关键词: 荻草谷网蚜, 唾液蛋白, 效应子, 亚细胞定位, 基因沉默

Abstract: 【Aim】The grain aphid, Sitobion miscanthi, is one of the most important pest insects in agricultural production in China. Its saliva contains many effector proteins with various functions, which are involved in aphid-plant interactions during the feeding process. The aim of this study is to clarify the functions of the salivary protein SmHMp1 in the processes of feeding and reproduction of S. miscanthi, and to explore the feasibility of using this gene as a silencing target to control S. miscanthi.【Methods】Based on salivary gland transcriptome data of S. miscanthi, the full-length cDNA sequence of SmHMp1 of S. miscanthi was cloned, and bioinformatically analyzed. The RT-qPCR was used to determine the expression levels of SmHMp1 in different development stages (1st-4th instar nymphs and wingless adult), tissues (salivary gland, head, thorax, abdomen, embryo and whole body) of wingless adults, adults of different wing morphs (winged and wingless adults) and wingless adults of S. miscanthi fed with different diets (artificial diet and wheat seedlings). Subcellular localization of the SmHMp1 protein was analyzed by Agrobacterium tumefaciens-mediated transient expression in tobacco. The recombinant virus vector of barley stripe mosaic virus (BSMV) was constructed and the host-induced gene silencing (HIGS) technology was used to silence SmHMp1 of S. miscanthi, and the silencing effects on the growth and development, reproduction and feeding of S. miscanthi were analyzed by anatomy, life table and electrical penetration graph (EPG) techniques. 【Results】 The full-length cDNA sequence of SmHMp1(GenBank accession no.: OP021692) of S. miscanthi was cloned. SmHMp1 has an open reading frame (ORF) of 426 bp in length, encoding 141 amino acid residues with the predicted molecular weight of 16.2 kD, theoretical isoelectric point of 8.74, and an N-terminal signal peptide with 19 amino acid residues. The phylogenetic analysis showed that SmHMp1 is most closely to the uncharacterized protein LOC100165393 precursor (GenBank accession no.: NP_001155659.1) of the pea aphid, Acyrthosiphon pisum. The RT-qPCR results showed that SmHMp1 of S. miscanthi was expressed at all developmental stages, and had the highest expression level at the 1st instar nymphal stage, showing an initial downward and later upward overall trend with the developmental time. The expression level of SmHMp1 in salivary glands of wingless adult was significantly higher than those in other tissues. There were no significant difference in the expression level of SmHMp1 in adults between both wing forms or between fed with both diets (artificial diet and wheat seedlings). Subcellular localization results showed that SmHMp1 protein is localized in the membrane and nucleus of tobacco cells. The expression level of SmHMp1 in S. miscanthi wingless adults fed with the wheat seedlings inoculated with the recombinant virus BSMV-SmHMp1 decreased extremely significantly to 43.64% of that of the control group (inoculated with the recombinant virus BSMV-GFP). When SmHMp1 was silenced, the number of aphids produced in 8 d and the number of embryos of S. miscanthi wingless adults decreased significantly (54.17% and 46.25% of the control, respectively), and the phloem-feeding time was significantly shortened to 64.95% of the control group.【Conclusion】The salivary protein SmHMp1 may play an important role in the feeding and reproduction processes of S. miscanthi, and has the potential to be used as a HIGS target to control S. miscanthi. This study is conducive to the deep understanding of aphid-host interactions at the molecular level and development of green control measures for S. miscanthi.

Key words: Sitobion miscanthi, salivary protein, effector, subcellular localization, gene silencing