Hattori Makoto, Komatsu Setsuko, Noda Hiroaki, Matsumoto Yukiko
National Institute of Agrobiological Sciences, 1-2 Ohwashi, Tsukuba, Ibaraki, 305-8634, Japan.
National Institute of Crop Science, 2-1-18 Kannondai, Tsukuba, Ibaraki, 305-8518, Japan.
PLoS One. 2015 Apr 24;10(4):e0123671. doi: 10.1371/journal.pone.0123671. eCollection 2015.
The green rice leafhopper, Nephotettix cincticeps, is a vascular bundle feeder that discharges watery and gelling saliva during the feeding process. To understand the potential functions of saliva for successful and safe feeding on host plants, we analyzed the complexity of proteinaceous components in the watery saliva of N. cincticeps. Salivary proteins were collected from a sucrose diet that adult leafhoppers had fed on through a membrane of stretched parafilm. Protein concentrates were separated using SDS-PAGE under reducing and non-reducing conditions. Six proteins were identified by a gas-phase protein sequencer and two proteins were identified using LC-MS/MS analysis with reference to expressed sequence tag (EST) databases of this species. Full -length cDNAs encoding these major proteins were obtained by rapid amplification of cDNA ends-PCR (RACE-PCR) and degenerate PCR. Furthermore, gel-free proteome analysis that was performed to cover the broad range of salivary proteins with reference to the latest RNA-sequencing data from the salivary gland of N. cincticeps, yielded 63 additional protein species. Out of 71 novel proteins identified from the watery saliva, about 60 % of those were enzymes or other functional proteins, including GH5 cellulase, transferrin, carbonic anhydrases, aminopeptidase, regucalcin, and apolipoprotein. The remaining proteins appeared to be unique and species- specific. This is the first study to identify and characterize the proteins in watery saliva of Auchenorrhyncha species, especially sheath-producing, vascular bundle-feeders.
稻绿蝽,电光叶蝉,是一种维管束取食者,在取食过程中会分泌水样和凝胶状唾液。为了解唾液在成功安全取食寄主植物方面的潜在功能,我们分析了电光叶蝉水样唾液中蛋白质成分的复杂性。唾液蛋白是从成年叶蝉通过拉伸的保鲜膜膜片取食的蔗糖饮食中收集的。蛋白质浓缩物在还原和非还原条件下使用SDS-PAGE进行分离。通过气相蛋白质测序仪鉴定出6种蛋白质,并使用LC-MS/MS分析参考该物种的表达序列标签(EST)数据库鉴定出2种蛋白质。通过cDNA末端快速扩增-PCR(RACE-PCR)和简并PCR获得了编码这些主要蛋白质的全长cDNA。此外,参考电光叶蝉唾液腺的最新RNA测序数据进行的无凝胶蛋白质组分析,又产生了63种额外的蛋白质种类。在从水样唾液中鉴定出的71种新蛋白质中,约60%是酶或其他功能蛋白质,包括GH5纤维素酶、转铁蛋白、碳酸酐酶、氨肽酶、钙调蛋白和载脂蛋白。其余蛋白质似乎是独特的且具有物种特异性。这是首次对沫蝉总科物种,特别是产生鞘的维管束取食者的水样唾液中的蛋白质进行鉴定和表征的研究。