Cloning,Prokaryotic Expression and Activity Research of Panax ginseng Thionins

doi:10.13560/j.cnki.biotech.bull.1985.2018-0702

Abstract

Abstract: Antibacterial peptides play an important role in bacteriostasis and resistance to stress in the growth process of ginseng. Studying the antibacterial activity of ginseng thionins may lay a foundation for large-scale production and biological pesticide research in the future. First,the gene sequence of ginseng thionins was analyzed and prokaryotic expression plasmid was constructed to induce the expression of the protein.. Then the affinity and purification of inclusion bodies was performed,the anti-fungal activity of the purified protein was detected by paper tablet diffusion method and membrane permeability assay. The ginseng thionins was 228 bp in length and encoded 75 amino acids,and its mature peptide region（29-75 aa）was selected for subsequent expression and purification. The prokaryotic expression plasmid pGEX-4T1-thionins was successfully constructed,the efficient expression of the target protein was obtained in Escherichia coli BL21,and the target protein was purified by inclusion body and then renatured and concentrated. The growth inhibition rate of ginseng thionins on Alternaria panax was 36.0% and 51.1% at 3 μmol/L and 9μmol/L,respectively. The half-inhibitory concentration of the fungal spores was 0.87 μmol/L. In conclusion,the ginseng thionins has a significant effect of inhibiting the fungus A. panax.

Key words: Panax ginseng, thionins, prokaryotic expression, protein purification, antifungal activity

ZHANG Peng-fei, BIAN Shuai, ZHAO Yue, ZHAO Yu, WANG Jia-wen. Cloning,Prokaryotic Expression and Activity Research of Panax ginseng Thionins[J]. Biotechnology Bulletin, 2019, 35(2): 53-57.

References

[1] 崔巍, 赵洪艳, 王燕嬉. 人参皂苷抗衰老的研究进展[J]. 中国老年学杂志, 2006, 26(11):1578-1581.
[2] 胡圣望, 胡勇, 王平. 人参皂苷Rg1对慢性应激大鼠空间学习能力的影响[J]. 四川中医, 2004, 22(3):14-15.
[3] Wesnes KA, Ward T, et al.The memory enhancing effects of a Ginkgo balboa/Panax ginseng combination in health middle aged volunteers[J]. Pschopharmacology, 2000, 152(4):353-361.
[4] 田建明, 李浩, 叶金梅, 等. 人参皂苷Rg2对大鼠化学性心肌缺血的影响[J]. 中国中药杂志, 2003, 28(12):1191-1192.
[5] 张秋梅, 张喆, 于德民, 等. 人参糖肽治疗2型糖尿病的临床观察[J]. 中国现代医学杂志, 2003, 13(6):59.
[6] Shergis JL, Di YM, et al.Therapeutic potential of Panax ginseng and ginsenosides in the treatment of chronic obstructive pulmonary disease[J]. Complement Ther Med, 2014, 22(5):944-953.
[7] Cai K, Wang J, Wang M, et al.Molecular cloning, recombinant expression, and antifungal functional characterization of the lipid transfer protein from Panax ginseng[J]. Biotechnology Letters, 2016, 38(7):1229-1235.
[8] Zhang H, Wang J, Li S, et al.Molecular cloning, expression, purifcation and functional characterization of an antifungal cyclophilin protein from Panax ginseng[J]. Biomedical Reports, 2017, 7(6):527-531.
[9] Tam JP, Wang S, Wong KH, et al.Antimicrobial peptides from plants[J]. Pharmaceuticals, 2015, 8(4):711-757.
[10] Ghosh M, Antifungal properties of haem peroxidase from Acorus calamus[J]. Annals of Botany, 2006, 98(6):1145-1153.
[11] Guida V, Cantarella M, Chambery A, et al.Purification and characterization of novel cationic peroxidases from Asparagus acutifolius L. with biotechnological applications[J]. Molecular Biotechnology, 2014, 56(8):738-746.
[12] Ajesh K, Sreejith K.A novel antifungal protein with lysozyme-like activity from seeds of Clitoria ternatea[J]. Applied Biochemistry and Biotechnology, 2014, 173(3):682-693.
[13] Candido Ede S, Pinto MF, Pelegrini PB, et al.Plant storage proteins with antimicrobial activity:novel insights into plant defense mechanisms[J]. FASEB Journal, 2011, 25(10):3290-3305.
[14] Wong JH, Ng TB, et al.Proteins with antifungal properties and other medicinal applications from plants and mushrooms[J]. Appl Microbiol Biotechnol, 2010, 87(4):1221-1235.
[15] Kulko AB, Kisl OV, Sadykova VS, et al.investigation of thionins from black cumin(nigella sativa L.)seeds showing cytotoxic, regulatory and antifungal activity[J]. Antibiol Khimioter, 2016, 61(9/10):8-16.
[16] Ji H, Gheysen G, et al.The role of thionins in rice defence against root pathogens[J]. Mol Plant Pathol, 2015, 16(8):870-881.
[17] Lee OR, Kim Y, Balusamy SRD, et al.Ginseng g-thionin is localized to cell wall-bound extracellular spaces and responsive to biotic and abiotic stresses[J]. Physiol Mol Plant Pathol, 2011, 76(2):82-89.
[18] Harper S, Speicher DW.Purification of proteins fused to glutathione S-transferase[J]. Methods Mol Biol, 2011, 681:259-280.
[19] Yu H, Li H, Gao D, et al.Secretory production of antimicrobial peptides in Escherichia coli using the catalytic domain of a cellulase as fusion partner[J]. J Biotechnol, 2015, 214:77-82.
[20] Barbosa PP, Del Sart RP, Silva ON, et al.Antibacterial peptides from plants:what they are and how they probably work[J]. Biochem Res Int, 2011, 2011(5):250349.