Transcriptome Analysis Of The Response Of Midgut In Pieris Papae L To CoTI

Pieris rapae L,a lepidoptera sulfur butterfly,is one of the main pests of cruciferous vegetables.The P.rapae obtains essential amino acids for their growth and development by degrading the food proteins by protease in midgut,and using the cytochrome P450 and other detoxification enzymes to degrade the insect resistant secondary metabolites of host plant origin.The preliminary study results indicated that the trypsin inhibitor of Cassia obtusifolia(CoTI),nonhost plant of P.rapae,showed strong resistance to P.rapae.In paper,the molecular biology and transcriptome sequencing and other techniques were applied to analyze the transcriptional level changes of digestive enzymes in the midgut of P,rapae after ingestion CoTI,laying a foundation for the study on insect-resistant mechanism of CoTI.At first,CoTI genes was cloned into prokaryotic expression vector pET28a and successfully constructed the recombinant plasmid pET28a/CoTI.The recombinant plasmid pET28a/CoTI were identified by PCR,dual-enzyme digestion and sequencing analysis,which indicated that the recombinant plasmid contained the target gene fragments and with successful construction.The protein was expressed and purified;the tested concentration was 920 ?g/ml.Then,larvae of P.rapae were fed on cabbage leaves containing CoTI.The larvae of P.rapae fed on normal cabbage leaves were used as control.The total RNA of midgut was extracted respectively and transcriptome sequencing was performed by Illumina 4000 sequencing platform,which produced transcriptome data of 17,16Gb.28,918,396 and 28,678,331 high-quality reads were obtained,respectively and therefore 51,544 unigenes were assembled by Trinity software.These unigenes were compared and annotated with the common database,including Nr,Swiss-Prot,GO,KOG,COG and KEGG,to produce 22,233 unigenes with functional annotations.The relevant enzyme for digestion and detoxification was found by comparing the Nr database,including 105 serine proteases(79 trypsin and 26 chymotrypsin,PrSPs),74 lipases(PrLPs),17 amylases(PrALs),113 cytochrome p450(PrCYP450s),46 carboxylesterases(PrCXs),54 UDP-glycosyltransferases(PrUGTs)and 41 glutathions-transferases(PrGSTs)were incuded.Based on the comparison between the transcriptome data in midgut of P.rapae fed with normal cabbage leaves and fed with cabbage leaves that contained CoTI,3,066 differentially expressed genes were found and analyzed in paper,including the GO analysis,COG analysis,KEGG pathway analysis and so on.Dozens of digestive enzymes were found to be differently expressed after ingestion of CoTI.By analyzing the KEGG pathway,it was found that the expression of some crucial genes Wnt,Hippo and Notch involved in the growth and development signaling pathways were different under different treatment conditions,indicating that CoTI could inhibit the normal growth and development of P.rapae.Furthermore,selection of qRT-PCR reference gene was performed.Eight relatively stable genes of Sec61,PSMD9,eIF2g,UPF3,LRE,NADH,hnRNP and THOC2 were screened out firstly from transcriptome database,and then three reference genes of RPL32,EF1 and GADPH were selected together as candidate reference genes to analyze their relative expression levels in different tissues,different developmental stages and different stress conditions.As a result,according to the analysis of GeNonn and NormFinder software,UPF3 expression level was found the most stable one.So the UPF3 was selected as a reference gene in follow-up quantitative analysis.