Molecular Cloning And Gene Expression Of Vitellogenin From Amblyseius Eharai Amitai&Swirskii (Acari:Phytoseiidae)

Amblyseius eharai,belonging to Arachnida,Acarina,Phytophthora,Amblyseiu,is an important predatory mite,which can prey on various agricultural pests such as leafhoppers,whiteflies and thrips.At present,researches on A.eharai are mainly focused on biology,ecology,and utilization.However,the studies on the gene involved in the reproductive regulation have not been reported.In this study,the full length cDNA sequence of vitellogenin gene from A.eharai was obtained using RT-PCR and RACE technology,and the sequence characteristics were analyzed using bioinformatics methods.The mRNA expression levels of vitellogenin gene in different developmental stages and different foods feeding were analyzed using real-time fluorescence quantitative PCR.The main results are as follows:1.The full-length cDNA of vitellogenin gene(AeVg,GenBank accession number: MH113158)was 1537 bp in length,and the open reading frame(ORF)is 5559 bp,which encodes 1852 amino acid residues.The predicted molecular mass is 211.69 KDa,and the theoretical isoelectric point(pI)is 9.13.Analysis of amino acid structure revealed three typical vitellogenin domains(LPD_N,DUF1943 and VWD),two protease cleavage sites RXXR,a typical GLCG signature motif,and the first 16 amino acid residues at the N-terminus are signal peptides.The results of amino acid sequence alignment showed that the highest similarity was between A.eharai and Amblyseius cucumeris,which was 90.6%,followed by 87.9% similarity with Neoseiulus barkeri,but the similarity to pest mites and ticks was lower.The phylogenetic tree was constructed according to the maximum likelihood method(ML).The results showed that A.eharai has the closest relationship with A.cucumeris and N.barkeri,which belong to the phytotiology family,followed by other mites and ticks in Arachnida.Arachnida,Insecta and Malacostraca form a juxtaposed relationship.2.The Vg expression levels in different developmental stages of A.eharai(egg,larvae,protonymph,duetonymph,adult female pre-oviposition,adult female oviposition,male mite)are quite different.No expression was observed in the eggs,larvae,protonymph and male mites,and the expression level of duetonymph was very low.The Vg in the female adults began to express a lot at the pre-oviposition.The expression in oviposition stage was the highest,which was 5.09 times that of the preoviposition period.These results indicated that the vitellogenin gene of A.eharai is closely related to yolk production,and it begins to express a large amount at the early stage of yolk,reaching the highest level during yolk development.3.The expression levels of Vg from A.eharai fed by different foods(Aleuroglyphus ovatus,Tetranychus cinnabarinus,tea pollen)was detected.The results showed that the highest level of expression of Vg was found that fed with teao pollen,which was 1.19 times higher than that fed with A.ovatus.There was no significant difference between the two groups(P>0.05).The lowest level of mRNA expression of vitellogenin gene was found that fed with T.cinnabarinus.It shows that using alternative foods such as A.ovatus or tea pollen when breeding A.eharai can achieve better results.In summary,the full length of the vitellogenin gene from A.eharai was cloned and the expression pattern of the gene was further analyzed,which laid the foundation for the subsequent study of the function of the gene,and also provides a theoretical basis for exploring the reproductive regulation of A.eharai.