Molecular cloning of a novel calcium-binding protein in the secreted saliva of the green rice leafhopper Nephotettix cincticeps

• Published in: Insect biochemistry and molecular biology 2012, Vol.42 (1), p.1-9
• Main Authors: Hattori, Makoto, Nakamura, Masatoshi, Komatsu, Setsuko, Tsuchihara, Kazuko, Tamura, Yasumori, Hasegawa, Tsuyoshi
• Format: Article
• Language: English
• Subjects: Amino Acid Sequence; amino acids; Animals; Blotting, Western; calcium chloride; Calcium-binding protein; Calcium-Binding Proteins - genetics; Calcium-Binding Proteins - isolation & purification; Calcium-Binding Proteins - metabolism; Cloning, Molecular; complementary DNA; diet; EF-hand motif; Feeding Behavior; Female; genes; Hemiptera - chemistry; Hemiptera - genetics; Hemiptera - metabolism; immunohistochemistry; in situ hybridization; ingestion; Insect Proteins - genetics; Insect Proteins - isolation & purification; Insect Proteins - metabolism; ions; molecular cloning; Molecular Sequence Data; Nephotettix cincticeps; Nephotettix nigropictus; open reading frames; Oryza - parasitology; Oryza sativa; Phloem; polyacrylamide gel electrophoresis; proteins; rice; RNA, Messenger - metabolism; saliva; Salivary glands; Salivary Glands - chemistry; Salivary Glands - metabolism; Salivary Proteins and Peptides - genetics; Salivary Proteins and Peptides - isolation & purification; Salivary Proteins and Peptides - metabolism; sap; secretion; Sequence Analysis, DNA; sieve tubes; signal peptide; stylets; two-dimensional gel electrophoresis; ultrafiltration; Watery saliva
• ISSN: 0965-1748; 1879-0240
• DOI: 10.1016/j.ibmb.2011.10.001

Green rice leafhoppers (Nephotettix cincticeps) secrete watery and coagulable saliva in the feeding process. In our study, the watery salivary secretion was concentrated by ultrafiltration from “fed diet” and subjected to SDS-PAGE. The N-terminal amino acid sequence of the most predominant band at 84kDa (designated NcSP84) was analyzed by Edman degradation. This sequence was completely consistent with the most abundant protein in the salivary gland extracts, which was separated by two-dimensional gel electrophoresis. Based on the N-terminal amino acid sequence, the complete cDNA of this protein was cloned by 5′- and 3′-RACE using degenerate primers. The deduced NcSP84 contained an open reading frame of 2061bp encoding a putative 687 amino acids with a putative signal sequence composed of 19 amino acids. The nucleotide and amino acid sequences of NcSP84 did not share statistically significant homology with any sequences in public databases. Motif search predicted that this protein had EF-hands, the most common motif found in Ca2+ -binding proteins. As predicted, NcSP84 exhibited Ca2+-binding activity. The SDS-PAGE mobility of purified NcSP84 bound to Ca2+ tended to decline discretely, depending on the concentration of CaCl2 with which it was mixed for 1h before adding SDS buffer. In situ hybridization and immunohistochemistry showed that the NcSP84 gene and gene product were expressed and stored in type III cells, which are the largest lobes in the primary salivary glands. The NcSP84 protein was detected in the phloem sap of rice exposed to leafhoppers, verifying that the NcSP84 protein was injected into the sieve tubes. These results suggest that NcSP84 could be secreted into the sieve tubes during feeding, which might bind Ca2+ ions that flow into sieve tubes in response to stylet puncturing. This might suppress sieve-element clogging and facilitate continuous ingestion from sieve tubes.