on many target genes including transcription ...

Claims

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Application Information

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IPC IPC(8): C12Q1/6809C12Q1/6841C12Q1/66
CPCC12Q1/6809C12Q1/6841C12Q1/66C12Q2535/122C12Q2539/103C12Q2537/165C12Q2525/207C12Q2563/107
Inventor 李英高志民
Owner INT CENT FOR BAMBOO & RATTAN
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