Molecular Mechanism Exploration Of MiR-318 Involved In Ovarian Development Of Bactrocera Dorsalis

Bactrocera dorsalis(Hendel)is a fruit and vegetable pest with important economic significance worldwide.It is difficult to control them with traditional chemicals because of its high fertility and strong adaptability.Therefore,it is urgent to find a new green control target.B.dorsalis is a typical r-strategy insect.It can produce 900-1600 eggs in its lifetime.Our previous constructed full transcriptome library of B.dorsalis ovary development stage indicated that miR-318 was highly expressed in the ovary of B.dorsalis.In this study,we used RT-q PCR,mimics injection,hyperfocal digital microscopy imiging system,gene editing via CRISPR-Cas9,high-throughput sequencing and other techniques to investigate the function of miR-318 in the ovarian development of B.dorsalis.The main results are as follows.1.The expression pattern of miR-318 and phenotype after mimics treatmentThe spatiotemporal expression patterns of miR-318 in B.dorsalis were detected via RT-q PCR.miR-318 was significantly high expressed in the abdomen of 6-day-old and 9-day-old females,which was determined from the relative expression of miR-318 in different days,gender and segment.Subsequently we detecting the expression of miR-318,it was found that miR-318 was significantly high expressed in ovaries.Then we detected the expression of miR-318 in ovaries of different ages of females and found that it was significantly high expressed in the ovaries of female after six days of age.47.2%(34/ 72)of female B.dorsalis showed ovarian dysplasia 72 h(6 days old)after treatment with miR-318 mimic.Using the hyperfocal digital microscopy imiging system,we found that the ovaries of the female treated with miR-318 mimic were significantly smaller than those of the control group.2.The effect of miR-318 knock-out on ovarian developmentUsing CRISPR-Cas9 system to knock out miR-318,we obtained strains with mutations at the mature sequence of miR-318,including-7 bp,-5 bp,+7 bp,+41 bp and other mutation types.The structure of pre-miR-318 of these mutation types is apparently different from that of wild type according to RNAfold Web Serve(http://rna.tbi.univ-ie.ac.at/cgi-bin/RNAWeb Suite/RNAfold.cgi)analysis.In the process of homozygote screening,we detected that the expression of miR-318 in heterozygotes was significantly down regulated,and the silencing efficiency was 96%.At the same time,it was observed that the mortality of all homozygotes was between 66% and 92%before they had developed to adult stage.We observed the phenotypic differences in three aspects: hatchability of eggs,morphological differences of eggs and morphological differences of ovaries.In terms of hatching rate,the homozygous females hardly lay eggs,and only 20 eggs were produced by the three homozygous females after multiple egg inducements,and the hatching rate was 10%,which was significantly lower than that of heterozygous and wild-type.The morphological observation of eggs showed that the eggs produced by homozygous females were dehydrated and collapsed;In terms of ovarian development,the ovarian size of homozygous females was observed at 15 days of age and it was significantly smaller than that of wild type.The average cross area of homozygous ovaries is 64% of wild type.3.Prediction and screening of miR-318 target gene candidatesBioinformatics software(miRanda,risearch,RNAhybrid and s RNATar Predictor)was used to predict the target genes of miR-318 in B.dorsalis.Their intersection contained 14 potential target genes.Using miR-318/ NC mimics treatment and RT-q PCR analyze,we didn’t find target gene in them which was negatively regulated by miR-318.Futher,mRNA transcriptome library was constructed after miR-318 mimic injection.The results of transcriptome analysis indicated that there were significant changes in the genes of lipid energy metabolism pathway and juvenile hormone synthesis pathway at 24 h and 48 h after miR-318 mimic injection,among which the gene of carnitine synthesis pathway gamma-butyrobetaine dioxygenase was down-regulated significantly both at 24 h and 48 h after injection.Bioinformatics prediction showed that miR-318 may directly target this gene,suggesting that miR-318 may down regulate lipid energy metabolism and promote its accumulation and storage.At the same time,glycerol kinase was down-regulated at 24 h after injection,which means that the energy metabolism pathway via glycolysis pathway was down-regulated.At 48 h after injection,the gene of juvenile hormone synthesis process,farnesol dehydrogenase,was significantly down-regulated.It was a potential target gene of miR-318 showed by bioinformatics analysis.Simultaneously,vg-like 3 was significantly up-regulated,suggesting that miR-318 was involved in the regulation of JH and ecdysone titer before oogenesis.In conclusion,this study demonstrated that miR-318 is involved in the regulation of ovarian development of B.dorsalis.miR-318 may reduce lipid energy metabolism process by targeting gamma-butyrobetaine dioxygenase and down regulate JH synthesis pathway by targeting farnesol dehydrogenase potentially,so as to provide a good environment for oogenesis.miR-318 could be new target in B.dorsalis control.This reaearch has guiding significance for the green prevention and control of B.dorsalis.