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

doi:10.1016/j.ab.2019.02.028

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

School of Pharmacy

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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 language	English
Journal	Analytical Biochemistry
Early online date	01 Mar 2019
DOIs	https://doi.org/10.1016/j.ab.2019.02.028
Publication status	Early online date - 01 Mar 2019

Keywords

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

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Cloning of a novel trypsin inhibitor from the Traditional Chinese medicine decoction pieces, Radix Trichosanthis
Copyright 2019 Elsevier. This manuscript is distributed under a Creative Commons Attribution-NonCommercial-NoDerivs License (https://creativecommons.org/licenses/by-nc-nd/4.0/), which permits distribution and reproduction for non-commercial purposes, provided the author and source are cited
Accepted author manuscript, 1.47 MBLicence: CC BY-NC-ND

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Sang, M., Ying, Y., Wu, Q., Ma, C., Xi, X., Zhou, M., Wang, L., Bininda-Emonds, O., & Chen, T. (2019). Cloning of a Novel Trypsin Inhibitor from the Traditional Chinese Medicine Decoction Pieces, Radix Trichosanthis. Analytical Biochemistry. https://doi.org/10.1016/j.ab.2019.02.028

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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.",

keywords = "Chinese medicine; Trichosanthes kirilowii; trypsin inhibitor peptide; molecular cloning; MS/MS sequencing",

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

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AU - Sang, Mengru

AU - Ying, Yuan

AU - Wu, Qinan

AU - Ma, Chengbang

AU - Xi, Xinping

AU - Zhou, Mei

AU - Wang, Lei

AU - Bininda-Emonds, Olaf

AU - Chen, Tianbao

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N2 - 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.

AB - 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.

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