Expression And Function Analysis Of Long Noncoding RNA BPHOGS10005591-OT2 Gene In Nilaparvata Lugens

The brown planthopper(BPH),Nilaparvata lugens,is a monophagous insect pest and feed only on rice.Here,we knocked out the BPHOGS10005591-OT2 by siRNA injection,suggesting BPHOGS10005591-OT2 might have a regulatory role in the brown planthopper.Glucose dehydrogenase(GLD)and BPHOGS10005591-OT2 were predicted and validated by PCR system,indicated thes genes participated in the regulating BPH reproductive development.1.The validation of lncRNA and protein-coding genesWe randomly selecte 17 IncRNAs to determine the transcription orientation with Strand-specific PCR.BPHOGS10035448-AS-RA was not amplified with a correct band.So,16 lncRNAs were successfully amplified and confirmed by sequencing.The results indicated that 12 IncRNA transcribed from the antisense strand and four from the sense strand.We validated the RT-PCR of alternatively spliced transcripts of BPHLNC-unc241.Isoform-specific primers were designed for ten isoforms.Five of them were successfully amplified followed by sequencing.The PCR product size and the sequencing result of variant J was not as expect.The expressions of five isoforms in the third instar to fifth instar nymph and adult were measured,suggesting transcript variants vary in their expression profiles.Amplification of three lncRNAs overlapped with reproduction-associated protein genes.Different combinations of primers pairs indicated that BP HOGS10005591-OT2 and BPHOGS10007976-OT were independently transcribed whereas BPHOGS10035598-OT might share a same transcript with its adjacent protein-coding gene BPHOGS10035598.2.The amplification of three lncRNAs overlapped with reproduction-associated protein genes.These lncRNAs overlapped with reproduction-associated protein genes encoding glucose dehydrogenase(GLD),gastrulation defective(gd),and GALNT7.We used the RACE technologe to amplifinate the overall length of these genes.The results showed that the length of protein genes encoding GLD and GALNT7 is 2540nt and 2733nt respectively.And the length of long non-coding RNA BPHOGS10007976-OT and BHHOGS10035598-OT is 2143nt and 1365nt respectively.3.Expression analysis of GLD and BPHOGS10005591-OT2The expressions of GLD and BPHOGS10005591-OT2 were analyzed in the female and male adults from the day 1st to 10th of the BPH using qRT-PCT.The results showed that these genes are differentially expressed in different day stages,suggesting that they might have important roles in regulating BPH reproductive development.4.RNA interfernee of GLD and BPHOGS10005591-OT2Glucose dehydrogenase is required for normal sperm storage and utilization in female Drosophila melaogaster.We chosed the one-days newly emerged females to knock down both GLD and BPHOGS10005591-OT2.The RNAi-treated individuals and males were kept in the beaker.The percentage of individuals of the offsprings were counted.And then we analysed the expression of each three sample at 24h,48h,72h.The results indicated that siGLD-treated and siBPHOGS10005591-OT2-treated group had obvious difference comparing with the control.In all,the results showed that BPHOGS10005591-OT2 have some effects on the regulation of reproductive development in the BPH.