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

Sang, Mengru, Ying, Yuan, Wu, Qinan, Ma, Chengbang, Xi, Xinping, Zhou, Mei, Wang, Lei, Bininda-Emonds, Olaf R.P., Chen, Tianbao
Analytical biochemistry 2019 v.578 pp. 23-28
Oriental traditional medicine, Trichosanthes kirilowii, air drying, ambient temperature, amino acids, cDNA libraries, complementary DNA, herbs, liquid chromatography, loci, messenger RNA, molecular cloning, peptides, sequence analysis, tandem mass spectrometry, transcriptomics, trypsin inhibitors
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.