Cloning of four oligosaccharide metabolism-related genes in Andrena camellia and their expression in Escherichia coli in vivo
Author of the article:XU Tian-Yu1, 2** LI Zhen3** ZENG Zhi-Jiang1, 2***
Author's Workplace：1. Honeybee Research Institute, Jiangxi Agricultural University, Nanchang 330045, China; 2. Jiangxi Province Key Laboratory of Honeybee Biology and Beekeeping, Nanchang 330045, China; 3. College of Life Science and Resources and Environment, Yichun University, Yichun 336000, China
Key Words：Andrena camellia; Camellia oleifera; honey bee; oligosaccharide metabolism; gene cloning; protein expression
Abstract：
[Aim] To clone four Andrena camellia oligosaccharide metabolism-related genes and
determine their expression in Escherichia coli in vivo. [Methods] Sequences of the coding regions of four
key genes; α-Galactosidase (α-GAL), Galactokinase (GALK), Galactose-1-phosphate
uridyltransferase (GALT) and Uridine diphosphate galactose-4'-epimerase (GALE),
were obtained through genome sequencing. Sequences of the coding regions of key
genes such as α-GAL, GALT, GALT and GALE were
enetically engineered to obtain the recombinant expression plasmids pET28a-GAL,
GALK, GALT, GALE, and the Escherichia coli strains pET28a-α-GAL-E.
coli BL21, pET28a-GALK-E. coli BL21, pET28a-GALT-E.
coli BL21, pET28a-GALE-E. coli BL21. [Results] α-GAL, GALK, GALT, and GALE were subjected to
induced expression and purification by Ni2+-NTA column
affinity chromatography. The molecular weights of the purified α-GAL, GALK,
GALT, and GALE were about 53, 55, 48 and 39 kD, respectively. Corresponding
concentrations of α-GAL, GALK, GALT, and GALE are 0.8, 1.5, 0.9 and 1.0 mg/mL,
respectively. [Conclusion] Genes
related to oligosaccharide metabolism of A. camellia were successfully
cloned and recombinant expressed proteins obtained. These results provide new
ideas for solving the problem of honey bees being poisoned by C.
oleifera, and provide technical support for the scientific use
of honey bees for the pollination of C. oleifera.