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Microbial Biotechnology (MB)  |  Protein Structure, Function, and Engineering

Microbiol. Biotechnol. Lett. 2021; 49(3): 305-315

https://doi.org/10.48022/mbl.2104.04002

Received: April 3, 2021; Revised: June 29, 2021; Accepted: July 22, 2021

Molecular Cloning, Protein Expression, and Regulatory Mechanisms of the Chitinase Gene from Spodoptera littoralis Nucleopolyhedrovirus

Norhan Yasser1, Reda Salem1, Maha Alkhazindar2, Ismail A. Abdelhamid2, Said A. S. Ghozlan2, and Wael Elmenofy1*

1Agricultural Genetic Engineering Research Institute, ARC, Giza 12619, Egypt 2Faculty of Science, Cairo University, Giza 12619, Egypt

Correspondence to :
Wael Elmenofy,  wael.elmenofy@ageri.sci.eg

The cotton leafworm, Spodoptera littoralis, is a major pest in Egypt and many countries worldwide, and causes heavy economic losses. As a result, management measures to control the spread of the worm are required. 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 reverse transcription polymerase chain reaction (RT-PCR) revealed that chitA transcripts were detected first at 12 h post infection (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 of 7.86-fold at 12 hpi in chitA transcription levels, which increased up to 71.4-fold at 120 hpi. An approximately 50 kDa protein fragment with chitinolytic activity was purified from ChitA-induced bacterial culture and detected by western blotting with an antirecombinant SpliNPV chitinase antibody. Moreover, purification of the expressed ChitA recombinant protein showed in vitro growth inhibition of two different fungi species, Fusarium solani and F. oxysporum, confirming that the enzyme assembly and activity was correct. 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

Graphical Abstract


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