摘要
通过转录组测序得到牛樟芝(Antrodia cinnamomea)cDNA序列,用合适的开放阅读框(open reading frame,ORF)进行BLAST得到羊毛甾醇合酶基因AcLSS的蛋白质编码区(sequence coding for amino acids in protein,CDS)序列,根据CDS序列设计引物进行PCR扩增,将AcLSS基因通过T4 DNA连接酶连接到载体pET28a上,通过原核表达得到重组蛋白,采用高效液相色谱(high performance liquid chromatography,HPLC)法检测体外酶促反应产物,验证其生物活性,并探讨AcLSS基因对不同环境因子的响应。结果表明:在2205 bp处有一条带,与CDS序列大小相符;编码734个氨基酸,相对分子质量大小约为80740,重组蛋白为可溶性蛋白质,能催化2,3-氧化角鲨烯生成羊毛甾醇,具有羊毛甾醇合酶的催化功能;与对照组(麸皮)相比,葡萄糖、蔗糖为碳源时,三萜含量降低,且组间差异均具有统计学意义,基因相对表达量组间差异均无统计学意义;与对照组(酵母提取物)相比,氮源为蛋白胨、硫酸铵时,三萜含量和基因相对表达量均降低,且组间差异均具有统计学意义;与对照组相比,添加金属离子时,三萜含量和基因相对表达量均下降,且组间差异均具有统计学意义;与对照组相比,茉莉酸甲酯、油酸、α-松油醇的添加均导致三萜含量下降,且组间差异均具有统计学意义,而油酸、α-松油醇的添加导致基因相对表达量增加,组间差异均具有统计学意义。
Through transcriptome sequencing and ORF BLAST analysis,the CDS of the lanosterol synthase gene AcLSS of Antrodia cinnamomea was identified,cloned and then expressed in E.coli.The resultant recombinant protein was first verified for its catalytic activity in vitro and then studied for its response to different carbon sources,nitrogen sources,metal ions and inducers,respectively.The results showed that the cloned AcLSS was 2205 bp,encoding 734 amino acids.The recombinant protein RAcLSS was soluble with a relative molecular weight of about 80740.It catalyzed the synthesis of lanosterol from 2,3-oxidized squalene,indicating that RAcLSS had the catalytic activity of lanosterol synthase.Compared with bran(control),glucose and sucrose decreased the content of triterpenoids regardless of the same AcLSS expression level.Compared with yeast extract(control),peptone and ammonium sulfate down-regulated AcLSS expression and also decreased the content of triterpenoids.Addition of Ca2+,Fe2+,Mn2+or Mg2+down-regulated AcLSS expression and also decreased the content of triterpenoids.Among different inducers,oleic acid andα-terpineol up-regulated AcLSS expression,but all the tested inducers decreased the content of triterpenoids.
作者
李甜甜
范智玉
梁运祥
梅余霞
LI Tiantian;FAN Zhiyu;LIANG Yunxiang;MEI Yuxia(State Key Laboratory of Agricultural Microbiology,College of Life Science and Technology,Huazhong Agricultural University,Wuhan 430070,Hubei,China)
出处
《食用菌学报》
CSCD
北大核心
2021年第5期55-63,共9页
Acta Edulis Fungi
基金
中央高校基本科研业务费(2662021FW006)
湖北省自然科学基金(2020CFB527)。
关键词
牛樟芝
羊毛甾醇合酶
酶促反应
三萜
基因表达
Antrodia cinnamomea
lanosterol synthase
enzymatic reaction
triterpenoid
gene expression
