Comparison Of Three Morphological Transcriptomes Of The Short-horned Striata And Mitochondrial Transcriptome Mapping

For the exploring molecular mechanisms of the orthopteran individual growth,development and sex differentiation and understanding gene expression characteristics of mitochondrial transcription,the transcriptomes of three samples of Xenocatantops brachycerus,the female adult(FA),male adult(MA)and male nymphs(MN),were sequenced using Illumina HiSeq4000 platform,and assembled by Trinity.The differentially expressed genes(DEGs)of three samples were annotated with GO and KEGG database.At the same time,qRT-PCR was used to test the expression level of 14 DEGs.Lastly,for analyzing the expression of mitochondrial gene,sequencing reads and Unigene to the X.brachycerus were mapped to mitochondria genome.Main conclusions were as following:(1)A total of 13.32Gb data was obtained from three samples of FA,NMA and MN(FA4.46Gb,MA4.43Gb,MN4.43Gb,respectively).Assembling without reference genome resulted in total number of 40,522 Unigene and total length 39,255,884bp for FA,20,187 Unigene and 14,320,681bp for MA,24,030 Unigene and 16,998,200bp for MN,respectively.Finally,combined assembly of the three samples obtained 43,187 All-Unigene with a total length of 41,642,132bp,with the average length 964bp and N50 1,799bp.(2)After the functional annotation of the 43,187 All-Unigene,there were 24,717(57.23%)assigned to at least one database.The Unigene annotated to the NR database up to 21,978,accounting for 50.89%,the NT database up to 12,071(27.95%),the COG up to 9,127(21.13%),the Interpro up to 15,099(34.96%),the KEGG up to 15,762(36.50%),the Swissprot up to 17,692(40.97%),the GO database up to the at least 3,272 Unigene(7.58%).The obtained data may provide a rich resource for study in the growth and development,sex determination mechanism and the molecular marker mining research for phylogenetic analysis of X.brachycerus.There was 42.77%Unigene without any annotation information to any database,this may be due to the lack of orthopteran genome data in the database.(3)DEGs analysis showed that there were 15,351 DEGs in FA-vs-MA,16,362 DEGs in FA-vs-MN,5,932 DEGs in MA-vs-MN,respectively.Of the 15,351 DEGs in FA-vs-MA,1,329 of them were up-regulated in MA,while the other 14,022 were up-regulated in FA.Of the 16,362 DEGs in FA-vs-MN,1,375 of them were up-regulated in MN,while the other 14,987 were up-regulated in FA.Of the 5,932 DEGs in MA-vs-MN,2,545 of them were up-regulated in MN,while 3,387 were up-regulated in MA.The GO annotation of these DEGs showed that the significant enrichment terms all belonged to the cell component class,more DEGs were up-regulated in adult than in nymph for the terms of extracellular complex,cytoplasmic components and ATPase complex.In the KEGG Pathway annotation,the spliceosome,DNA replication,nitrogen synthesis,base excision repair and fatty acid metabolism were significantly enriched in different comparison samples.Given the fact that X.brachycerus overwinters as adult,it needs to accumulate more fat as food,more expressed genes were required for lipid synthesis.(4)The qRT-PCR was used to carry out experimental verification of 14 DEGs.The experimental results were consistent with the results of RNA-Seq,which proved the reliability of the transcriptome data.The 14 DEGs used to validate all had a strong relationship with the sex determining mechanism of X.brachycerus.(5)The results of read mapping to complete mitochondrial genomes using the Clean Reads of three samples showed that 95%of the base sites was covered.After transcription,it pruduced 5 transcripts(J1,J2,N1,N2 and N3).Three samples of X.brachycerus were basically the same with each other in the mitochondrial expression patterns and expression trends,L-rRNA,COX1,COX2,COX3 and CYTB genes were the highest coverage genes,while the ND6,ND4L and S-rRNA genes were the lowest coverage genes.The reason for the highest coverage of L-rRNA gene may be the independent and efficient transcription start point,while the lowest coverage of S-rRNA gene may be caused by the short poly(A)tail which resulted in inefficient sequencing.This is the first time to study the transcriptome sequence of X.brachycerus.The large number of DEGs obtained from the three samples comparisons would provide the gene level information for the exploration of transcriptional regulation mechanism.The identification of some key genes involved in the growth,development and the sex regulation and a large number of SNP of X.brachycerus can provide valuable information for future study.