Isolation And Expression Analysis Of Floral Fragrance Related Genes PmCFATs In Prunus Mume

Floral fragrance is one of the important characteristics of ornamental plants.It is of great importance to the survival and reproduction of plants,and its products have important economic value in food,health care,chemical industry and so on.Mei(Prunus mume)is a famous traditional flower,which can release pleasant aroma and is loved by Chineses people.Our previous study showed that eugenol was one of the characteristic aroma components of P.mume.Coniferyl alcohol acyltransferase(CFAT)and eugenol synthase(EGS)take part in the last two steps of the biosynthesis pathway of eugenol.CFAT is a key gene apart eugenol metabolism and lignin metabolism.However,there are few studies on CFAT.Study on eugenol metabolism of P.mume has just started.In this study,the genome-wide analysis of candidate in P.mume were performed.Then,coniferyl alcohol acyltransferase genes were isolated from P.mume 'Sanlun Yudie'.Their functions were explored by bioinformatics analysis,subcellular localization,genes expression patterns analysis,prokaryotic expression,construction and transformation of eukaryotic vectors.The main results are as follows:1.86 CFAT candidate genes were screened from P.mume genome database,and its evolution was divided into five branches.According to sequence alignment,conserved motifs analysis and phylogenetic analysis,Pm030674,Pm013138,Pm030672 and Pm023001 belonging to branch II were preliminary predicted as the homologous genes of CFAT in P.mume.Then,three full length cDNAs named PmCFAT1a,PmCFAT1b,PmCFAT2 were cloned from P.mume 'Sanlun Yudie'.Their sequence was 1383bp,encoding 460 amino acids belonging to unstable acidic protein.Their predicted molecular weight was 51.61 kDa,51.54 kDa and 50.92 kDa,respectively.PmCFAT1a and PmCFAT1b are allelic gene variants of Pm030674.Sequence alignment and phylogenetic analysis showed that PmCFATs were homologous with identified genes of coniferyl alcohol acyltransferase2.The GFP fusion expression vectors of PmCFATs were constructed and transiently expressed in tobacco leaves.The results showed that PmCFAT1a,PmCFATlb and PmCFAT2 were all located in cytoplasm and nucleus.3.The expression of PmCFAT1 was correlated with the release of eugenol.PmCFAT1 mainly expressed in stamens and petals with no expression in leaves,and its expression increased as the flowers opening up,achieved the maximum at stage 4,then begined to decline,suggesting that PmCFAT1 may take part in the biosynthesis of eugenol as an important upstream gene of PmEGS2.The relative expression of PmCFAT2 was higher in leaves than that in flowers,and showed no correlation with the eugenol synthesis and release in flowering process.4.Prokaryotic expression vectors of PmCFAT1a,PmCFAT1b,PmCFAT1 were constructed using pET-28b,and proteins about 52 kDa,52 kDa,51 kDa respectively expressed in Escherichia coli,which were consistent to the predicted molecular mass.The fusion protein expressed in the form of inclusion bodies.Though protein solubility improved slightly when they were induced using 0.2 mM IPTG at 16 C with 14 hours,most of them still expressed as inclusion bodies.The expression vector pCHF3301 were used to build single gene expression vectors of PmCFAT1a,PmCFAT1b,PmCFAT2,PmEGS2 and double gene expression vectors of pCHF330 1-PmEGS2-PmCFAT1a,pCHF330]-PmEGS2-PmCFAT1b,pCHF3301-PmEGS2-PmCFAT2.Besides,hygromycin resistance expression vectors named pSuper1300-PmCFAT1a,pSuper1300-PmCFAT1b,pSuper1300-PmCFAT2 were constructed.Preliminary transformation and Basta screening of Petunia hybrida were carried out.In this study,three coniferyl alcohol acyltransferase cDNAs were isolated from P.mume 'Sanlun Yudie',genes expression patterns were analysised and their functions were preliminary explored.This study lays the foundation for further analysis of PmCFATs functions,metabolic pathway of eugenol,as well as the depth mining of BAHD genes.