油茶地蜂中四个寡糖代谢相关基因克隆及其在大肠杆菌体内表达
Cloning of four oligosaccharide metabolism-related genes in Andrena camellia and their expression in Escherichia coli in vivo
徐天羽1, 2** 李 震3** 曾志将1, 2***
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DOI：10.7679/j.issn.2095-1353.2025.084
作者单位：1. 江西农业大学蜜蜂研究所，南昌 330045；2. 蜜蜂生物学与饲养江西省重点实验室，南昌 330045； 3. 宜春学院生命科学与资源环境学院，宜春 336000
中文关键词：油茶地蜂；油茶；蜜蜂；寡糖代谢；基因克隆；蛋白表达
英文关键词：Andrena camellia; Camellia oleifera; honey bee; oligosaccharide metabolism; gene cloning; protein expression
中文摘要：
【目的】 油茶授粉效率很低，存在“百花一果”现象，严重影响油茶产业发展。人工饲养的东方蜜蜂Apis cerana和西方蜜蜂Apis mellifera虽具广泛授粉适应性，却受油茶花中寡糖影响，出现中毒症状。野生油茶地蜂Andrena camellia能为油茶有效授粉，可以依靠自身肠道分泌的4种关键酶消化油茶蜜、粉中的寡糖，但数量有限。为了解决油茶授粉难题，我们克隆了油茶地蜂体内4种寡糖代谢酶。【方法】通过基因组测序成功获得了α-半乳糖苷酶（α-Galactosidase, α-GAL）、半乳糖激酶（Galactokinase,
GALK）、半乳糖-1-磷酸尿苷转移酶（Galactose-1-phosphate uridyltransferase,
GALT）和尿苷二磷酸半乳糖-4′-变位酶（Uridine diphosphate
galactose-4′-epimerase, GALE） 4个与寡糖代谢相关的关键基因编码区序列，并构建了重组表达质粒pET28a-α-GAL、pET28a-GALK、pET28a-GALT、pET28a-GALE及大肠杆菌Escherichia coli表达工程菌株pET28a-GAL-E. coli BL21、pET28a-GALK-E. coli BL21、pET28a-GALT-E. coli BL21和pET28a-GALE-E. coli BL21。【结果】 对α-GAL、GALK、GALT和 GALE进行了诱导表达和Ni2+-NTA 柱亲和层析纯化，获得纯化后的α-GAL、GALK、GALT和 GALE的蛋白分子量分别约53、55、48和39 kD，浓度分别为0.8、1.5、0.9和1.0 mg/mL。【结论】 本研究成功克隆油茶地蜂寡糖代谢相关基因并获得重组表达蛋白，研究结果为破解蜜蜂访问油茶后中毒的“瓶颈”提供了新思路和方法，为科学利用蜜蜂为油茶授粉提供了技术支撑。
英文摘要：
[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.