Genome-wide Analysis And Cloning And Expression Of Toxin Genes Of Pyemotes Zhonghuajia Yu,Zhang & He

Pyemotes zhonghuajia Yu,Zhang & He is a unique natural enemy mite in China,which can parasitize a variety of agricultural and forestry pests in vitro,such as Lepidoptera,Coleoptera,Homoptera,and Diptera.Pyemotes zhonghuajia can parasitize and kill pests more than 680,000 times its body weight.In the process of parasitizing the host,P.zhonghuajia injects toxins into the host to paralyze and kill the host,then selects a suitable site on the host surface to parasitize,and obtains nutrition for itself and its offspring by feeding on the host.How can a mite so tiny in size kill a host 680,000 times larger than itself? What is the main component of toxin proteins? In this study,we explored the whole genome of P.zhonghuajia,the types of toxin proteins,and the effects of toxin proteins on Spodoptera frugiperda to provide a theoretical basis for the lethal mechanism of P.zhonghuajia to the host and the development of insecticidal proteins.1.Sequencing and analysis of the whole genome of Pyemotes zhonghuajiaIn this study,PacBio Sequel II platform was used to sequence the whole genome of P.zhonghuajia,the size of it was 71.943 Mb,and containing 3 chromosomes.By annotating the genome,a total of 214 non-coding RNAs,77,050 repeats,and 11,183protein-coding genes were obtained.Phylogenetic analysis and the evaluation of differentiation time indicated that P.zhonghuajia was sister to Tetranychus urticae,both of which originated from the Triassic(223.43-247.66 Mya).Referring to the toxin reference sequences of Chelicerata and Myriapoda,the dermonecrotic toxin family,neurotoxin family,and agatoxins family were annotated.2.Bioinformatics analysis of the toxin genes in Pyemotes zhonghuajiaThen we conducted a bioinformatics analysis on these toxin genes of P.zhonghuajia.The neurotoxin gene family was distributed on three chromosomes,while the dermonecrotic toxin family and agatoxins family were located on chromosome 3.Except for Pz DNT1,which has no signal peptide,all toxin proteins had signal peptide.Through the analysis of the physicochemical properties of the toxin proteins,it was found that the physicochemical properties of all the toxin proteins were quite different,and the expression of most of the toxin genes was higher in the one-day-old than seven-day-old P.zhonghuajia.3.Expression and bioactivity of the toxin proteins of Pyemotes zhonghuajiaWe expressed the proteins Pz NT1 、 Pz NT10 、 Pz NT11 and Pz Ag3 using Escherichia coli.Under the induction of 1 m M IPTG,we obtained the recombinant proteins.Finally,the biological activity of the toxin proteins were measured,and the effects of the toxin proteins on the mortality rate,pupal weight and developmental of each stage of S.frugiperda were observed by feeding method.Among the four toxin proteins,Pz NT1 and Pz NT11 at a concentration of 10 mg/m L had a significant effect on the lethality of S.frugiperda(37.9%,31.2%)compared to lower concentrations of the proteins;Pz NT10 and Pz NT11 significantly reduced the pupal weight of S.frugiperda;the larval stage was significantly reduced after Pz NT11 treatment compared to the control(20.4 days);the pupal stage was significantly increased after four proteins treatments compared with the control.