| Ceracris nigricornis is the type species of the genus Ceracris,distributed in Asia.It is a serious pest that harm bamboo resouces,which has seriously hindered the development of forestry.The Vitellogenin(Vg)can provide nutrients and functional substances such as protein,fat,vitamins,carbohydrates,phosphorus and sulfur for developing embryos.Vitellogenin receptor(VgR)is not only closely related to vitellogenesis,ovarian activation,egg formation,but also plays a crucial role in insect information communication,behavioral construction,social differentiation and immune regulation,and has become a potential new target for pest control.In this study,we sequenced the transcriptomes of gonad(ovary and testis)of C.nigricornis using Illumina high-throughput sequencing technology,the main results and conclusions are as follows:1)Using high-throughput sequencing,we finally obtained 264,449,130 raw reads from the testis and ovary of C.nigricornis.After data filtering,260,628,928 clean reads were yielded,and the ratio of Q20 reached 97.37%.All sequences were assembled into 251,354 Unigenes.Of them,a total of 73,101 Unigenes were annotated in at least one transcriptome database,with an annotation rate of 29.08%,which indicates that a large number of transcripts still need to be annotated.A total of 43,453 sequences were assigned to 55 clusters of gene ontology(GO)according to biological processes,molecular functions,and cell components.In addition,21,386 Unigenes were assigned to 26 clusters of KOG,in which the "General function prediction" makes up the largest proportion.In total,14,487 Unigenes were mapped onto 229 pathways using the KEGG database.In addition,we identified 32,801 short sequences as potential SSRs;for SNP markers,a total of 389,026 SNPs were found.2)In the study of differential expression analysis between the testis and ovary transcriptomes of C.nigricornis,we screened 1,924 differentially expressed genes,of which 34.8%were up-regulated and 65.2%down regulated.Among the all genes upregulated,we identified 10 Vg sequences and 12 VgR sequences through transcriptome annotation and Blast.Phylogenetic trees were generated using the Neighbor-joining(NJ)method in MEGA 7.0 software,based on the sequence choosed in the transcriptome and published in NCBI,including Orthoptera,Blattaria,Lepidoptera,Coleoptera,Hymenoptera,Isoptera,Anoplura,Diptera and Hemipteran.The result of cluster analysis showed that both Vg and VgR were conserved,and each order was clearly clustered into independent branches.Moreover,in the whole branch,the relationship between Orthoptera and Blattaria are closer.3)The VgR gene of C.nigricornis was cloned for the first time with the technology of RACE.The full-length of VgR cDNA is 5,675 bp with an ORF of 5,427 bp encoding 1,808 amino acids.And we analysised its physical and chemical properties.The ML-tree and BI-tree were constructed based on the amplified full-length VgR sequence and other insects’ homologous sequences.And the result showed that species of each order had a separate branch.At the same time,the results of selective pressure analysis showed that the insect VgR gene were general under negative selection.In addition,we analyzed the gene expression profiling of VgR and the data showed that the gene is mainly expressed in ovarian tissue and is almost not expressed in other tissues.In conclusion,our research has build a database of C.nigricornis gonadal transcriptome,which has obtained a large number of reproductive information.We have successfully cloned the full-length of VgR gene for the first time,and analyzed the sequence information,molecular structure,the phylogenetic analysis and expression pattern.The research not only lays a theoretical foundation for exploring the physiological function of insect VgRs,but also provides a basis for developing new methods of pest control. |
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