| Phenacoccus solenopsis Tinsley is a worldwide eurytrophic invasive pest.P.solenopsis had damaged a number of cash crops and horticultural plants in many countries.Fatty acid and its derivatives play an important role in energy supplying,pheromone synthesis,epidermal formation,environmental adaptability in insect.In this study,we conducted a transcriptome analysis from different developmental stages of P.solenopsis using Illumina HiSeq? 2000 platform.A number of unigenes was annotated after being filtrated and assembled.We analyzed the expression of lipid synthesis related genes of P.solenopsis and validated these expression levels by qRT-PCR.Base on the transcriptome,we obtained complete encoding sequences of Elongase of very long chain fatty acid(Elovl)and Far Fatty acyl-coenzyme reductase(Far)with RACE technique.We analyzed molecular characterization of those genes.In addition,we compared the expression levels of two fatty acyl-coenzyme reductases of P.solenopsis after treated with spirotetramat.Our study lays basis for lipid synthesis mechanism.The main results are as follows:1.We analyzed the transcriptome of different developmental stages of P.solenopsis and identified related genes in lipid synthesis.Six samples from different developmental stages were running on Illumina sequence platform,and a total of 360,978,854 raw reads were obtained respectively.The clean reads of each sample were pooled and assembled into 132,753 unigenes by Trinity software.57,200 unigenes(43.09%)were annotated at least once by either of the five public database(NR protein database,Uniprot database,COG database,Pfam database and EST database).Heatmap of unigenes annotated in fatty acid biosynthesis pathway analysis found that 2nd nymph,3rd nymph,female adults and oviposition of female have similar gene expression patterns.The expression levels of acetyl-CoA carboxylase(Acc),fatty acid synthase(Fas),Elovl and Far,which are key genes in the pathway of lipid fatty acid synthesis,elongation and the fatty alcohol synthesis were analyzed and and verified by qRT-PCR.The expression levels of those genes in nymph stages are higher than those in the adult stages.2.A ELOVL gene of P.solenopsis was cloned using RACE.The length of open reading frame of PsElovl7 is 852 bp,and encodes a protein with 283 amino acides.TMHMM and Euk-mPLoc Software predicted that PsELOVL7 has 6 membrane spaning domains and located in endoplasmic reticulum.Four conservative motifs,including KXXE/DXXDT、HXXHH、HXXMYXYY、TXXQXXQ,were discovered in PsELOVL7 by sequence alignment.Evolutionary tree analysis shows: P.solenopsis has a close relationship with Acyrthosiphon pisum and Diuraphis noxia;Result of qRT-PCR revealed that PsElovl7 remarkably expressed in 1st nymph of P.solenopsis.3.Two FAR genes of P.solenopsis were cloned using RACE.Analysis sequence of PsFar Ⅰ and PsFar Ⅱ found the length of the open reading frames are 1584 bp and 1515 bp respectively,and encode amino acid of 527 aa and 504 aa.TMHMM and Euk-mPLoc Software predicted that both PsFAR I and PsFAR II had two membrane spaning domains which located in endoplasmic reticulum.Two conservative motifs were discovered in Rossmann folding region by sequence alignments,including a NADPH combining motif,TGXXGG,and an active site motif,YXXXK.Evolutionary tree analysis showed that P.solenopsis had a close relationship with Acyrthosiphon pisum and Diuraphis noxia.Expression of PsFar Ⅰ and PsFar Ⅱ at different development stages showed that there were differences expression levels.Expression levels of two Fars in 1st nymph were significantly higher than other stages.Expression level of PsFar Ⅰ in the male adult is dramatically higher than 2nd nymph nymphy,3rd nymph,female adult and female adult during oviposition.However,expression level of PsFar Ⅱ in male adult is remarkably lower than other stages.Spirotetramat treatment at 125 mg/L prominently increased the expression of PsFar Ⅰ in 3rd instar nymphs,but there was no difference in the expression of PsFar Ⅱ. |
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