灵芝端粒解旋酶基因的克隆与生物学功能  

Cloning and biological function of telomere-linked helicase gene in Ganoderma lingzhi

作　　者：冷豆豆 尚丽 邹明 朱云龙 武忠伟[1] 靳荣线[2] 张广 LENG Doudou;SHANG Li;ZOU Ming;ZHU Yunlong;WU Zhongwei;JIN Rongxian;ZHANG Guang(College of Life Science and Technology,Henan Institute of Science and Technology,Xinxiang 453000,Henan,China;The Institute of Edible Fungi,Xinxiang Academy of Agricultural Sciences,Xinxiang 453003,Henan,China)

机构地区：[1]河南科技学院生命科技学院,河南新乡453000 [2]河南省新乡市农业科学院食用菌研究所,河南新乡453003

出　　处：《菌物学报》2023年第8期1718-1729,共12页Mycosystema

基　　金：国家自然科学基金青年基金(31902090);河南省科技攻关项目(222102110431)。

摘　　要：端粒解旋酶(telomere-linked helicase,Tlh)在维持端粒长度和基因组稳定性中起关键作用。除其基础作用外,解旋酶还在涉及DNA/RNA代谢的细胞过程中担当重要的角色。本研究通过同源序列比对在灵芝中获得Tlh基因,分析发现Tlh基因编码750个氨基酸,其蛋白含有DEXHC-RecQ、DEAD和HELICc结构域,与担子菌中的Tlh蛋白保守性较高。进一步构建灵芝Tlh沉默菌株(Tlhi-9和Tlhi-21),检测Tlh在灵芝中的生物学功能。结果显示,与野生型相比,Tlhi-9和Tlhi-21菌株菌丝生长减缓,菌落直径分别降低23%和24%,生物量分别降低41%和43%;相较野生型,Tlhi-9和Tlhi-21菌株中CMCase活性分别降低39%和43%,而木聚糖酶活性均降低40%;Tlhi-9和Tlhi-21菌株中胞内多糖含量较野生型分别降低38%和40%;Tlhi-9和Tlhi-21菌株中灵芝酸含量较野生型分别降低51%和56%。研究结果说明,Tlh基因参与调控灵芝生长、纤维素酶与木聚糖酶活性及次生代谢。Telomere-linked helicase(Tlh)plays crucial role in maintaining telomere length and genomic stability.In addition,helicase is also involved in cellular processes concerning DNA/RNA metabolism.In this study,Tlh gene was obtained by homologous sequence alignment in Ganoderma lingzhi.Sequence analysis revealed that the Tlh gene encoded 750 amino acids with a high evolutionary conservation in basidiomycetes,and its protein contained DEXHC-RecQ,DEAD and HELICc domains.The Tlh-silenced strains(Tlhi-9 and Tlhi-21)were constructed to explore the biological functions of Tlh in G.lingzhi.The results showed that the mycelia of Tlhi-9 and Tlhi-21 strains grew slower than those of wild type strain(WT).Comparison of Tlhi-9 and Tlhi-21 with WT showed that the colony diameter of Tlhi-9 and Tlhi-21 decreased by 23%and 24%,the biomass decreased by 41%and 43%,and the CMCase activities decreased by 39%and 43%,respectively;the xylanase activities of both Tlhi-9 and Tlhi-21 were reduced by 40%.The content of intracellular polysaccharides in Tlhi-9 and Tlhi-21 was reduced by 38%and 40%,respectively,compared with that in WT.The content of ganoderic acids in Tlhi-9 and Tlhi-21 was reduced by 51%and 56%,respectively,compared with that in WT.These results indicate that the Tlh gene is involved in the regulation of fungal growth,cellulase and xylanase activities and secondary metabolism.

关 键 词：端粒解旋酶 灵芝酸 纤维素酶 次生代谢 

分 类 号：S567.31[农业科学—中草药栽培]