Abstract:

In this study,three invertase genes were cloned from Hemerocallis fulva‘Athlone’,named HfCIN3,HfCWIN1 and HfVIN1. The ORFs of the three invertase genes were 1 941,1 701,1 920 bp,encoding 646,566 and 639 amino acids,respectively,with the similarity of 18.81%-41.86%. The amino acid sequence of HfCIN3 was 75.16%-82.79% similar to those of dendrobium and wild banana,HfCWIN1 was 58.95%-68.88% similar to asparagus and oil palm,and HfVIN1 was 73.4% similar to agave. HfCIN3 had a signal peptide and typical glycoside hydrolases family 100 domain,HfCWIN1 and HfVIN1 had glycosyl hydrolases family 32 domain. HfCWIN1 also contained a 72 amino acid Malectin domain related to cell wall sugar recognition. HfVIN1 contained a possible vacuolar motif“ILPD”and had a typical transmembrane domain at the N terminus. Both HfCWIN1 and HfVIN1 possessed the N-glycosylation and O-β-GlcNAc sites,whereas HfCIN3 only had the O-β-GlcNAc sites. Phylogenetic analysis showed that monocotyledons and dicotyledons could clearly be distinguished regarding different types of invertase;CWIN and VIN were closely related,and HfCIN3,HfCWIN1 and HfVIN1 were clustered in monocots. Transient expression analysis showed 35S:HfCIN3-GFP was expressed in chloroplasts;35S:HfVIN1-GFP was expressed in vacuoles;and 35S:HfCWIN1-YFP was detected in cell walls and guard cells. The expression levels of HfCIN3,HfCWIN1 and HfVIN1 were significantly higher in leaves than that of the root and flower tissues;the expression levels in leaves were HfVIN1 > HfCWIN1 > HfCIN3. After 24 h treatment at 0 ℃,the expression of HfVIN1 was increased by 2.2 times compared with 5 ℃,and the expression level of HfCWIN1 was increased significantly at 0 ℃ compared with the other temperatures. The highest expression of HfVIN3 was recorded at 0 ℃. After 24 h treatment with 5% or 10% PEG,the expression levels of HfCIN3 and HfVIN1 were not significantly different from the control,but that of HfCWIN1 was significantly lower than the control. The CIN activity and gene expression were similar in different tissues,but CWIN and VIN were not consistent with the gene expression. As temperature decreased,CIN declined,CWIN increased at first and then decreased,and VIN decreased at first then increased,and then decreased;PEG induced VIN activity. The enzyme activity and gene expression were not synchronized after the low temperature and osmotic stress treatment,possibly because the gene post-transcriptional modifications played an important role in invertase activity. The results of this study established the differences in subcellular localizations,gene expression patterns and the responses to abiotic stresses among the three various types of Hemerocallis fulva invertase,HfVIN1 responded to low temperature,whereas HfCWIN1 responded to osmotic stress.

Key words: Hemerocallis fulva, sucrose, invertase, abiotic stress, gene expression