Molecular Cloning And Expression Profiling Of Vitellogenin Receptor Gene In The Diamondback Moth, Plutella Xylostella

The diamondback moth, Plutella xylostella (Lepidoptera, Plutellidae), is one of the most distribution pests, which has a destructive damage to Brassicaceous crops. It brings huge difficulties to prevention and treatment, because of short generation cycle, reproductive ability, high resistance, host range. However, there are still no effective strategies to control the P. xylostella. Arounding of reproductive regulation deciding to insects reproductive potential and adaptability, we employed many techniques and methods to examine vitellogenin receptor (VgR), which is the fundamental substance of vitellogenesis and oogenesis, in insects. Four experiments will be conducted. First, the morphology of reproductive structure and developmental dynamic of oogenesis were observed, in P. xylostella. Second, the molecular characteristics and phylogenetic relationships with other insects of VgR in P. xylostella (PxVgR) were analyzed. Third, the expression profiles of PxVgR in different developmental stages and tissues were surveyed. Finally, the RNAi efficiency of PxVgR were studied. We believe that this study will provide further understanding of reproductive system, functions of VgR and the reproductive regulatory mechanisms, in P. xylostella. The main results were as follows:1. The morphology of female reproductive structure and its dynamic of ovarian development, in P. xylostellaThe female reproductive system of P. xylostella consists of a pair of ovaries (each has four fallopian tubes), a pair of lateral oviducts, median oviduct, a pair of accessory glands, afferent duct, spermatheca, bursa copulatrix. Yolk sedimentary emerges at 2d pupa and matured eggs were found in newly emerged female, Oogenesis of P. xylostella could be divided into three stages:the previtellogenic stage, the vitellogenic stage and the yolk maturation stage, on the basis of egg morphology and yolk sedimentary in ovary.2. The molecular characteristics of PxVgRThe open reading frame (ORF) of PxVgR consists of 4338 base pairs (bp) that encodes 1446 amino acids, with a proposed molecular mass of 161 kDa and a predicted isoeletric point of 5.33. Sequence analysis reveals that Aspartic (119, up to 8.2%), Alanine (110, up to 7.6%), Cysteine (103,7.1%) were more than other amino acids. Signal peptide locates in N-terminal 1-20. The comparative analysis with genome shows that PxVgR has 10 amino acid sites mutation. PxVgR consists of five typical conserved domain:ligand-binding domain (LBD), epidermal growth factor precursor homology domain (EGFD), transmembrane domain (TMD), O-linked sugar domain (OLSD), cytoplasmic domain (CPD). PxVgR was predicted to contain 79 phosphorylation sites and 26 glycosylation sites. The phylogenetic analysis shows that PxVgR is a separate branch, similarly to Spodoptera litura (up to 35.02%), Helicoverpa armigera (up to 34.99%), Danausplexippus (up to 33.94%).3. The expression profiles of PxVgRGene analysis of different developmental stages shows that PxVgR could only express in egg stage and female adult stage. The expression of PxVgR in 0-6d adult is significantly higher than that in egg stage. It rises to the peak after emergence 24h and then decline, and also express at 6d adult, with the time dely. To further study on fat body, intestine, cuticle, head and ovary of P. xylostella, we found that PxVgR only express in ovary, specifically, which indicated to related to reproductive regulation.4. Analysis of VgR RNAi efficiency in P. xylostellaInjection of PxVgR-siRNA into 2 day female pupa caused severe suppression of VgR mRNA expression within 24h.