摘要
【目的】荻草谷网蚜Sitobion miscanthi为我国小麦Triticum aestivum主产区麦蚜优势种;Sm13498蛋白是在荻草谷网蚜唾液腺中特异表达的唾液蛋白。本研究旨在探析荻草谷网蚜功能未知的Sm13498在调节植物防御反应中的潜在作用。【方法】基于荻草谷网蚜唾液腺转录组测序数据,PCR克隆Sm13498的cDNA全长序列,并进行生物信息学分析;采用RT-qPCR测定Sm13498在取食小麦叶片不同时间的荻草谷网蚜无翅成蚜中的表达动态;通过酵母分泌系统验证Sm13498蛋白信号肽的分泌功能;利用根癌农杆菌Agrobacterium tumefaciens介导在本氏烟Nicotiana benthamiana中瞬时表达技术鉴定Sm13498蛋白功能及亚细胞定位。【结果】克隆获得了荻草谷网蚜Sm13498 cDNA全长序列(GenBank登录号:MW346655),开放阅读框(ORF)全长783 bp,编码260个氨基酸,预测蛋白分子量28.01 kD,第1-22位氨基酸为N端信号肽。系统进化树显示,Sm13498与豌豆蚜Acyrthosiphon pisum的功能未知蛋白LOC100159087 precursor(GenBank登录号:NP_001313548.1)亲缘关系最近(氨基酸序列一致性为71.7%)。RT-qPCR结果表明,Sm13498在荻草谷网蚜无翅成蚜取食小麦叶片12 h时表达水平达到最高。含有Sm13498信号肽片段的酿酒酵母Saccharomyces cerevisiae YTK12可在YPRAA培养基正常生长,并可将无色2,3,5-氯化三苯基四氮唑(TTC)还原为不可溶的暗红色的氯化三苯基四氮唑(TTF),证实其信号肽具有分泌活性。经根癌农杆菌介导在本氏烟瞬时表达Sm13498蛋白可抑制Bcl-2相关X蛋白(Bcl-2-associated X protein,BAX)及病原菌激发子INF1诱导的程序性细胞死亡。亚细胞定位结果表明,Sm13498-GFP融合蛋白定位于本氏烟叶片细胞膜。【结论】结果说明荻草谷网蚜唾液蛋白Sm13498可抑制植物防御反应。本研究为发掘荻草谷网蚜唾液中效应子,深入解析麦蚜对小麦品种强适应性奠定了基础。
【Aim】The grain aphid,Sitobion miscanthi,is a dominant cereal aphid species in the major growing areas of wheat(Triticum aestivum)of China.Sm13498 is a salivary protein specifically expressed in the salivary glands of S.miscanthi.This study aims to investigate the potential role of the functionally unknown salivary protein Sm13498 of S.miscanthi in modulating plant defense.【Methods】Based on the sequencing data of the salivary gland transcriptome of S.miscanthi,the full-length cDNA sequence of Sm13498 gene was cloned by PCR and analyzed by bioinformatics.The expression levels of Sm13498 in apterous adults of S.miscanthi feeding on T.aestivum leaves for different time were determined by RT-qPCR.The secretion function of the signal peptide of Sm13498 was verified by yeast secretory system.The function of Sm13498 and its subcellular localization in Nicotiana benthamiana was examined using Agrobacterium tumefaciens-mediated transient expression technique.【Results】The full-length cDNA sequence of Sm13498 of S.miscanthi was cloned(GenBank accession no.:MW346655).Its open reading frame(ORF)is 783 bp in length,encoding 260 amino acids with the predicted molecular weight of 28.01 kD and amino acids 1-22 predicted to be N-terminal signal peptide.Phylogenetic analysis showed that Sm13498 was most closely related to LOC100159087 precursor,an uncharacterized protein of Acyrthosiphon pisum,deposited in GenBank under the accession no.NP_0013135481,sharing 71.7%amino acid sequence identity.The RT-qPCR results revealed that the expression level of Sm13498 reached the peak in apterous adults of S.miscanthi feeding on wheat leaves for 12 h.Saccharomyces cerevisiae strain YTK12 containing the signal peptide fragment of Sm13498 grew normally on the YPRAA medium in the yeast secretion system,and catalyzed the conversion of colorless 2,3,5-triphenyltetrazolium chloride(TTC)to insoluble dark-red-colored triphenylformazan(TTF),confirming the secretion activity of the predicted signal peptide.The transiently expressed Sm13498 in
作者
付裕
王倩
张勇
陈巨莲
FU Yu;WANG Qian;ZHANG Yong;CHEN Ju-Lian(Biology of Plant Diseases and Insect Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100193, China)
出处
《昆虫学报》
CAS
CSCD
北大核心
2021年第9期1009-1019,共11页
Acta Entomologica Sinica
基金
国家自然科学基金项目(31871979,31901881)
国家重点研发计划(2017YFD0201700,2017YFD0200900,2016YFD0300700)。
关键词
荻草谷网蚜
唾液蛋白
效应子
基因克隆
信号肽
亚细胞定位
Sitobion miscanthi
salivary protein
effector
gene cloning
signal peptide
subcellular localization
