水稻干尖线虫14-3-3(Ab-14-3-3-a)基因克隆与功能分析

Characterization of a 14-3-3 gene from the white tip nematode Aphelenchoides besseyi

水稻干尖线虫是水稻上重要的寄生线虫,严重危害水稻安全生产。14-3-3蛋白调控生物体的细胞代谢、生长发育和逆境适应等一系列生物过程。本研究通过c DNA末端快速扩增技术(rapid amplification of c DNA ends,RACE)分离获得了一个水稻干尖线虫(Aphelenchoides besseyi)14-3-3基因,命名为Ab-14-3-3-a。Ab-14-3-3-a全长c DNA序列含有59 bp的5'非翻译区(UTR)、编码251个氨基酸756 bp的开放阅读框。Ab-14-3-3-a DNA序列包含4个外显子和3个内含子。Ab-14-3-3-a蛋白与其他植物寄生线虫14-3-3蛋白氨基酸序列高度相似,系统发育树显示与植物寄生线虫位于同一进化分支。原位杂交显示Ab-14-3-3-a特异定位于成虫的背食道腺细胞中,表明其在线虫取食和寄生过程中发挥潜在功能。qRT-PCR显示,Ab-14-3-3-a在水稻干尖线虫各个龄期均表达,其中在成虫表达水平最高,而在发育过程中幼虫表达水平最低。当线虫取食不同寄主时,Ab-14-3-3-a的表达水平具有差异性,线虫取食感病水稻早期,Ab-14-3-3-a表达水平上升1.5倍,而取食灰葡萄孢(Botrytis cinerea)时Ab-14-3-3-a表达可提高数百倍。利用体外RNA干扰技术将Ab-14-3-3-a的表达进行有效抑制,结果显著影响了线虫的产卵、成虫发育以及线虫种群数量。综上,Ab-14-3-3-a参与调控水稻干尖线虫的取食行为和发育进程。本研究相关结果有助于拓展植物寄生线虫14-3-3蛋白功能,为深入研究线虫与植物互作机制提供了理论依据。

The ectoparasite Aphelenchodies besseyi,agent of the white tip disease of rice,causes substantial loss of yield in most rice-growing areas of the world.14-3-3 proteins are an important component in biological pathways involved in cell metabolism,development progress and transcriptional alterations in response to environmental stimuli.In our study,a new 14-3-3 gene（Ab-14-3-3-a） was cloned and functionally characterized from A.besseyi.The cDNA of Ab-14-3-3-a（KP190141） encodes a deduced 251 amino acids,the molecular weight and theoretical isoelectric point were 28.45 and 4.81 respectively.The signal peptide and transmembrane region were absent in Ab-14-3-3-a protein.The qRT-PCR test supported the finding that the expression of Ab14-3-3-a is highest in the adult nematode,followed by the developmental eggs and then the juveniles.In-situ hybridization showed that Ab-14-3-3-a is specifically located in the dorsal esophageal gland cell,suggesting its potential role in parasitism.The expression levels of Ab-14-3-3-a were upregulated when the nematode feeding on rice shootings and on fungal hyphae （Botrytis cinerae ） at 1 day after inoculation.The silencing of Ab-14-3-3-a in the nematodes soaked in Ab-14-3-3-a dsRNA resulted in a significant,reproducible decrease in the number of eggs,juveniles and adults,demonstrating a potentially important role for Ab-14-3-3-a in A.besseyi for responding to feeding and fertility.