National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki 305-8634, Japan.
Insect Biochem Mol Biol. 2012 Jan;42(1):1-9. doi: 10.1016/j.ibmb.2011.10.001. Epub 2011 Oct 19.
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 Ca(2+) -binding proteins. As predicted, NcSP84 exhibited Ca(2+)-binding activity. The SDS-PAGE mobility of purified NcSP84 bound to Ca(2+) tended to decline discretely, depending on the concentration of CaCl(2) 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 Ca(2+) ions that flow into sieve tubes in response to stylet puncturing. This might suppress sieve-element clogging and facilitate continuous ingestion from sieve tubes.
绿盲蝽在取食过程中会分泌水样和凝固的唾液。在我们的研究中，水样唾液分泌通过超滤从“喂食饲料”中浓缩，并进行 SDS-PAGE。84kDa 最主要带的 N 末端氨基酸序列（命名为 NcSP84）通过 Edman 降解进行分析。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。基于 N 末端氨基酸序列，使用简并引物通过 5'和 3'-RACE 克隆该蛋白质的完整 cDNA。推导的 NcSP84 包含一个 2061bp 的开放阅读框，编码一个假定的 687 个氨基酸，具有由 19 个氨基酸组成的假定信号序列。NcSP84 的核苷酸和氨基酸序列与公共数据库中的任何序列均没有统计学上显著的同源性。基序搜索预测该蛋白质具有 EF 手，这是在 Ca(2+)结合蛋白中最常见的基序。如预测的那样，NcSP84 表现出 Ca(2+)结合活性。纯化的 NcSP84 与 Ca(2+)结合的 SDS-PAGE 迁移率根据与 SDS 缓冲液混合前 1h 混合的 CaCl2浓度离散下降。原位杂交和免疫组织化学显示，NcSP84 基因和基因产物在 III 型细胞中表达和储存，III 型细胞是初级唾液腺中最大的叶。暴露于盲蝽的水稻韧皮部汁液中检测到 NcSP84 蛋白，验证了 NcSP84 蛋白被注入筛管。这些结果表明，NcSP84 可能在取食过程中分泌到筛管中，这可能结合了由于刺吸作用而流入筛管的 Ca(2+)离子。这可能抑制筛管堵塞并促进从筛管中连续摄入。
