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An effective homologous cloning method for isolating novel miR172s from Phalaenopsis hybrida
Ying Ying Han1, Qin Hua Yan2, Feng Ming2
1State Key Laboratory of Genetic Engineering , Institute of Genetics , Institute of Plant Biology , School of Life Sciences , Fudan University , Shanghai , China . ; Center of Systematic Biomedical Research , College of Medical Appliance and Food Sciences , University of Shanghai for Science and Technology , Shanghai , China .
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.
