RESUMEN
The biosynthetic pathways of phytosterols and steroidal saponins are located in two adjacent branches which share cycloartenol as substrate. The rate-limiting enzyme S-adenosyl-L-methionine-sterol-C24-methyltransferase 1 (SMT1) facilitates the metabolic flux toward phytosterols. It catalyzes the methylation of the cycloartenol in the side chain of the C24-alkyl group, to generate 24(28)-methylene cycloartenol. In this study, we obtained two full-length sequences of SMT1 genes from Pari polyphylla, designated PpSMT1-1 and PpSMT1-2. The full-length cDNA of PpSMT1-1 was 1369 bp long with an open reading frame (ORF) of 1038 bp, while the PpSMT1-2 had a length of 1222 bp, with a 1005 bp ORF. Bioinformatics analysis confirmed that the two cloned SMTs belong to the SMT1 family. The predicted function was further validated by performing in vitro enzymatic reactions, and the results showed that PpSMT1-1 encodes a cycloartenol-C24-methyltransferase, which catalyzes the conversion of cycloartenol to 24-methylene cycloartenol, whereas PpSMT1-2 lacked this catalytic activity. The tissue expression patterns of the two SMTs revealed differential expression in different organs of Paris polyphylla plants of different developmental stage and age. These results lay the foundation for detailed genetic studies of the biosynthetic pathways of steroid compounds, which constitute the main class of active substances found in P. polyphylla.
Asunto(s)
Melanthiaceae/enzimología , Melanthiaceae/genética , Metiltransferasas/genética , Secuencia de Bases , Catálisis , Clonación Molecular , ADN de Plantas/química , ADN de Plantas/genética , Medicamentos Herbarios Chinos , Isoenzimas/genética , Isoenzimas/metabolismo , Modelos Moleculares , Estructura Molecular , Sistemas de Lectura Abierta , Fitosteroles/metabolismo , Triterpenos/metabolismoRESUMEN
The study was aimed to establish a stable, accurate site specific PCR identification system to identify Manis pentadactyla and its adulterants using DNA molecular identification. The genomic DNA was extracted from experimental samples using the DNA extraction kit. The Cytb and CO â genes were amplified using PCR and sequenced bi-directionally. Obtained sequences were assembled using the BioEdit software. The neighbor-joining tree was constructed by MEGA 6.0. Specific identification primers were designed according to the specific allelets, and PCR reaction system was optimized. The results indicated that the Cytb and CO â sequence both were able to be used to identify M. pentadactyla and its adulterants. With the specific primers CO â -S10/A5, the M. pentadactyla could be amplified a 400 bp DNA band when the annealing temperature ranged from 55 to 60 â and the amount of DNA template ranged from 3 to 100 ng within 35 PCR cycles. However, other adulterants displayed no relevant bands. So that primers CO â - S10 / A5 can be used to identify the M. pentadactyla with the adulterants.