Expression And Functional Analysis Of Long Non-coding RNA MSTRG.28822 Gene In Aphis Gossypii Glover

Long non-coding RNAs(lncRNAs)are a class of nc RNAs larger than 200 nt in length with no obvious protein coding potential.It can participate in a variety of biological processes and has gradually become a hot area of scientific research.The research of lncRNA involved in the regulation of resistance of drugs is still in its infancy,and there are few related reports.There is no report on the involvement of lncRNA in the resistance of cotton aphids to spirotetramat.As a worldwide pest,the cotton aphid(Aphis gossypii Glover)has broad-spectrum resistance to many insecticides.Therefore,it is of great significance to study the resistance mechanism of cotton aphid.In this study,we used high-throughput sequencing technology to analyze and identify the differentially expressed lncRNA genes between spirotetramat-resistant and sensitive cotton aphids,and the number of lncRNA fragments obtained by sequencing was 7025.The quality of transcriptome data reaching Q30 exceeds 90%,which proves that the transcriptome data obtained from this high-throughput sequencing is true and reliable.Taking the SS strain as a control,in the SR strain,there were 945 non-coding genes differentially expressed,of which 472 were up-regulated non-coding genes and 473 were down-regulated non-coding genes.The enrichment analysis of differentially expressed lncRNA target genes shows that in terms of function,the differentially expressed lncRNA target genes are significantly enriched in metabolic processes,cell processes,cell membranes,cell membrane components,binding functions,and catalytic activities.In terms of pathways,differential lncRNA target genes are enriched in fatty acid extension,polytype O-glycan biosynthesis and ether lipid metabolism pathways,indicating that differential lncRNAs may be involved in the regulation of genes in these pathways,and these regulatory pathways may be opposed to cotton aphid Spirotetramat resistance is related.The target of Spirotetramat is Acetyl-Co A carboxylase(ACC),which inhibits the expression of ACC and reduces the biosynthesis of total lipids.We sorted out and predicted the lncRNA involved in the regulation of ACC,and found the non-coding RNA MSTRG.28822 that may be involved in the cis-regulation of ACC,which is located on the 3’end of the antisense strand of ACC and has 5 transcripts.We quantitatively analyzed the lncRNA of the SS and SR strains of cotton aphid,and the results showed that the expression of MSTRG.28822 gene was 0.78 times that of the sensitive strain,which was consistent with the transcriptome data.We synthesized three si RNAs and used the sandwich method to feed the SR strain of cotton aphid to target the knockout of MSTRG.28822.Fluorescence quantitative PCR was performed to verify the interference efficiency of the three si RNAs on lncRNA MSTRG.28822.The relative expression of lncRNA MSTRG.28822 was inhibited by 43.23%,48.24%,and 59.13%,respectively,and the relative expression of ACC was increased by51.32%,55.70%,and 11.99%,respectively.Experiments show that MSTRG.28822 is negatively correlated with the expression of ACC,that is,MSTRG.28822 may be involved in inhibiting the expression of ACC.We performed real-time fluorescence quantification of ACC downstream genes in the fatty acid synthesis pathway,and the four FAS genes were up-regulated to varying degrees;the resistance of cotton aphids to spirotetramat after silencing MSTRG.28822 was tested,and the cotton aphid in the treatment group was against spirotetramat Decreased sensitivity.Preliminary experiments indicate that MSTRG.28822 may be involved in the resistance of cotton aphid to spirotetramat.In order to study the mechanism of lncRNA transcription regulation,the experiment found the binding sites of related transcription factors by analyzing the upstream 5’flanking sequence of MSTRG.28822.We screened out 7transcription factors and knocked out related transcription factors through RNAi.The results showed: when the expression levels of C/EBP and C/EBPzeta decreased to0.68 times and 0.80 times,the transcription level of lncRNA MSTRG.28822 decreased to 0.47 times and 0.50 times,respectively.It can be seen from the experimental results that the two C/EBP and C/EBPzeta Transcription factors may be involved in the transcriptional regulation of cotton aphid MSTRG.28822.This experiment provides a theoretical basis for the subsequent verification of related transcription factors and the analysis of promoter activity.The results of this study are helpful to understand the role of non-coding RNA in cotton aphid resistance and the related molecular mechanisms of cotton aphid spirulina resistance.