Cloning of a Novel Trypsin Inhibitor from the Traditional Chinese Medicine Decoction Pieces, Radix Trichosanthis

Mengru Sang, Yuan Ying, Qinan Wu, Chengbang Ma, Xinping Xi, Mei Zhou, Lei Wang, Olaf Bininda-Emonds, Tianbao Chen

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Abstract

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 allelic 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.
Original languageEnglish
JournalAnalytical Biochemistry
Early online date01 Mar 2019
DOIs
Publication statusEarly online date - 01 Mar 2019

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Keywords

  • Chinese medicine; Trichosanthes kirilowii; trypsin inhibitor peptide; molecular cloning; MS/MS sequencing

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