RESUMO
Selection of transgenic plants by using genes encoding screenable markers of plant origin with health benefit properties, such as anthocyanin is an important aim in plant genetic engineering. In this study, Malus domestica MYB10 (MdMYB10) gene, was used for Agrobacterium tumefaciens-mediated transformation of two SBS-02 and SBS-04 sugar beet lines. The impact of different light regimes on plant tissue culture from a combination of light, dark/light and dark was investigated. The results of this study showed that the MdMYB10 gene was successfully integrated into the selected purple transgenic lines, suggesting that the expression of MdMYB10 gene in sugar beet shoots can be used as a screenable markers for transformation, possibly replacing antibiotic resistant genes. Furthermore, the results of the antibacterial activity of transgenic plants extracts showed that the total extract obtained from transgenic lines significantly (P < 0.01) inhibited the growth and development of Enterococcus faecium and Enterococcus faecalis bacteria compared to the non-transgenic plants. The results of this study showed that the combination of betalain with vancomycin demonstrated a synergistic antimicrobial effect, also, suggesting that the expression of MdMYB10 may play a dual role by accumulating betalain and exhibiting a screenable markers function. SUPPLEMENTARY INFORMATION: The online version contains supplementary material available at 10.1007/s13205-022-03120-7.
RESUMO
Antimicrobial peptides have been long known to confer resistance to plant pathogens. In this study, new recombinant peptides constructed from a dermaseptin B1 (DrsB1) peptide fused to a chitin-binding domain (CBD) from Avr4 protein, were used for Agrobacterium tumefaciens-mediated transformation of tobacco plants. Polymerase chain reaction (PCR), semi-quantitative RT-PCR, and western blotting analysis demonstrated the incorporation and expression of transgenes in tobacco genome and transgenic plants, respectively. In vitro experiments with recombinant peptides extracted from transgenic plants demonstrated a significant (P<0.01) inhibitory effect on the growth and development of plant pathogens. The DrsB1-CBD recombinant peptide had the highest antifungal activity against fungal pathogens. The expression of the recombinant peptides greatly protected transgenic plants from Alternaria alternata, Alternaria solani, Fusarium oxysporum, and Fusarium solani fungi, in comparison to Pythium sp. and Pythium aphanidermatum. Expression of new recombinant peptides resulted in a delay in the colonization of fungi and appearance of fungal disease symptoms from 6 days to more than 7 weeks. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, cling together, and crushed following the antimicrobial activity of the recombinant peptides. Greenhouse bioassay analysis showed that transgenic plants were more resistant to Fusarium and Pythium infections as compared with the control plants. Due to the high antimicrobial activity of the recombinant peptides against plant pathogens and novelty of recombinant peptides, this report shows the feasibility of this approach to generate disease resistance transgenic plants.
RESUMO
Dermaseptin B1 (DrsB1), an antimicrobial cationic 31 amino acid peptide, is produced by Phyllomedusa bicolor. In an attempt to enhance the antimicrobial efficacy of DrsB1, the DrsB1 encoding 93 bp sequence was either fused to the N or C terminus of sequence encoding chitin-binding domain (CBD) of Avr4 gene from Cladosporium fulvum. Tobacco leaf disk explants were inoculated with Agrobacterium rhizogenes harboring pGSA/CBD-DrsB1 and pGSA/DrsB1-CBD expression vectors to produce hairy roots (HRs). Polymerase chain reaction (PCR) was employed to screen putative transgenic tobacco lines. Semi-quantitative RT-PCR and western blotting analysis indicated that the expression of recombinant genes were significantly higher, and recombinant proteins were produced in transgenic HRs. The recombinant proteins were extracted from the tobacco HRs and used against Pectobacterium carotovorum, Agrobacterium tumefaciens, Ralstonia solanacearum, and Xanthomonas campestris pathogenic bacteria and Alternaria alternata and Pythium sp. fungi. Two recombinant proteins had a statistically significant (p < 0.01) inhibitory effect on the growth and development of plant pathogens. The CBD-DrsB1 recombinant protein demonstrated a higher antibacterial effect, whereas the DrsB1-CBD recombinant protein demonstrated greater antifungal activity. Scanning electron microscopy images revealed that the structure of the fungal mycelia appeared segmented, adhered to each other, and crushed following the antimicrobial activity of the recombinant proteins. Due to the high antimicrobial activity of the recombinant proteins against plant pathogens, this strategy can be used to generate stable transgenic crop plants resistant to devastating plant pathogens.
Assuntos
Proteínas de Anfíbios/metabolismo , Anti-Infecciosos/metabolismo , Peptídeos Catiônicos Antimicrobianos/metabolismo , Expressão Gênica , Nicotiana/metabolismo , Plantas Geneticamente Modificadas/metabolismo , Proteínas Recombinantes de Fusão/metabolismo , Alternaria/efeitos dos fármacos , Alternaria/crescimento & desenvolvimento , Proteínas de Anfíbios/genética , Peptídeos Catiônicos Antimicrobianos/genética , Bactérias/efeitos dos fármacos , Bactérias/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Testes de Sensibilidade Microbiana , Plantas Geneticamente Modificadas/genética , Proteínas Recombinantes de Fusão/genética , Nicotiana/genéticaRESUMO
ETHNOPHARMACOLOGICAL RELEVANCE: Complementary and alternative medicine has been increasingly used to treat chronic illnesses, such as diabetes mellitus. However, various limitations in terms of their application and efficacies exist. Furthermore, there is still much to be done to discover the right herbal medicine for diabetes. AIM OF THE STUDY: This paper aims to evaluate previous herbal studies on the management of diabetes mellitus, to address their strengths and weaknesses and propose a general framework for future studies. APPROACH AND METHODS: Data sources such as PubMed, ScienceDirect, Scopus, SpringerLink, and Wiley were searched, limited to Iran, using 36 search terms such as herbal, traditional, medicine, and phytopharmacy in combination with diabetes and related complications. Reviewed articles were evaluated regarding the use of botanical nomenclature and included information on (1) identity of plants and plant parts used, (2) the processing procedure, and (3) the extraction process. The main outcomes were extracted and then surveyed in terms of the efficacies of herbs in the management of diabetes mellitus. Then a comparative study was performed between Iranian and non-Iranian studies with respect to herbs best studied in Iran. RESULTS: Of the 82 herbs studied in Iran, only six herbs were endemic and 19 were studied in detail. Although most of the reviewed herbs were found to decrease the level of blood glucose (BG) and/or glycated hemoglobin (HbA1C) in both Iranian and non-Iranian studies, information on their pharmacological mechanisms is scarce. However, the level of HbA1C was measured in a limited number of clinical trials or animal studies. Available information on both short- and long-term use of studied herbs on diabetes related complications and functions of involved organs as well as comorbid depression and/or simultaneous changes in lifestyle is also insufficient. Furthermore, little or no information on their phytochemical, toxicological, and herb-drug interaction properties is available. It is worth noting that the efficacy of the reviewed herbs has been studied scarcely in both humans and animals regarding both Iranian and non-Iranian studies. A significant number of reviewed articles failed to cite the scientific name of herbs and include information on the processing procedure and the extraction process. CONCLUSIONS: Treatment of diabetes mellitus as a multifactorial disease using herbal medicines requires a comprehensive approach. In order to discover the right herbal medicine for the management of diabetes many other important factors than the levels of BG, HbA1C and insulin should be considered. According to our criteria, all the reviewed herbs suffered from inadequate investigation in human, animal and in vitro models in this respect, whereas they are worth investigating further. However, more research on endemic plants and the traditional history of herbal medicine is warranted. In our opinion, the pharmacological, toxicological, and phytochemical information should be obtained before clinical trials. Furthermore, information such as botanical scientific nomenclature, side effects, and toxicity will improve the quality and validity of publications in herbal research. In particular, designing a database covering all valid information about herbs and/or diseases will decrease unnecessary costs and increase the efficiency of research.