Isolation And Functional Analysis Of Pistil Development Related Gene In Japanese Apricot

Japanese apricot (Prunus mume Sieb. et Zucc.) originated in China belongs to Rosaceae. The flowers of which have high ornamental value, the fruit of which was rich in nutrients. However, the phenomenon of pistil abortion widely occurs in Japanese apricot and has seriously affected the yield in production. We used'daqiandi'and'longyan'grown in the 'National Field Genbank for Japanese apricot' as experimental material. The PmKNAT2 and PmCCoAOMT were isolated through Rapid Amplification of cDNA Ends (RACE). Gene structural characteristics were analyzed by the bioinformatics software; Subcellular localization experiment of onion epidermal cells and quantitative real-time PCR (qRT-PCR) were carried out. The PmCCoAOMT gene functiong was preliminaries identified by transferred it into tobacco (Nicotiana). The main research results are as follows:1 Isolation, subcellular localization and expression analysis of PmKNAT2 gene. Specific primers were designed based on the peach sequence (EF093491) in NCBI, which is the highest homologue with peach gene KNOPE2. Isolate the KNAT2 homologous gene from Japanese apricot (Prunus mume Sieb. et Zucc.) cv 'Daqiandi', named as PmKNAT2.The full length of PmKNAT2 cDNA was 1402bp and contained 47bp 5'UTR, 293bp 3'UTR and a 1062bp ORF which encoded a 353 amino acid 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 (KNOX? and KNOX?) and HD area, which indicated that it was belonged 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. Subcellular localization results showed that the PmKNAT2 protein was localized in the 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 was 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 differences in September, October and November; in contrast with the trend of gene expression. Expression analysis of November showed that PmKNAT2 expressed in all the tissues, the expression level of imperfect flower (pistil brown, pistil deformity and no pistil) was 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.2 Isolated and bioinformatics analysis of PmCCoAOMT genes. Specific primers were designed based on the peach sequence (DW351650.1) in NCBI, the PmCCoAOMTcDNA was obtained by using RT-PCR and RACE. The structural characteristics were analyzed by the bioinformatics software. The results showed that the PmDCCoAOMT gene ORF which encoded a 247 amino acid polypeptide with a calculated molecular weight of 27.91 kD and an isoelectric point of 5.42, the PmLCCoAOMT gene ORF which encoded a 247 amino acid polypeptide with a calculated molecular weight of 27.89k kD and an isoelectric point of 5.42. Pro/hydrophobicity analysis showed that the protein is hydrophilic proteins, signal peptide analysis found that the protein without signal peptide, subcellular localization prediction showed that PmCCoAOMT protein might be localizedin the cytoplasm, similarity analysis showed that the predictive amino acid sequence of PmCCoAOMT compared with other sequences in GenBank shared 88%-96% in homology.3 Expression analysis and subcellular localization experiment of onion epidermal cells. The fusion expression vector PJIT166-PmCCoAOMT-GFP was constructed. Subcellular localization results showed that the PmCCoAOMT protein was localized in the cell cytoplasm. Real-time PCR analysis showed that PmCCoAOMT gene expressed in two cultivars, in the prophase development of pistil the expression level of 'daqiandi' were higher than 'longyan', in the late period development of pistil the expression level of 'longyan'were higher than'daqiandi', the expression level of perfect flower(perfect pistil) and imperfect flower (pistil brown), were higher than imperfect flower (pistil deformity and no pistil).4 Functional analysis of PmCCoAOMT. We constructed an overexpression vector of PBI121-PmCCoAOMT and transferred it into tobacco by Agrobacterium tumefaction, obtained 4 Kan positive tobacco plants. Analyses of GUS straining, PCR and RT-PCR showed that obtained 4 positive tobacco plants. The observer of transgenic tobacco lines and wild-type lines showed that under the same culture conditions, compared with the control, transgenic tobacco plants were higher, the flowers were bigger, the petal color became dark, the pistil became longer, respectively.