One vector-based method to verify predicted plant miRNAs, target sequences, and function modes.

Area of Science:

• Plant molecular biology
• Biotechnology
• Genetics

Background:

• Experimental verification of predicted microRNAs (miRNAs) is challenging due to a lack of efficient methods.
• Small RNA sequencing data has predicted numerous plant miRNAs, but validation remains limited.

Purpose of the Study:

• To develop a simple and reliable dual gene expression cassette vector-based method for verifying predicted plant miRNAs.
• To confirm the authenticity of a predicted plant miRNA (hvu-miRX) and identify its target sequence.
• To elucidate the mechanism of miRNA-mediated gene silencing in plants.

Main Methods:

• Construction of dual gene expression vectors containing known and predicted miRNAs, non-miRNA genes, and predicted target/non-target sequences fused to a green fluorescent protein (GFP) reporter.
• Bombardment of constructs into plant cells to assess GFP expression.
• Stem-loop reverse-transcription polymerase chain reaction (RT-PCR) to detect mature miRNAs.
• Northern blot analysis to detect GFP messenger RNA (mRNA) degradation.

Main Results:

• Constructs with predicted plant miRNAs (osa-miR528, hvu-miRX) and their complementary sequences failed to produce GFP fluorescence.
• Mature osa-miR528 and hvu-miRX were detected in plant cells using stem-loop RT-PCR.
• Northern analysis revealed degradation of GFP mRNA when co-expressed with complementary miRNA sequences.
• The results confirmed hvu-miRX as an authentic miRNA and identified its complementary sequence as the target.

Conclusions:

• The developed dual gene expression vector method is effective for verifying predicted plant miRNAs and their target sequences.
• hvu-miRX functions as a genuine miRNA, similar to osa-miR528.
• Plant miRNAs silence gene expression, specifically GFP, through mRNA cleavage mediated by complementary target sequences.