| Trehalose is a very stable non-reducing disaccharide and constituted with two glucose molecules by a ?,?-1,1-glycosidic bond.Trehalose is present in a wide variety of organisms including plants,algae,fungi,yeasts,bacteria,insects and other invertebrates.Especially,in the animal kingdom,trehalose was first reported in insects.Trehalose,is called hemolymph sugar in insect,have revealed multiple physiological roles in insect,not only acting as a storage molecule,but also protecting against abiotic stresses,Therefore,trehalose are pivotal for the growth,development and survival of insects.Trehalose-6-phosphate synthase(TPS)is also widely studied as a key enzyme in trehalose biosynthesis.At present,there are more reports about TPSs of bacteria and fungi,but it is less studied about its functions in insects.In this study,Heortia vitessoides Moore was selected as the research object,and partial TPS gene sequences is screening on the basis of transcriptome of H.vitessoides,then 3 'RACE technology were applied to clone to abtain compeleted ORF.Bioinformatics analysis of the sequence was carried out.The expression pattern of TPS in different tissues and different stages of development of H.vitessoides was studied.Furthermore,We used an in vivo RNAi method that allowed us to efficiently disrupt the Hv TPS gene function in order to help elucidate its role in developmental processe of H.vitessoides,including phenotypic observation,development time,mortality,larval chitin and lipid biosynthesis.The main achievements are as follows:(1)Cloning and sequence analysis of TPS gene from H.vitessoidesBase on H.vitessoides transcriptome data,cloning and identification of the whole ORF of Hv TPS(Gen Bank accession number:MG787167)were facilitated in this study.Hv TPS c DNA contains an open reading frame of 2496 nucleotides,which encodes a protein of 831 amino acids with a predicted mass of approximately 93.48 k Da and a p I of 7.06.Hv TPS shares 70-96% identity with other known TPSs.BLAST analysis and the Multiple sequence alignment of TPSs from different insects revealed that there were two putative catalytic domains and two signature motifs(HDYHL and DGMNLV).(2)Tissue-specific and development-stage expression patterns of Hv TPS RT-qPCR experiments were performed to determined the expression patterns of Hv TPS in tissue-specific and development-stage.Tissue expression analysis revealed that the expression of Hv TPS was detected in all the examined tissues in this study,and fat body expressed the highest level among these tissues.Development-stage expression patterns showed that Hv TPS was expressed in all tested stages,but highly expressed after pupating or before molting in H.vitessoides.(3)RNAiAmong the injection concentration gradient(1?g/?l,2?g/?l,3?g/?l,4?g/?l,5?g/?l,injection volume is 1?l),concentration of 3?g/?l ds RNA can cause the highest silence efficiency.When 24 h or 36 h after injection of ds Hv TPS with 3?g,trehalose content and Hv TPS enzyme activity decreased,and glucose content increased.It was speculated that this change may be caused by decrease of the expression level of Hv TPS.After RNAi was carried out successfully,it showed abnormal phenomenon during the pupal and adult stages,and the survival rate decreased significantly compared with the control,suggesting that Hv TPS was involved in metamorphosis of H.vitessoides.At the same time,chitin content and the expression level of key enzyme participated in the biosynthesis of chitin were decreased.It also found that fat body mass and size after dissection and the expression level of two genes related to lipogenic pathway increased,but the expression level of gene relevant to lipid degradation decreased.So it indicated that Hv TPS was involved in the biosynthesis of chitin and lipid H.vitessoides. |
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