| Japanese apricot(Prunus mume Sieb.et Zucc),which belongs to Rosaceae,contains many amino acids that benefit the body metabolism and is known as the healthy food.Pistil is the basis of fruit formation.In production,pistils often develop abortion.In order to discover the mechanism of pistil development in Japanese apricot,the Pm-miR319a precursor and its target gene were isolated on the basis of previous studies.The cleavage of Pm-miR319a to the target gene Pm-TCP4 was clarified.The structure and function of Pm-TCP4 were analyzed by bioinformatics.Meanwhile,the expression of Pm-miR319a and its target gene Pm-TCP4 in different stages of pistil development were analyzed.The main results are as follows:1.It was found that miR319a may be associated with pistil development in Japanese apricot according to previous studies.The 229bp precursor of Pm-miR319a was cloned,sequenced and analyzed,with whose secondary structure was predicted.The precursor sequence and mature sequence of miR319a homologs in 19 other plant species were available from the miRbase 21.Through sequence alignment and phylogenetic analysis,mature miR319a was found to be highly conserved.Among the 20 species,miR319a has interspecific difference in evolutionary relationships.The Pm-miR319a target gene were predicted.The results showed that the target gene contained Pm-TCP4,which was consistent with Arabidopsis thaliana,indicating that the miR319a target gene was conserved.2.In order to explore the way of Pm-miR319a participating in pistil development in Japanese apricot,the ORF sequence was cloned by RT-PCR in the flower buds of the cultivar 'Daqiandi'.The results showed that Pm-TCP4 encodes 439 amino acids,which are consistent with the predicted sequence on NCBI.Pm-miR319a was confirm to cleave Pm-TCP4 by 5'RACE.Pm-TCP4 for biological information analysis showed that Pm-TCP4 encoded protein was a hydrophilic protein,with no signal peptide and located in the nucleus,and has a transmembrane structure.It can be speculated that it located on the nucleus membrane.This provided the foundation for exploring the role of Pm-miR319a in pistil development.3.In order to explore the role of Pm-miR319a and its target gene Pm-TCP4 in pistil development in Japanese apricot,we used qRT-PCR to detect the expression pattern of Pm-miR319a and Pm-TCP4 in two varieties 'Daqiandi' and 'Longyan' and different pistils(normal pistil and browning pistil)in 'Daqiandi'.The results showed that Pm-miR319a similarly expressed in the 'Daqiandi' and 'Longyan' in Japanese apricot,and the expression level of Pm-miR319a in 'Daqiandi' was higher than that of 'Longyan'.Observed Pm-TCP4 expression between the two varieties showed that Pm-TCP4 in the 'Daqiandi' was significantly higher than the 'Longyan'.The expression of Pm-miR319a in the normal pistil and browning pistil was not significantly different,and the expression of Pm-TCP4 in the pistil of browning was higher than that in the normal pistil.This indicated that Pm-TCP4 may play a role in pistil browning,which is relate to the occurrence of pistil abortion.For the downstream gene prediction of Pm-TCP4 transcription factor,16 genes related to flower development were found.Pm-TCP4 may affect the development of pistils through those genes.In summary,Pm-miR319a indirectly regulates the development of pistil by regulating its target gene Pm-TCP4. |
PDF Full Text Request