cDNA cloning, heterogeneous expression and biochemical characterization of a novel trypsin-like protease from Nilaparvata lugens.

A reverse transcription-polymerase chain reaction (RT-PCR) strategy was used to clone diverse trypsin-like protease gene transcripts from midguts of the brown planthopper Nilaparvata lugens Stål (Homoptera: Delphacidae). Six individual trypsin-like protease transcripts were identified. On the basis of one nucleotide sequence of the six clones, a full-length cDNA sequence (1902 bp) was obtained by rapid amplification of cDNA ends (RACE). The cDNA contained an 1128-bp open reading frame encoding a putative protein of 375 amino acids with typical features of the trypsin-like protease. Heterogeneous expression of the coding sequence for the mature peptide in Escherichia coli cells showed that the expressed protease with a molecular weight of 27.0 is active, for its BApNAse activity assayed by using BApNA (N-benzoyl-D,L-arginine-p-nitroanilide) as substrate. The protease had its maximum activity at pH 8.0 and 35 degrees C. A much better stability was observed at pH values above 4.0 and temperatures below 40 degrees C. The enzyme was strongly inhibited by serine protease inhibitor. The trypsin-like protease is therefore likely one of the major digestive proteases responsible for protein hydrolysis in N. lugens gut, and multiple gene families encoding digestive proteases may help in adaptation of this sap-sucker to different rice varieties.