The Identification Of Long Non-coding RNA In The Growth Of Pieris Rapae

The cabbage butterfly,Pieris rapae(Lepidoptera: Pieridae)is one of the most destructive agricultural pests in the Brassicaceae family,causing considerable economic loss in China.As said by Gilbert in the preface of his book “Insect Development”,studies on insect development is invaluable for insect control.If the key gene involved in insect development is found,many methods like RNA interaction could be used directly into the pest control,and provide important reference for pest control strategy explore.In this study,we focused on Pieris rapae.Firstly,two DNA libraries(300bp and 500 bp insert size)were constructed for high-throughput sequencing.Totally,102.129 G base were generated by Illumina Hi-Seq machine.After quality control,101.652 G base were filtered out.Different depth of raw reads were used assemble the genome by using the Platanus software.We obtained the assembled genome with the N50 of 5Kb,which was used in the following lncRNA annotation.Then,the Pieris rapae transcriptome previously assembled in our lab was used to screen lncRNA in the following steps.First,the sequences of the transcriptome were blast against non-redundant protein database,rRNA database,miRNA database,and tRNA database to exclude homologous mRNA,rRNA,miRNA,and tRNA sequences.Second,within the remaining sequences,the sequence length estimation and ORF prediction were conduct by using CPC software.The sequence with length less than 200 nt and with ability to encode more than 100 amino acids,were screened again using Portait software.Finallly,1194 lncRNAs were filtered out with high credibility.Next,the basic characteristic of these lncRNA was analyzed.Compared with mRNA,thelength of P.rapae lncRNAs were relatively short.The length of the most lncRNA is between200 to 700 bp,while a few lncRNAs are over 1 kb.Similarly,the length of ORFs of the most lncRNA are less than 100 nt,while a few of them are over 100 nt.In the meantime,expression of lncRNA in different stages were estimated by RSEM and edgeR.The result suggests that the number of the expressed of lncRNA in six stages are similar,which is about 300.However,the number of stage specific lncRNA is quite different.There are280 stage specific lncRNA in egg stage and about 60 stage specific lncRNA in other stages.After the differential expression analysis,we obtained 97 differential expression lncRNA and the result suggests that the differential expression lncRNA were mostly occurred from the stage of egg to first instar larva and these lncRNAs had high expression level in egg stage.In other stages,only a few lncRNAs were found to be differential expressed between adjacent stages.After the genome sequence obtained,we also map the lncRNA and mRNA to the genome to annotate lncRNA.According to the position of lncRNA in the genome,we identified 635 intergenic lncRNAs,212 intronic lncRNAs,347 sense and antisense lncRNAs.In the meantime,We found 2164 mRNA which were located near the lncRNA,then KEGG,GO annotation and differential expression analysis were processed.In order to know the interaction of lncRNA and miRNA,we used software to predict 36 lncRNA were the target of miRNA.Finally,nine differentially expressed lncRNAs were random selected to evaluated the expression level by the qPCR method.Quantitative results showed that the expression level detected by RNA-seq and qPCR methods were quite consistent,which indicated the accuracy of the bioinformatics results.In short,P.rapae draft genome,1194 lncRNA and 2164 lncRNA neighoring mRNA were obtained by using both RNA-seq and DNA-seq technology.Furthermore,we got 97 lncRNA and1671 mRNA which were differential expressed in the growth of P.rapae.Using miRNA target predicted software,36 lncRNA may interact with miRNA.Our findings will lay the foundation for the study of P.rapae in the future.