Wang Xu Tong, Sun Ting Ting, Sun Jian, Wang Shixin, Zou Li
College of Forestry, Northeast Forestry University, Harbin 150040, China.
Department of Food Engineering, Harbin University, Harbin 150086, China.
Protein Pept Lett. 2022;29(1):37-45. doi: 10.2174/0929866528666210922103059.
Sanghuangporus baumii is a traditional Chinese medicine with anti- cancer, anti-tumor, and anti-inflammatory effects. Triterpenoids are one of the main medicinal ingredients found in S. baumii. However, the dynamic changes of triterpenoids content and its molecular regulation mechanism are still unclear.
Some studies have shown that Lanosterol synthase ( LS) is a key enzyme involved in the mevalonate pathway (MVA pathway) to produce lanosterol, which is a precursor for synthesizing S. baumii triterpenoids. Therefore, the study of LS gene and expression characteristics can provide clues for the further study of triterpenoids synthesis.
The PCR, RACE PCR, RT-PCR, homologous recombination and prokaryotic expression technology were used to research the gene characteristic and dynamic changes of LS transcription level.
The S. baumii LS sequence included a 5'-untranslated region (129 bp), a 3'-untranslated region (87 bp), and an open reading frame (2,229 bp) encoding 734 amino acids. The S. baumii LS protein was expressed in E. coli BL21 (DE3). The transcription start site of the S. baumii LS promoter sequence ranged from 1 740 bp to 1790 bp. The LS promoter contained 12 CAAT-boxes, 5 ABREs, 6 G-Boxes, 6 CGTCA-motifs, and so on. The LS transcription levels were the highest on day 11 in mycelia (1.6-fold), and the triterpenoids content also gradually increased. The transcription levels began to decrease on day 13, but the triterpenoids content still increased.
The S. baumii LS was cloned and characterized to help to understand the mechanism of triterpenoids synthesis. In addition, we studied the relationship between LS transcription level and triterpenoid dynamic accumulation, and we found that they had a certain correlation.
鲍氏层孔菌是一种具有抗癌、抗肿瘤和抗炎作用的传统中药。三萜类化合物是鲍氏层孔菌中发现的主要药用成分之一。然而，三萜类化合物含量的动态变化及其分子调控机制仍不清楚。
一些研究表明，羊毛甾醇合酶（LS）是甲羟戊酸途径（MVA途径）中产生羊毛甾醇的关键酶，羊毛甾醇是合成鲍氏层孔菌三萜类化合物的前体。因此，对LS基因及其表达特性的研究可为进一步研究三萜类化合物的合成提供线索。
采用PCR、RACE PCR、RT-PCR、同源重组和原核表达技术研究LS的基因特性和转录水平的动态变化。
鲍氏层孔菌LS序列包括一个5'非翻译区（129 bp）、一个3'非翻译区（87 bp）和一个编码734个氨基酸的开放阅读框（2229 bp）。鲍氏层孔菌LS蛋白在大肠杆菌BL21（DE3）中表达。鲍氏层孔菌LS启动子序列的转录起始位点范围为1740 bp至1790 bp。LS启动子包含12个CAAT盒、5个ABRE、6个G盒、6个CGTCA基序等。菌丝体中LS转录水平在第11天最高（1.6倍），三萜类化合物含量也逐渐增加。转录水平在第13天开始下降，但三萜类化合物含量仍在增加。
克隆并鉴定了鲍氏层孔菌LS，有助于了解三萜类化合物的合成机制。此外，我们研究了LS转录水平与三萜类化合物动态积累之间的关系，发现它们具有一定的相关性。
