Vacuolar invertases in sweet potato: molecular cloning, characterization, and analysis of gene expression.
Two cDNAs (Ib beta fruct2 and Ib beta fruct3) encoding vacuolar invertases were cloned from sweet potato leaves, expressed in Pichia pastoris, and the recombinant proteins were purified by ammonium sulfate fractionation and chromatography on Ni-NTA agarose. The deduced amino acid sequences encoded by the cDNAs contained characteristic conserved elements of vacuolar invertases, including the sequence R[G/A/P]xxxGVS[E/D/M]K[S/T/A/R], located in the prepeptide region, Wxxx[M/I/V]LxWQ, located around the starting site of the mature protein, and an intact beta-fructosidase motif. The pH optimum, the substrate specificity, and the apparent K(m) values for sucrose exhibited by the recombinant proteins were similar to those of vacuolar invertases purified from sweet potato leaves and cell suspensions, thus confirming that the proteins encoded by Ib beta fruct2 and Ib beta fruct3 are vacuolar invertases. Moreover, northern analysis revealed that the expression of the two genes was differentially regulated. With the exception of mature leaves and sprouting storage roots, Ib beta fruct2 mRNA is widely expressed among the tissues of the sweet potato and is more abundant in young sink tissues. By contrast, Ib beta fruct3 mRNA was only detected in shoots and in young and mature leaves. It appears, therefore, that these two vacuolar invertases play different physiological roles during the development of the sweet potato plant.
Wang
Li-Ting
LT
Institute of Microbiology and Biochemistry, National Taiwan University, Taipei 106, Taiwan.
Wang
Ai-Yu
AY
Hsieh
Chang-Wen
CW
Chen
Chih-Yu
CY
Sung
Hsien-Yi
HY
D000595:Amino Acid Sequence; D001483:Base Sequence; D003001:Cloning, Molecular; D018076:DNA, Complementary / Q000737:chemistry / Q000302:isolation & purification; D015870:Gene Expression; D027723:Ipomoea batatas / Q000201:enzymology* / Q000648:ultrastructure; D008969:Molecular Sequence Data; D018515:Plant Leaves / Q000201:enzymology; D035281:Plant Tubers / Q000201:enzymology; D016415:Sequence Alignment; D014617:Vacuoles / Q000201:enzymology*; D043324:beta-Fructofuranosidase / Q000737:chemistry / Q000235:genetics*
D018076:DNA, Complementary; D043324:beta-Fructofuranosidase