| Aphids are major agricultural pests which cause significant yield losses of the crop plants each year by inflicting damage both through the direct effects of feeding and by vectoring debilitating plant viruses. Annual worldwide crop losses due to aphids are estimated at hundreds of millions of dollars. The application of nitrogen fertilizer and elevation of atmospheric CO2concentration exacerbated aphid infestation. For example, in2010-2011crop seasons,62.5%of the26million hectares of the Chinese wheat growing area suffered severe aphid infestations. For many crops, insecticides provide a simple and effective strategy for aphid control. However, the application of such chemicals is not desirable in the long term, because of the development of insecticide resistance and the potential negative effects on non-target organisms, and the need for more sustainable agricultural practices with fewer external chemical inputs.Among the major aphid species infesting wheat in China such as grain aphid (Sitobion avenae F), greenbug (Schizaphis graminum Rondani), bird-cherry oat aphid (Rhopalosiphum padi Linnaeus) and rose-grain aphid (Metopolophium dirhodum Walker), grain aphid is the most dominant and destructive one, seriously affecting wheat production in Yellow Huai and the Northern China Plain, the Southwest, Northwest and the Middle Yangtze River regions. Grain aphid is also the major pest of wheat in Europe and North America. In some wheat production areas, it has damaging infestations every year, causing the wide-spread use of chemicals and as much as15to60%of severe reduction in wheat yield. For a couple of decades, conventional breeding programs have been undertaken and considerable efforts have been expended in searching for aphid resistance in small grain germplasm worldwide. However, due to the complexity of plant-aphid interactions and the rapid development of resistant pest biotypes, outbreak of aphids causing substantial losses of wheat are reported regularly. Breeders and growers are still struggling to find an efficient strategy for aphid control in wheat.Expression of double-stranded RNA (dsRNA) directed against suitable insect target genes in transgenic plants has been shown to give protection against pests through RNA interference (RNAi), opening the way for a new generation of insect-resistant crops. However, the genome and transcriptome data of grain aphid have never been reported so far, which make the massive selection of RNAi targets unfeasible. In addition, the data of grain aphid transcriptome or genome can be used for comparative genome study to understand the evolutional diversity among aphid species and host plant specialization which could provide a basis for aphidcontrol in the future.In this research, Roche GS-FLX454pyrosequencing, in which DNA is sequenced in a parallel manner and more than1million reads are produced with a single sequencing run reaction within a limited time, was adopted to sequence the transcriptome of a kind of wheat aphid (Sitobion avenae Fabricius) using an mRNA sequencing method in our laboratory. Ultimately, a total of1,106,696ESTs with an average length of380bp, totaling401.7Mb, was obtained from one pico-plate reaction using GS-FLX Titanium Sequencing Reagents. In total,31,198contigs and1,078singletons with an average length of866bp and369bp, respectively, were obtained after being assembled by mira3and gsAssembler v2.3. In addition, functional annotation, classification and comparative sequences analysis of unigenes (contigs and singletons) were performed using public database resources and BLAST tools. Of these unigenes,25,389,21,635and16,211unigenes matched the Nucleic Acid Database (Nt), Non-redundant Database (Nr), and Swiss-Prot databases respectively. The comparative analysis of grain aphid transcriptome data with mRNA sequences of pea aphid and Ka/Ks analysis of orthologs between the two species indicate that genes participating in "amino acids and other biomolecular synthesis and metabolism","polysaccharide and saccharide metabolism","insecticide resistance and detoxification" and so forth are very important to evolution, transcriptome diversity and host plant specificity between the two aphid species. Among the total32,277unigenes,66were selected by some criteria and were then subjected to double-stranded RNA synthesis and an oral feeding test of aphids.4unigenes, which encode cytochrome c oxidase subunit Ⅶ c precursor, secreted salivary peptide, salivary protein MYS2and serine protease1, were screened out. Their effects on the mortality of aphids, which were caused by suppression of the expression of homologous genes in the aphid body, were verified using qPCR. Furthermore, Cy3-dCTP labeled dsRNA tracking experiments showed that the double-stranded RNAs of the unigenes were absorbed in the digestive system and then permeated into the whole body of the aphid fed with double-stranded RNAs after72h. This work sets up the fundamental basis for improving the resistance of wheat to aphid ruin by genetic transformation with constructs of double-stranded RNA of aphid genes. |
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