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The cloning and expression analysis of mevalonate pyrophosphate decarboxylase gene cDNA sequence from Sanghuangporus baumii
LIU Zengcai, SUN Tingting, WANG Shixin, MA Yisha, WANG Xutong, SUN Jian, ZOU Li
JOURNAL OF NANJING FORESTRY UNIVERSITY ›› 2020, Vol. 44 ›› Issue (4) : 79-85.
PDF(2297 KB)
PDF(2297 KB)
The cloning and expression analysis of mevalonate pyrophosphate decarboxylase gene cDNA sequence from Sanghuangporus baumii
To clone and characterize a mevalonate pyrophosphate decarboxylase (MVD) gene involved in the triterpenoids biosynthesis pathway in Sanghuangporus baumii is to understand the regulation mechanism of the triterpenoids biosynthesis pathway.
The characteristics of the MVD gene sequence were determined using a series of bioinformatic tools. The prokaryotic expression vector was constructed using homologous recombination. Moreover, qRT-PCR was performed to measure the MVD gene transcript level, and spectrophotometry was used to determine the content and variation of triterpenoids at different developmental stages of S. baumii.
The sequence analysis showed that the MVD gene cDNA sequence length was 1 209 bp and encoded 402 amino acids. The molecular weight of the protein was predicted to be 43.43 ku, and it was named SbMVD (GenBank number MK977617). Amino acid sequence alignment revealed that the SbMVD protein shared the greatest homology with Ganoderma sinense and Grifola frondosa. SDS-PAGE electrophoresis showed that the target protein band was located at approximately 65 ku (including a 21 ku tag protein), which was consistent with the predicted protein. Furthermore, qRT-PCR and spectrophotometry results showed that the SbMVD gene transcript level and triterpenoids content increased before decreasing dynamically at different developmental stages. The variation trends were basically the same.
The cloning and analysis of the SbMVD gene indicated that it might play an important role in the triterpenoids biosynthesis pathway. Our results lay a foundation for further elucidating the triterpenoids biosynthesis function of the SbMVD gene in S. baumii.
Sanghuangporus baumii / mevalonate pyrophosphate decarboxylase / gene cloning / bioinformatic analysis / expression analysis
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