Molecular Cloning And Expression Profiling Of Vitellogenin Receptor Of Calliptamus Italicus Based On Transcriptome Dataset

Calliptamus italicus is a major plague pest in the arid and semi-arid grasslands of Xinjiang,with a lack of information of its reproduction related genes.Vitellogenin receptors(VgR),members of the low-density lipoprotein receptor(LDLR)superfamily,are responsible for taking vitellogenin(Vg)into developing oocytes.The transcriptome of C.italicus was sequenced using the Illumina HiSeq/MiSeq platform and was bioinformatically analyzed.By further screening and identification,gonadal development related genes of C.italicus.VgR was cloned using RACE according to previous transcriptomic data of C.italicus and analyzed by bioinformatic tools,and the mRNA levels of VgR in different developmental stages and adult tissues were detected via real-time PCR and RCR.The results are as follows:(1)Transcriptome sequencing and analysisA total of 718 872 unigenes were obtained,with a mean length of 631 bp and an N50 length of 1 186 bp.Through a similarity search,254 597 unigenes were annotated.Most unigenes were annotated to Nr database(28.87%),and the unigenes of C.italicus had the highest similarity(10.80%)with those of Lasius niger in species distribution of unigenes in Nr database.In KEGG database,a total of 23 666 unigenes were assigned to 251 metabolic pathways,of which many pathways are involved in the reproductive system development,including oocyte meiosis,insulin signaling pathway,Wnt signaling pathway,GnRH signaling pathway,TGF-?signaling pathway,ubiquitin mediated proteolysis,progesterone-mediated oocyte maturation,and insect hormone biosynthesis.By further screening and identification,gonadal development related genes of C.italicus,including Vg,VgR,sox 21b,were obtained(2)Cloning of vitellogenin receptor geneThe complete cDNA of CiVgR is 5 589 bp in length with a 86 bp 5'-untranslated sequence,a 238 bp 3'-untranslated region and a putative ORF of 5265 bp.The putative mature VgR has 1754 amino acids with a molecular weight of 193.19 kDa and isoelectric point is 5.15.The signal peptide at the N-terminal end contains 27 amino acids.CiVgR has 4 typical conserved domains of LDLR family,including ligand-binding domain,epidermal growth factor precursor homology domain,transmembrane domain and cytoplasmic domain.It does not have O-linked carbohydrate domain.The phylogenetic tree analysis shows significant similarity with Periplaneta Americana(3)Expression analysis of CiVgRCiVgR was specifically expressed in the ovary.The expression of VgR started at the 4th nymph of a locust,increased rapidly after adult eclosion,hit the peak in previtellogenic stage,and decreased subsequently in vitellogenic stageBased on the sequencing of transcriptome,CiVgR was cloned and molecular structure and expression profile was analysed.The results showed that CiVgR function is closely related to vitellogenesis and provide valuable information to ascertain the regulation mechanism of vitellogenesis in C.italicus.