Molecular Cloning and Functional Identification of Invertase Isozymes from Green Bamboo Bambusa oldhamii

• Published in: Journal of agricultural and food chemistry 2006-04, Vol.54 (8), p.3101-3107
• Main Authors: Hsieh, Chang-Wen, Liu, Li-Ka, Yeh, Sheng-Hsiung, Chen, Chi-Fu, Lin, Hsin-I, Sung, Hsien-Yi, Wang, Ai-Yu
• Format: Article
• Language: English
• Subjects: acidity; Amino Acid Sequence; amino acid sequences; Bambusa - enzymology; Bambusa oldhamii; beta-fructofuranosidase; beta-Fructofuranosidase - analysis; beta-Fructofuranosidase - chemistry; beta-Fructofuranosidase - genetics; Biological and medical sciences; cell walls; Cloning, Molecular; complementary DNA; cytochemistry; DNA, Complementary - genetics; enzyme activity; Food industries; Fundamental and applied biological sciences. Psychology; Gene Expression; isoelectric point; Isoenzymes - analysis; Isoenzymes - chemistry; Isoenzymes - genetics; isozymes; messenger RNA; Molecular Sequence Data; nucleotide sequences; Phylogeny; Pichia - genetics; Recombinant Proteins; Reverse Transcriptase Polymerase Chain Reaction; Sequence Alignment; shoots; substrate specificity; vacuoles
• ISSN: 0021-8561; 1520-5118
• DOI: 10.1021/jf052711s

Three Boβfruct cDNAs encoding acid invertases were cloned from shoots of the green bamboo Bambusa oldhamii. On the basis of the amino acid sequences of their products and phylogenetic analyses, Boβfruct1 and Boβfruct2 were determined to encode cell wall invertases, whereas Boβfruct3 encodes a vacuolar invertase. The recombinant proteins encoded by Boβfruct2 and Boβfruct3 were produced in Pichia pastoris and purified to near homogeneity using ammonium sulfate fractionation and immobilized metal affinity chromatography. The pH optima, pI values, and substrate specificities of the isolated enzymes were consistent with those of plant cell wall or vacuolar invertases. The growth-dependent expression of Boβfruct1 and Boβfruct2 in the base regions of shoots underscores their roles in sucrose unloading and providing substrates for shoot growth. Its high sucrose affinity suggests that the Boβfruct2-encoded enzyme is important for maintaining the sucrose gradient between source and sink organs, while the predominant expression of Boβfruct3 in regions of active cell differentiation and expansion suggests functions in osmoregulation and cell enlargement. Keywords: Bamboo (Bambusa oldhamii); cell-wall invertase; vacuolar invertase; cDNA cloning; recombinant invertase; gene expression