过表达天麻GS1基因促进蜜环菌在低温条件下的生长  

Overexpression of Gastrodia elata GS1 Gene Promotes the Growth of Armillaria mellea Under Low Temperature

作　　者：陈必连 周春艳 蔡金龙 胥鹏 徐慧妮[1] 李昆志[1] CHEN Bi-Lian;ZHOU Chun-Yan;CAI Jin-Long;XU Peng;XU Hui-Ni;LI Kun-Zhi(Faculity of Life Science and Technology,Kunming University of Science and Technology,Kunming 650500,China)

机构地区：[1]昆明理工大学生命科学与技术学院,昆明650500

出　　处：《农业生物技术学报》2023年第12期2599-2611,共13页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金项目(31960071)。

摘　　要：天麻(Gastrodia elata)是中国传统大宗药材,是一种药食兼用植物。天麻无根无叶,只能通过消化侵染的蜜环菌(Armillaria mellea)获取营养。氮素营养是天麻和蜜环菌生长所必需的,谷氨酰胺合成酶(glutamine synthetase,GS)在氮素代谢方面起着重要作用。天麻自然生长周期长,常受到冬天和早春低温胁迫。研究表明,过表达细胞质GS1基因能提高植物氮素利用、植物发育和盐胁迫耐性,但GS1基因对低温胁迫的抗性作用还不清楚。本研究从天麻转录组数据库中筛选到GS1基因5'端CDS序列,通过巢式PCR技术克隆其全长,并构建其过表达载体pH2GW7.0-35s-GS1和原核表达载体pET-32a-GS1。pET-32a-GS1转化大肠杆菌(Escherichia coli)Bl21进行蛋白表达纯化获得GS1酶蛋白。该酶的最适pH为4,最适温度为50℃。低浓度5 mmol/L Ca^(2+)、Mg^(2+)和K^(+)条件下对GS1酶活性有促进作用,9 mmol/L高浓度金属离子对GS1酶活性有抑制作用。过表达GS1蜜环菌与野生蜜环菌相比,在低温13℃条件下能显著提高与谷氨酸和谷胱甘肽合成有关的GS、谷氨酸合成酶(glutamate synthase,GoGAT)、谷氨酸脱氢酶(glutamate dehydrogenase,GDH)、谷胱甘肽还原酶(glutathione reductase,GR)和谷胱甘肽过氧化物酶(glutathione peroxidase,GPX)基因表达水平,提高脯氨酸、谷胱甘肽和可溶性糖含量,降低H2O2和丙二醛(malondialdehyde,MDA)含量,明显促进蜜环菌生长。研究结果表明,过表达天麻GS1基因能够提高蜜环菌在低温条件下的生长能力,本研究为进一步提高天麻产量提供了参考。Gastrodia elata is a traditional Chinese medicinal herb that can be used for both medicine and food.G.elata has no roots or leaves,and can only obtain nutrients through the digestion of infected Armillaria mellea.Nitrogen nutrition is essential for the growth of G.elata and A.mellea,and glutamine synthetase(GS)plays an important role in regulating nitrogen metabolism.Previous studies have shown that overexpression of the cytoplasmic GS1 gene can improve plant nitrogen utilization,plant development,and salt stress tolerance,but the resistance of the GS1 gene to low temperature stress is still unclear.In this study,the 5'CDS of GS1 gene was screened from the transcriptome database of G.elata,and it's full length was cloned by Nested-PCR.The overexpression vector pH2GW7.0-35s-GS1 and prokaryotic expression vector pET-32a-GS1 were constructed.pET-32a-GS1 was transformed into Escherichia coli Bl21 for protein express and purification to obtain GS1 enzyme protein.The optimal pH of GS1 enzyme was 4 and the optimal temperature was 50℃.Under low concentration of 5 mmol/L Ca^(2+),Mg^(2+)and K^(+)had a promoting effect on GS1 enzyme activity.While at high concentration of 9 mmol/L metal ions had a inhibitory effect on GS1 enzyme activity.Compared with wild A.mellea,overexpression of GS1 significantly enhanced the expression levels of GS,glutamate synthase(GoGAT),glutamate dehydrogenase(GDH),glutathione reductase(GR)and glutathione peroxidase(GPX)genes related to glutamate and glutathione synthesis under low temperature conditions of 13℃,increased the content of proline,glutathione and soluble sugar,and reduced the content of H2O2 and malondialdehyde(MDA),significantly promoted the growth of A.mellea.These results indicated that overexpression of G.elata GS1 gene could enhance the growth ability of A.mellea under low temperature conditions.This study provids a reference for further research on enhancing growth and yield of G.elata under low temperature.

关 键 词：谷氨酰胺合成酶(GS) 蜜环菌 低温胁迫 

分 类 号：S182[农业科学—农业基础科学]