摘要
油茶因自交不亲和导致授粉效率低下,而利用蜜蜂授粉时,其花蜜中的棉籽糖家族寡糖(raffinose family of oligosaccharides,RFOs)会引发蜜蜂中毒.本研究旨在通过重构巴斯德毕赤酵母(Pichia pastoris)表达油茶地蜂(Andrena camellia)的3种半乳糖代谢关键酶(半乳糖激酶,galactokinase,GALK;半乳糖-1-磷酸尿苷转移酶,galactose-1-phosphate uridyltransferase,GALT;尿苷二磷酸-4′-变位酶,uridine diphosphate galactose-4′-epimerase,GALE),探究其对蜜蜂寡糖解毒能力的提升效果.通过质粒重组技术构建p PIC9K-GALK/GALT/GALE表达载体,电转化至巴斯德毕赤酵母(GS115)中,经诱导表达获得重组酶.通过Western blot验证蛋白表达,并检测酶活性.体外实验评估酶对半乳糖的代谢效率,同时以添加重组酶的糖水饲喂西方蜜蜂(Apis mellifera)工蜂,统计其存活率及中位生存期.成功实现GALK,GALT和GALE在巴斯德毕赤酵母中的高效表达.体外消化实验表明,三酶联用可使半乳糖含量显著降低(P<0.05),并检测到Leloir途径代谢产物(UDP-葡萄糖).饲喂实验显示:饲喂添加3种酶的糖水的工蜂平均寿命较对照组显著延长34.43%(P<0.05).巴斯德毕赤酵母表达的GALK,GALT和GALE可有效代谢油茶寡糖中的半乳糖,显著缓解蜜蜂中毒症状.本研究为生物工程法改良蜜蜂代谢能力提供技术支撑,对推动油茶产业可持续发展具有重要意义.
Due to self-incompatibility,Camellia’s pollination efficiency is low.When honey bees are introduced for pollination,the raffinose oligosaccharides in the nectar induce bee poisoning.The purpose of this study is to reconstruct Pichia pastoris to express three key enzymes of galactose metabolism(GALK,GALT,GALE)in Andrena camellia to explore their effect on the detoxification ability of oligosaccharides in honeybees.The pPIC9K-GALK/GALT/GALE expression vector was constructed using plasmid recombination technology,which was then electrotransformed into Pichia pastoris GS115 and induced to produce the recombinant enzyme.Protein expression was verified by Western blot.In in vitro experiments,we fed honey bees sugar water supplemented with the recombinant enzymes.We then evaluated the metabolic efficiency of the enzymes in galactose,the survival rate,and the median survival of the bees.The results showed that the efficient expression of GALK,GALT,and GALE in P.pastoris was successfully achieved.In in vitro digestion experiments,the combination of three enzymes could significantly reduce the content of galactose(P<0.05),and we detected Leloir pathway metabolites(such as UDP-glucose).The feeding experiment showed that the median survival of the worker bees supplemented with three enzymes was 34.43%longer than that of the control group(P<0.05).GALK,GALT,and GALE expressed by Pichia pastoris can effectively metabolize galactose in Camellia oleifera oligosaccharides,significantly alleviating the symptoms of bee poisoning.This study provides technical support for improving honey bee metabolism using the bioengineering method,which is of great significance for promoting the sustainable development of the Camellia oleifera industry.
作者
徐天羽
李震
颜伟玉
吴小波
曾志将
XU TianYu;LI Zhen;YAN WeiYu;WU XiaoBo;ZENG ZhiJiang(Honeybee Research Institute,Jiangxi Agricultural University,Nanchang 330045,China;College of Life Science and Resources and Environment,Yichun University,Yichun 336000,China;Jiangxi Province Key Laboratory of Honeybee Biology and Beekeeping,Nanchang 330045,China)
出处
《中国科学:生命科学》
北大核心
2025年第11期2286-2296,共11页
Scientia Sinica(Vitae)
基金
国家蜂产业技术体系(批准号:CARS-44-KXJ15)
国家自然科学基金(批准号:32460137)
江西省重点实验室基金项目(批准号:2024SSY04151)资助。
关键词
巴斯德毕赤酵母
质粒重组
半乳糖代谢酶
油茶寡糖
蜜蜂解毒
Pichia pastoris
plasmid recombination
galactose metabolizing enzymes
oligosaccharide metabolism
honeybee detoxification
