灰飞虱卵表面分泌液中蛋白质组分鉴定及分析

Identification and analysis of proteins in the egg-associated secretions of Laodelphax striatellus(Hemiptera:Delphacidae)

【目的】鉴定和分析灰飞虱Laodelphax striatellus卵表面分泌液中的蛋白质组分,为后续灰飞虱效应子筛选和功能研究提供基础。【方法】在显微镜下从水稻叶鞘中收集灰飞虱完整卵,用磷酸缓冲液洗脱并收集完整卵表面的分泌液;利用液相色谱-串联质谱(liquid chromatography-tandem mass spectrometry,LC-MS/MS)联用仪测定卵表面分泌液蛋白质组,将其中的35个蛋白与10种节肢动物唾液蛋白质进行比较分析,推测这35种蛋白的功能保守性和特异性;采用qPCR检测10个基因(LsHSP70,LsPDIA3,LsHSP90,LsPPIase,LsADPRF1,LsHSP68,LsEF 1α,LsATPS,LsG3PD和RZF49036)在灰飞虱不同发育阶段(卵、1-5龄若虫和成虫)和雌成虫组织(输卵管、唾液腺、肠道和脂肪体)中的表达量。【结果】在灰飞虱卵表面分泌液中鉴定到149个蛋白,其中有35种蛋白检测到2个及以上的唯一肽段。在这35个蛋白中,发现6个蛋白在至少5种节肢动物的唾液蛋白质组中也普遍存在,推测它们在取食和产卵中具有相似功能;另有9个蛋白未在节肢动物唾液蛋白质组中发现,这些蛋白可能特异性地参与了飞虱的产卵过程。qPCR检测结果显示,LsHSP90和LsEF 1α在雌成虫中高表达,且LsEF 1α在雄成虫中不表达;LsPDIA3和LsHSP90在输卵管中高表达;其余基因在灰飞虱各个发育阶段和组织中均有不同程度的表达。【结论】在灰飞虱卵表面分泌液中鉴定到149个蛋白,其中一些蛋白与节肢动物唾液蛋白相比既有普遍性也有特异性,不同编码基因在不同发育阶段和组织中的表达模式也存在不同程度的差异。

【Aim】To identify and analyze proteins in the egg-associated secretions of the small brownplanthopper,Laodelphax striatellus,so as to lay a foundation for screening effectors of L.striatellus and studying their functions.【Methods】Intact eggs of L.striatellus were collected in the rice leaf sheath under a microscope,then were washed using phosphate buffer solution to get the egg-associated secretions.Liquid chromatography-tandem mass spectrometry(LC-MS/MS)was applied to identify the proteome of the egg-associated secretions.The functional conservation and specificity of 35 proteins from the proteome of the egg-associated secretions of L.striatellus were analyzed through comparing the 35 proteins with the salivary proteins from 10 arthropod species.The expression levels of 10 genes(LsHSP70,LsPDIA3,LsHSP90,LsPPIase,LsADPRF1,LsHSP68,LsEF 1α,LsATPS,LsG 3 PD and RZF 49036)in different developmental stages(egg,1st-5th instar nymphs and adult),and tissues(oviduct,salivary gland,gut and fat body)of female adults of L.striatellus were determined by qPCR.【Results】A total of 149 proteins in the egg-associated secretions of L.striatellus were finally identified,and among them,35 proteins with two or more unique peptides were detected.Among these 35 proteins,six proteins were found to be commonly present in the saliva proteomes of at least five arthropod species,implying their similar functions in both feeding and oviposition,and other nine proteins were not found in the saliva proteomes of arthropods,implying that these nine proteins may be specifically involved in the oviposition process of L.striatellus.qPCR results showed that LsHSP90 and LsEF 1αwere highly expressed in female adults,but LsEF 1αwas not expressed in male adults.LsPDIA3 and LsHSP90 were highly expressed in the oviduct,and other genes exhibited various expression levels in different developmental stages and female adult tissues of L.striatellus.【Conclusion】A total of 149 proteins have been identified in the egg-associated secretions of L.striatellus,some of them may have conservation or specificity compared with the salivary proteins from arthropod species,and their expression patterns in different developmental stages and tissues also show some variations.