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Microbiology and Biotechnology Letters

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Microbial Biotechnology  |  Protein Structure, Function, and Engineering

Microbiol. Biotechnol. Lett.

Received: April 3, 2021; Revised: June 29, 2021

Molecular cloning, protein expression, and regulatory mechanisms of the chitinase gene from Spodoptera littoralis nucleopolyhedrovirus

Norhan Yasser 1, Reda Salem 1, Maha Alkhazindar 2, Ismail Abdelhamid 2, Said Ghozlan 2 and Wael Elmenofy 1*

1Agricultural Genetic Engineering Research Institute, 2Faculty of Science, Cairo University

Correspondence to :
Wael Elmenofy, Agricultural Genetic Engineering Research Institute (AGERI), Agricultural Research Center (ARC), 12619, Giza, Egypt. [12619]
Fax : 00201203212056, E-mail : wael.elmenofy@ageri.sci.eg

Abstract

The cotton leafworm, Spodoptera littoralis, is an important economic pest in Egypt and many countries worldwide and requires control management measures. S. littoralis nucleopolyhedrovirus (SpliNPV) is one of the most promising bioagents for the efficient control of insect pests. In this study, a chitinase gene (chitA) of a 1.8 kb DNA fragment was cloned and fully characterized from SpliNPV-EG1, an Egyptian isolate. A sequence of 601 amino acids was deduced when the gene was completely sequenced with a predicted molecular mass of 67 kDa for the preprotein. Transcriptional analyses using real-time polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h postinfection (hpi) and remained detectable until 168 hpi, suggesting their transcriptional regulation from a putative late promoter motif. In addition, quantitative analysis using quantitative RT-PCR showed a steady increase in chitA transcription levels of 7.86-fold at 12 hpi and increased up to 71.4-fold at 120 hpi. A ~50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an anti-recombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and Fusarium oxysporum, confirming the correct enzyme assembly and activity. The results supported the potential role and application of the SpliNPV-ChitA protein as a synergistic agent in agricultural fungal and pest control programs.

Keywords: Spodoptera littoralis NPV, chitinase gene A, qRT-PCR, protein expression, antifungal activity

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