Plant microRNA expression vector, construction method and application thereof

Abstract

The present invention relates to a construction method of a vector and particularly relates to a plant microRNA expression vector, the construction method and an application thereof. A target gene promoter sequence and a target gene promoter sequence with downstream containing a segment of microRNA combination are cloned by an F / R1 primer and an F / R2 primer, respectively. Nco I / Nco I double enzymedigestion and T4 DNA ligase ligation are used to respectively replace a 35S sequence upstream of GFP on a pGFPGUSplus vector to construct two vectors; the 1# vector miRNA target gene promoter drivesthe GFP, and the 2# vector microRNA binding target gene promoter sequence drives the GFP; transgenic lines transformed by the two vectors are selected, GFP signals of a primary root tip meristem region are observed, and the GFP signals are compared and analyzed to clearly show expression modes of the plant microRNA before and after target gene regulation. The construction method simplifies operation steps of conventional vector construction methods, reduces cost, and improves work efficiency.

Description

technical field [0001] The present invention relates to a construction method of a vector, in particular to a plant microRNA expression vector, construction method and application. Background technique [0002] microRNAs (miRNAs) are one of the hot issues in epigenetics research, and are widely involved in the regulation of plant growth, development and stress tolerance and other life activities. The function of miRNAs mainly depends on the post-transcriptional regulation of related target genes. Therefore, to analyze the mechanism of miRNAs regulating related life activities, the first task is to clarify the regulation mode of miRNAs on their target genes in related life activities. [0003] At present, the research methods for the regulation mode of miRNAs to their target genes mainly include: qRT-PCR, RNA in situ hybridization, and the construction of vectors that drive target genes / anti-splicing target genes and GFP reporter genes from target gene promoters. Comparing t...

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Problems solved by technology

Comparing the above three methods, the qRT-PCR method is difficult to locate and analyze the expression pattern of the target gene at the cellular level before and after transcription; RNA in situ hybridization can realize the in situ expression analysis of miRNAs and their target genes, but the operation is cumbersome and the workload is heavy The conventional vector construction method can display the expression level of the target gene before and after transcription by comparing the signal of the reporter gene, but as far as the vector construction is concerned, it is necessary to clone the promoter of the gene, the gene coding sequence and the mutated coding sequence at the same time, and the workload In addition, it is unavoidable that the protein encoded by the gene itself will affect the spatial conformation of the reporter gene protein, and it is easy to have a well-constructed carrier, but the signal of the reporter gene cannot be seen

[0004] To sum up, the problems existing in the prior art are that the conventional vector construction method has cumbersome steps and heavy workload, and the influence of the protein of the gene itself on the space conformation of the reporter gene protein leads to the construction of the vector, and the signal of the reporter gene cannot be seen.

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