Abstract: 【Objective】 This paper aims to isolate the PmKNAT2 gene from Japanese apricot (Prunus mume Sieb. et Zucc.) cv ‘Daqiandi’, and analyze the structure and expression pattern of this gene, for further studying the molecular mechanism of Japanese apricot pistil abortion and molecular breeding. 【Method】 Specific primers were designed based on the peach sequence (EF093491) in NCBI, which is the highest homologue with peach gene KNOPE2. The improved CTAB method was used to isolate total RNA and the full length of PmKNAT2 cDNA was obtained by using RT-PCR and RACE. The sequencing data were assembled by DNAMAN software; BLASTn and BLASTp in NCBI were used to do the similarity analysis. PmKNAT2 gene structural characteristics were analyzed by the following software: DNAMAN was used to analyze the ORF and amino acid sequences and MEGA4.0 was used to create the phylogenetic tree; the protein molecular weight and isoelectric point were speculated by using Bioxm2.6; the conserved domain structure of protein was predicted by Conserved Domains program in NCBI; the protein secondary structure was predicted by using SOPMA program. The fusion expression vector PJIT166-PmKNAT2-GFP was constructed and then introduced into onion epidermal cells by the particle bombardment method; green fluorescence was monitored under a laser scanning confocal microscope. Quantitative real-time PCR (qRT-PCR) was performed to determine the expression pattern of PmKNAT2 in different developmental stages of flower buds and different flower organs. 【Result】 The full length of PmKNAT2 cDNA was 1 402 bp and contained 47 bp 5′UTR, 293 bp 3′UTR and a 1 062 bp ORF which encoded a 353 amino acids polypeptide with a calculated molecular weight of 40.14 kD and an isoelectric point of 4.85. Protein structure analysis showed that PmKNAT2 contained two kinds of domain namely MEINOX area (KNOXⅠand KNOXⅡ) and HD area, which indicated that it belongs to the KNOX protein. Similarity analysis showed that the predictive amino acid sequence of PmKNAT2 compared with other sequences of KNOX in GenBank shared 50%-100% in homology. The phylogenetic tree analysis showed that PmKNAT2 was clustered together with peach KNOX protein, which was consistent with the morphological classification. In addition, the predictive secondary structure showed that PmKNAT2 protein was made up of 47.14% alpha-helix, 3.43% beta-turn, 3.14% extended strand and 46.29% random coil. Subcellular localization results showed that the PmKNAT2 protein localized in cell nucleus and cell membrane. Real-time PCR analysis showed that the expression level of PmKNAT2 was various in different stages of flower buds of ‘Daqiandi’ , the highest level in November. There were no significant difference in September, October and November. However, there was a significant difference between December and January. As for the determination of auxin content, the results showed that the highest level in January, and there was no significant difference in September, October and November; in contrast with the trend of gene expression. The expression analysis of flower buds in November showed that PmKNAT2 expressed in all the tissues, the expression level of imperfect flower (pistil brown, pistil deformity and no pistil) were higher than perfect flower. There was no tissue specific expression of PmKNAT2 gene between perfect flower and imperfect flower. The expression level in the sepal was higher than that in the stamen and the expression level in the stamen was higher than that in the petal. The lowest expression level of pistil was in perfect flower. 【Conclusion】 The abnormal expression of this gene might be related to pistil abortion in ‘Daqiandi’.

Key words: Japanese apricot , pistil abortion , PmKNAT2 , gene cloning , characterization , expression pattern