| The α-glucosidases are a class of starch hydrolyzing enzymes,the main function of which is to hydrolyze α-1,4 glycosidic bonds and release glucose.They are indispensable enzymes in the sugar metabolism pathway of insect.Myzus persicae is an important economic pest with a wide range of host species.They feed on phloem sap with high sugar content.The study on carbohydrate metabolism of Myzus persicae is an important aspect to analyze the feeding behavior of aphids.In our previous study,we found that α-glucosidase genes were the main common differentially expressed gene in the feeding-associated transcriptomes of three aphids.Therefore,genome-wideα-glucosidase genes of Myzus persicae,Acyrthosiphon pisum and Aphis citricidus were identified,and the expression pattern and function of α-glucosidase genes of Myzus persicae were analyzed.The main results were as follows:1.Genome-wide identification of α-glucosidase genes of three aphids speciesIn order to understand the gene numbers,sequence structure and evolutionary relationship of aphid α-glucosidase genes,by genome-wide identification,we found 13,15 and 8 α-glucosidase genes in green peach aphid,pea aphid and brown orange aphid.Compared with other insects,the number of α-glucosidase genes in aphid was expanded.All aphid α-glucosidase genes had Aamy catalytic domain,most of them contained signal peptides,and some of them had transmembrane regions.The α-glucosidase gene sequence had high homology in the multi-sequence alignment,and there were no deletion in the four conserved regions(CSR I-IV)and key catalytic sites necessary for the catalytic function.Phylogenetic analysis divided aphid α-glucosidases into five categories:Category I,Category II,Category III,Category IV.Among them,Category I aggregatedα-glucosidase genes of Drosophila melanogaster and Bombyx mori.Category II was suspected to be salivary protein aggregation;Category III was aphid specific clustering and did not aggregate with salivary protein genes.Category IV was a unique cluster of stinger-sucking insects,and was far fromα-glucosidase members of Drosophila melanogaster and Bombyx mori.Myzus persicae distributed in Category II,Category III,Category IV.According to the above information,each α-glucosidase gene of Myzus persicae were named.2.Expression pattern analysis of α-glucosidase gene in green peach aphidIn order to investigate the differences in the expression patterns of suspected salivary protein and non-salivary protein α-glucosidase genes in aphids,four α-glucosidase genes(Mp Ag C2-2,Mp Ag C3-1,Mp Ag C3-3,and Mp Ag C3-4)being differentially expressed in the feeding-associated transcriptomes were studied in this chapter.First of all,RT-q PCR was used to analyze the expression patterns of four α-glucosidase genes in different developmental stages and tissues of Myzus persicae.Mp Ag C2-2 which belonged to Category II was highly expressed in adult aphids,and had a high expression level in salivary glands and intestines.Secondly,Western Blot showed that Mp Ag C2-2might be secreted into the plant tissues,suggesting that Mp Ag C2-2 may be involved in the feeding process of aphids as a salivary protein.Mp Ag C3-1,Mp Ag C3-3,and Mp Ag C3-4 which belonged to Category III were all highly expressed in the intestinal tract.These three genes have different expression pattern in various developmental stages.For example,Mp Ag C3-1 was highly expressed in adult stage,and Mp Ag C3-3 was highly expressed in 1st instar nymph and adult stage.Mp Ag C3-4was highly expressed in 4th instar nymph stage.There might be functional complementarity among the three genes.In summary,we speculated that Mp Ag C2-2 as a salivary protein but others(Mp Ag C3-1,Mp Ag C3-3,and Mp Ag C3-4)as intestinal proteins play different functions in feeding process.3.The involvement of Mp Ag C2-2 in the feeding of in green peach aphidIn order to explore whether the α-glucosidase Mp Ag C2-2 was involved in the feeding of Myzus persicae in the form of salivation protein,first of all,we observed that RNAi-based silencing of Mp Ag C2-2 induced the mortality of adult aphid,and decreased the total aphid number as well as body weight and body size.In addition,the specific activity of α-glucosidase and the glucose content of aphids were significantly decreased.These results indicated that Mp Ag C2-2 as an α-glucosidase played an important role in the feeding process of peach aphid.It may inhibit the process ofα-glucosidase digestion and decrease the ability to obtain nutrients.For instance,EPG parameters showed that the aphids that interfered with Mp Ag C2-2 had longer probing time and lower feeding efficiency than the control group.Meanwhile,the total duration of E wave was significantly shorter than that of the control group,indicating that the aphids that silencing of Mp Ag C2-2 had shorter feeding time in phloem,resulting in lower fitness to host plants.Finally,transient expression of Mp Ag C2-2 in tobacco leaves rescued the aphid mortality from RNAi-based silencing of Mp Ag C2-2.Meanwhile,when Mp Ag C2-2 was expressed in tobacco leaves,the total aphids numbers was significantly higher than that the other groups,indicating that the plant-mediated expression of Mp Ag C2-2 enhanced the aphid’s performance.Therefore,we could conclude that the α-glucosidase Mp Ag C2-2 was a salivary protein involved in feeding.In conclusion,the results of this thesis have clarified the expression pattern of α-glucosidase genes in feeding process,and explored the role of Mp Ag C2-2 in the form of salivary proteins,which provides a reference for understanding the function of α-glucosidase genes in aphids and the types of salivary protein in the interaction between aphids and plants. |
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