Generation of RNAi plasmid corresponding to the m1l1 gene (ID 140500) belonging to myosin class i in the fungus Mucor circinelloides

To investigate biological roles of Myosin class I in the morphology of M. circinelloides and identify good candidates for further analyses of Mucor pathogenesis, we are going to inhibit their expression using RNAi technique. We named the two myosin class I like genes as M1L1 (ID 140500) and M1L2 (ID 116620). In the present study, we report the results of generation of RNAi plasmid to inhibit the expression of M1L1 gene.
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Generation of RNAi plasmid corresponding to the m1l1 gene (ID 140500) belonging to myosin class i in the fungus Mucor circinelloidesHNUE JOURNAL OF SCIENCENatural Sciences, 2020, Volume 65, Issue 4A, pp. 27-32This paper is available online at http://stdb.hnue.edu.vn GENERATION OF RNAi PLASMID CORRESPONDING TO THE m1l1 GENE(ID 140500) BELONGING TO MYOSIN CLASS I IN THE FUNGUS Mucor circinelloides Le Ngoc Mai1, Le Phuong Thao1, Pham Thi Thu Trang 2 and Trieu Anh Trung1 1 Faculty of Biology, Hanoi National University of Education 2 Nguyen Trai Gifted High School, Hai Duong province Abstract. Mucormycosis has emerged as a second most common filamentous fungal infection in human caused by species belonging to order Mucorales, especially in immunocompromised patients, with a high mortality ratio. Our understanding of its virulence determinants is limited, leading to the lacking of efficient therapies. Dimorphism is one of the phenotypic characteristics that can be used as a marker for virulence screening. Mucor circinelloides, a dimorphic fungus, has become an attractive model for studies on various studies, including mucormycosis. Our previous study identified Mcmyo5 protein belonging to Myosin class V which plays an important role in Mucor pathogenesis. We have found three protein families with high similarities of amino acid sequences compared to Mcmyo5, including Myosin class I, II and V, in the Mucor genome. To investigate the possible roles of those genes in Mucor pathogenesis using RNAi techniques, we generated the RNAi plasmid which is corresponding to endogenous gene, m1l1 (ID 140500). In the present study, we show the results of isolation and cloning a fragment of the target gene m1l1 into the vector pMAT1812 to obtain RNAi plasmid. This recombinant RNAi plasmid was checked by using the digestion of restriction enzyme and DNA sequencing techniques. Keywords: mucormycosis, myosin, RNAi, Mucor circinelloides.1. Introduction Mucormycosis (zygomycosis) is the rare but lethal fungal infection, especially inimmunocompromised patients, such as those with diabetes, organ-transplanted, AIDS,hematologic malignancies, or trauma [1-3]. Mucormycosis caused by species belonging to orderMucorales [4]. The most common pathogenic species found in the families Rizopus, Mucor,Lichtheimia, Apophysomyces, Rhizomucor and Cunninghamella. Mucormycosis is moredifficult to treat than more common mycoses due to restriction of efficient therapies by lackingof knowledge about the disease and the organisms that cause the infection [5]. Mucor circinelloides is an emerging opportunistic human pathogen that causesmucormycosis. It has become an outstanding model species for various studies, including RNAimechanism [6], carotenogenesis [7], lipid production [8], and mucormycosis [9] due to itsavailable for genetic transformation. We have applied RNAi techniques to investigate genefunctions in this fungus, based on well-studied RNA silencing mechanisms. In our knowledge,there is no study on mucormycosis and RNAi mechanism in M. circinelloides in Vietnam.Received March 27, 2020. Revised May 4, 2020. Accepted May 11, 2020Contact Trieu Anh Trung, e-mail address: trungta@hnue.edu.vn 27 Le Ngoc Mai, Le Phuong Thao, Pham Thi Thu Trang and Trieu Anh Trung Dimorphism is a process in which they can grow like hyphae or like yeast, depending onthe environmental conditions. This yeast-hyphae transition contributes to fungal virulence. M.circinelloides is also a dimorphic fungus that grows as hyphal mycelium in aerobic conditionand as yeast in anaerobic/high CO2 condition. The transition between those two forms in thisfungus was demonstrated to be involved in virulence, as the yeast phase is not pathogenic [9]. Inaddition, the size of sporangiospores is also related to virulence in M. circinelloides, beingpathogenic only the strains producing large multinucleated spores [10]. These results suggestedthat dimorphism and other phenotypic characteristics could be used as markers for a preliminaryevaluation of virulence in this fungus. We have identified mcmyo5 gene (ID 51513), encoding Mcmyo5 protein belonging toMyosin class V, involved in morphogenesis and pathogenesis of fungus M. circinelloides [11].Myosin class V is a widely conserved protein family that belongs to the myosin superfamily.Myosin superfamily contains actin-based motor proteins that play diverse functions in cells,including cytokinesis, cell adhesion, endocytosis, exocytosis, movement of mRNA, movementof pigment granules and cell motility [12]. As occurs in mo ...