Genome Sequencing And Analysis Of Epicauta Chinensis And Verification Of The Correlation Between EchiCYP72 And Cantharidin Synthesis

Cantharidin is a defensive toxin in insects such as Meloidae,with efficient insecticidal,bacteriostatic,tumor suppressor,and antiviral activities.In recent years,the biosynthetic pathway of cantharidin has become a research hotspot.Previous work of our laboratory has made great progress in the in the synthesis of cantharidin in Epicauta chinensis,but the lack of genomic data limits further research.In this context,we carried out the whole genome sequencing of E.chinensis.Through comparative genomics analysis,we obtained many specific to Meloidaea and rapidly expanding gene family of E.chinensis.and analyzed gene families that may be related to cantharidin synthesis and transport at the genome-wide level.We further screened the Cytochrome P450(CYP450)specific to Meloidae,and verified the correlation between cantharidin synthesis and a P450 gene(EchiCYP72)specific to Meloidae.The main findings are as follows:(1)The sequencing and comparative genomics analysis of the whole genome of E.chinensisWe performed high-throughput sequencing of E.chinensis by the third-generation sequencing platform(Pac Bio).Then,we obtained the high throughput sequencing of 151.88 Mb genome data and prepared the genome sketch(GC content was 33.08%,contig number was 870,N50 length was 659.43 KB,and the longest scaffold was 5.89Mb)and 12520 high quality protein-coding gene sets were annotated.The results of BUSCO showed that the genome integrity,duplication rate and deletion rate were 98.7%,2.9% and 0.9%,respectively.The results of genome analysis and enrichment analysis showed that 45 gene families,such as Odorant receptor and CYP450,were significantly expanded.Through reviewing and analyzing previous studies,it was found that CYP450 family has a great correlation with cantharidin biosynthesis.(2)The identification and analysis of CYP450 specific to MeloidaeThe study identified the CYP450 genes of 7 species that can not produce cantharidin,such as Tribolium castaneum,Aethina tumida,Anoplophora glabripennis,Sitophilus oryzae,Dendroctonus ponderosae,Leptinotarsa decemlineata,Agrilus planipennis and 3 species that produce cantharidin such as Hycleus cichorii,Hycleus phaleretus and E.chinensis.A total of 899 CYP450 genes were obtained(including 116 CYP450 genes of E.chinensis).Among them,25 CYP450 genes belong to CYP4、CYP6and CYP9,respectively were identified by local bidirectional Blast.We found that there is only one EchiCYP72 which is specific to Meloidae in the CYP9 subfamily,and its correlation with cantharidin biosynthesis has not been verified before.(3)The verification of correlation between EchiCYP72 and cantharidin synthesisIn order to verify the correlation between EchiCYP72 and cantharidin synthesis,we detected the expression of EchiCYP72 at different periods and different tissues.Additionally,we determined the content of cantharidin in E.chinensis by combined with real-time quantitative PCR and gas chromatography after EchiCYP72 was knock-down.The results showed that injection of ds RNA of this gene significantly inhibited the expression of EchiCYP72,and the interference efficiency is more than74.05%.The cantharidin content was respectively inhibited by 49.12%,31.67%,40.59%,and 60.59% after 1d,3d,5d,and 7d,indicating that EchiCYP72 is closely correlated with the synthesis of cantharidin in E.chinensis.(4)The identification of other rapidly expanding gene families in E.chinensisThis study also found that taste receptors,transporters,carboxylic acid metabolism and immune genes were involved in the annotation of the expansion gene family in E.chinensis.In order to further explore the recognition and mechanism of cantharidin and its immune defense function,the study identified Odorant receptors(ORs),Ionotropic receptors,(IRs),Ionotropic glutamate receptors,(i Glu Rs),Chemosensory proteins(CSPs)Odorant binding proteins(OBPs),Sensory neuron membrane proteins(SNMPs),ATP-binding cassette transporters(ABCs),Glutamate S-transferases(GSTs)and Immune genes.A total of 150 GR,80 OR,30 IR,17 i Glu R,9 CSP,47 OBP,10 SNMP,32 GST,71 ABC and 281 Immune genes were obtained.