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

Abstract: 【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 N. benthamiana mediated by A. tumefaciens could suppress the programmed cell death induced by Bcl-2-associated X protein (BAX) and pathogen elicitor INF1. Subcellular localization results indicated that the fusion protein  Sm13498-GFP was localized in the cytomembrane of N. benthamiana leaves. 【Conclusion】 The results suggest that the salivary protein Sm13498 of S. miscanthi may be involved in the suppression of plant defense responses. This study lays a foundation for identifying the salivary effectors of S. miscanthi and understanding the high adaptability of wheat aphids to wheat varieties.

Key words: Sitobion miscanthi, salivary protein, effector, gene cloning, signal peptide, subcellular localization