[关键词]
[摘要]
目的 为了更好地挖掘牛樟芝Antrodia cinnamomea菌丝体三萜合成和代谢相关基因、筛选互作蛋白和牛樟芝指纹图谱分析,构建了牛樟芝菌丝体cDNA文库并分析其表达序列标签(EST)序列。方法 以牛樟芝菌丝体为材料,采用Gateway法构建其cDNA文库,对部分EST序列进行生物信息学分析、功能注释和EST-SSR分析。结果 成功构建了牛樟芝菌丝体cDNA文库,经鉴定文库重组率高达95%,文库滴度为6.1×106cfu/mL,总克隆数为1.2×107cfu,插入片段大小为300~2 000 bp,平均长度达1 000 bp。随机挑选单克隆进行测序获得65个有效EST序列,其中1个重叠群,64个单一序列,比对结果显示有45条序列有明确功能注释,18条为未知功能基因,GO功能注释结果表明序列涉及细胞组成、转运、催化活性、调控等功能。所有EST序列共含有271个SSR,牛樟芝核苷酸重复类型丰富,其中二核苷酸和三核苷酸重复基元占总重复基元的94.23%。结论 初步明确牛樟芝菌丝体cDNA文库及其EST序列相关生物信息,为牛樟芝菌丝体基因组学研究奠定理论基础。
[Key word]
[Abstract]
Objective To excavate the terpenoid synthesis and metabolism-related gene function and screen the interaction protein and fingerprint analysis of Antrodia cinnamomea mycelium, a cDNA library from A. cinnamomea mycelia was constructed and the EST sequences were analyzed. Methods The cDNA library from the A. cinnamomea mycelium was constructed by the Gateway technique. A part of EST sequences about the bioinformatics, functional annotation and EST-SSR were analyzed. Results The cDNA library of the A. cinnamomea mycelium was constructed successfully. The recombinant rate of the cDNA library was 95%, the titer of the library was 6.1×106 cfu/mL, the total cloning number was 1.2×107 cfu, the length of cDNA was between 300-2 000 bp with an average length of 1 000 bp. The clones were randomly sequenced and 65 valid ESTs were obtained. After being compared in the Genbank database, 45 ESTs had a definite annotation, and 18 ESTs were unnamed and hypothetical protein. The results with GO functional annotation showed that the ESTs involved the cell composition, transport, catalytic activity, regulation functions and etc. It contained 271 SSRs of all the ESTs in total. The nucleotide repeats in A. cinnamomea were abundant, among which dinucleotide and trinucleotide repeat units were more common accounting for 94.23%. Conclusion The cDNA library from the A. cinnamomea mycelium and its ESTs related biological information were preliminarily identified, which will provide a theoretical foundation for research the mycelium genomics of A. cinnamomea.
[中图分类号]
R282.12
[基金项目]
福建省教育厅中青年科技项目“牛樟芝深层发酵及其三萜成分差异研究”(JT180121)