Cloning of a novel trypsin inhibitor from the Traditional Chinese medicine decoction pieces, Radix Trichosanthis

Most herbs of traditional Chinese medicine (TCM) are used as air-dried decoction pieces that are manufactured and kept at ambient temperature for long periods. Given the ability of some desiccation-tolerant plants to conserve RNA, it could be worthwhile to isolate mRNA from TCM decoction pieces as p...

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Veröffentlicht in:Analytical biochemistry 2019-08, Vol.578, p.23-28
Hauptverfasser: Sang, Mengru, Ying, Yuan, Wu, Qinan, Ma, Chengbang, Xi, Xinping, Zhou, Mei, Wang, Lei, Bininda-Emonds, Olaf R.P., Chen, Tianbao
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Sprache:eng
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Zusammenfassung:Most herbs of traditional Chinese medicine (TCM) are used as air-dried decoction pieces that are manufactured and kept at ambient temperature for long periods. Given the ability of some desiccation-tolerant plants to conserve RNA, it could be worthwhile to isolate mRNA from TCM decoction pieces as part of a transcriptomic strategy to identify new substances with potential pharmaceutical application. Here, we report the molecular cloning of a novel trypsin inhibitor (as the probable alleleic variants TKTI-2 and TKTI-3) from the decoction piece of Radix Trichosanthis, representing the dried root of Trichosanthes kirilowii. From this material, the total RNA was extracted and a cDNA library was constructed from the isolated mRNA from which the cDNAs of two precursors were successfully cloned and sequenced. TKTI-3 showed an amino-acid substitution in the otherwise highly-conserved P1-P1′ reaction site of the mature peptide, which we confirmed to not be an artefact. Subsequent analysis using LC-MS/MS detected the presence of specific tryptic peptides expected from TKTI-3, confirming the presence and expression of this locus in Radix Trichosanthis. More generally, this study indicates that mRNA can persist in decoction pieces and so could present a viable option for the molecular cloning from other TCMs. •Success of isolation of RNA using decoction pieces of Chinese medicine, Radix Trichosanthis.•The probable allelic variants of a novel trypsin inhibitor were successfully cloned and sequenced using the cDNA library.•Parallel analysis using LC-MS/MS to determine the amino acid mutation of trypsin inhibitor peptide.
ISSN:0003-2697
1096-0309
DOI:10.1016/j.ab.2019.02.028