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An effective homologous cloning method for isolating novel miR172s from Phalaenopsis hybrida.

ABSTRACT: MiR172 is an important microRNA that regulates floral development in various plants and downregulates AP2 family members to relieve the stress on floral determinacy, leading to phase transition from vegetative to reproductive growth. In this work, PCR with primers designed based on the rice miR172 sequence was used to isolate two miR172-like transcripts from Phalaenopsis hybrida (PhmiR172-1 and PhmiR172-2) that were very similar to Oryza miR172d and Arabidopsis miR172b. RT-PCR indicated that the levels of these two transcripts were negatively correlated with the level of the Phalaenopsis AP2 (PhAP2) gene in stem, root, pedicel and sepal, and that both were co-expressed with PhAP2 in young buds. Overproduction of PhmiR172-2 in Arabidopsis led to early flowering. The homologous cloning method used to isolate the Phalaenopsis miR172-like transcripts can be used to isolate miRNAs from other species. These PhmiR172 transcripts may be used to accelerate the flowering of orchids.

SUBMITTER: Han YY

PROVIDER: S-EPMC4094608 | biostudies-literature | 2014 Jun

REPOSITORIES: biostudies-literature

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An effective homologous cloning method for isolating novel miR172s from Phalaenopsis hybrida.

Han Ying Ying YY Yan Qin Hua QH Ming Feng F

Genetics and molecular biology 20140601 2

MiR172 is an important microRNA that regulates floral development in various plants and downregulates AP2 family members to relieve the stress on floral determinacy, leading to phase transition from vegetative to reproductive growth. In this work, PCR with primers designed based on the rice miR172 sequence was used to isolate two miR172-like transcripts from Phalaenopsis hybrida (PhmiR172-1 and PhmiR172-2) that were very similar to Oryza miR172d and Arabidopsis miR172b. RT-PCR indicated that the ...[more]

PMID: 25071407

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Project description:BackgroundViruses of the genus Begomovirus (Geminiviridae) are emerging economically important plant viruses with a circular, single-stranded DNA genome. Previous studies have shown that geminiviruses and RNA viruses exhibit similar mutation frequencies, although geminiviruses are replicated by host DNA polymerases and RNA viruses by their own virus-encoded error-prone RNA-dependent RNA-polymerase. However, the phenotypic effects of naturally occurring mutations have never been extensively investigated in geminiviruses, particularly because, to be infectious, cloned viral genomes usually require sub-cloning as complete or partial tandem repeats into a binary vector from Agrobacterium tumefaciens.ResultsUsing Tomato yellow leaf curl virus (TYLCV), we show here that infectivity can be obtained when only a 41-nucleotide region containing a highly conserved stem-loop is repeated. A binary vector containing this 41-nt region and a unique restriction site was created, allowing direct cloning of infectious monomeric viral genomes provided that they harbour the same restriction site at the corresponding nucleotide position. This experimental system, which can be transferable to other geminiviruses, was validated by analysis of the phenotypic effect of mutations appearing in TYLCV genomes in a single tomato host plant originally inoculated with a unique viral sequence. Fourteen full-length infectious genomes extracted from this plant were directly cloned and sequenced. The mutation frequency was 1.38 x 10-4 mutation per nucleotide sequenced, similar to that found previously for another begomovirus by sequencing PCR-amplified partial sequences. Interestingly, even in this minimal pool of analysed genomes, mutants with altered properties were readily identified, one of them being fitter and reducing plant biomass more drastically than the parental clone.ConclusionThe cloning strategy presented here is useful for any extensive phenotyping of geminivirus variants and particularly of artificially generated mutants or recombinants.

Dataset Information

An effective homologous cloning method for isolating novel miR172s from Phalaenopsis hybrida.

Publications

An effective homologous cloning method for isolating novel miR172s from Phalaenopsis hybrida.

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