Molecular cloning of a novel calcium-binding protein in the secreted saliva of the green rice leafhopper Nephotettix cincticeps
Molecular cloning of a novel calcium-binding protein in the secreted saliva of the green rice leafhopper Nephotettix cincticeps
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DOI：
10.1016/j.ibmb.2011.10.001
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发表时间：
2012-01-01
影响因子：
3.8
通讯作者：
Hasegawa, Tsuyoshi
中科院分区：
文献类型：
--
作者：
Hattori, Makoto;Nakamura, Masatoshi;Hasegawa, Tsuyoshi
绿色稻叶蝉（Nephotettix cincticeps）在取食过程中分泌水状和可凝固的唾液。在我们的研究中，水样唾液分泌物浓缩从“喂养饮食”，并进行SDS-PAGE。通过Edman降解分析84 kDa处最主要条带（命名为NcSP 84）的N-末端氨基酸序列。该序列与唾液腺提取物中最丰富的蛋白质完全一致，该蛋白质通过二维凝胶电泳分离。根据N-末端氨基酸序列，使用简并引物通过5 '-和3'-RACE克隆了该蛋白的完整cDNA。NcSP 84的开放阅读框为2061 bp，编码687个氨基酸，信号肽由19个氨基酸组成。NcSP 84的核苷酸和氨基酸序列与公共数据库中的任何序列都没有统计学上显著的同源性。模体搜索预测，这种蛋白质EF手，最常见的模体中发现的钙离子结合蛋白。如所预测的，NcSP 84表现出Ca 2+结合活性。与Ca 2+结合的纯化NcSP 84的SDS-PAGE迁移率倾向于离散地下降，这取决于在加入SDS缓冲液之前与其混合1 h的CaCl 2的浓度。原位杂交和免疫组化结果表明，NcSP 84基因和基因产物的表达和存储在III型细胞，这是最大的叶在初级唾液腺。在接触叶蝉的水稻韧皮部汁液中检测到NcSP 84蛋白，验证了NcSP 84蛋白被注射到筛管中。这些结果表明，NcSP 84可以分泌到筛管在进食过程中，这可能会结合Ca 2+离子流入筛管响应探针穿刺。这可以抑制筛元件堵塞并促进从筛管的连续摄取。(C)2011爱思唯尔有限公司版权所有。
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 84 kDa (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 2061 bp 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 1 h 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 sieveelement clogging and facilitate continuous ingestion from sieve tubes. (C) 2011 Elsevier Ltd. All rights reserved.