IC4R007-lncRNA-2015-26437919

Project Title

• Genome-wide identification of long noncoding RNA genes and their potential association with fecundity and virulence in rice brown planthopper, Nilaparvata lugens

The Background of This Project

• The functional repertoire of long noncoding RNA (lncRNA) has been characterized in several model organisms, demonstrating that lncRNA plays important roles in fundamental biological processes. However, they remain largely unidentified in most species. Understanding the characteristics and functions of lncRNA in insects would be useful for insect resources utilization and sustainable pest control.

Plant Culture & Treatment

• The rice brown planthoppers were collected from rice fields in Nanjing area, Jiangsu Province, China and maintained on rice seedlings at 27 ± 1 °C, under a 16-h light/8-h dark photoperiod and 70–80 % relative humidity. The insects were transferred to fresh seedlings every 5–7 days to ensure sufficient nutrition.

Research Findings

• A computational pipeline was developed to identify lncRNA genes from the N. lugens transcriptome (Fig. 1).This pipeline was applied on 12 different N. lugens transcriptome datasets and yielded 2439 transcript isoforms corresponding to 1882 loci from 12 N. lugens RNA-seq datasets.

'Fig. 1 The computational pipeline for identifying lncRNA genes in N. lugens from RNA-seq data.'

• The individual datasets were analyzed separately using the computational pipeline. In total, 948 and 1562 lncRNA genes were found in the 2-day-old adults of the LFP and HFP populations, respectively, whereas 1324 and 1563 lncRNAs were identified in the fifth instar larvae of the LFP and HFP populations. A higher number of lncRNAs(1798–2081) were discovered in the fat body, salivary gland,and antennae of the virulence-associated Mudgo and TN1 populations. 1618, 1806, and 1721 lncRNAs were found in the wild, I87i, and C89i populations, respectively (Table 1).

• To confirm the reliability of the identified lncRNA genes,the researchers selected 20 lncRNAs for RT-PCR validation. seventeen lncRNAs were successfully amplified (Additional file 3:Figure S2), suggesting that a high percentage of lncRNAs detected by this pipeline were reliable in terms of expression. The transcription orientation of these 17 lncRNAs were determined by strand-specific RT-PCR. Sixteen out of them were successfully amplified. The results demonstrated that four lncRNAs were transcribed from the sense strand whereas 12 lncRNAs from the antisense strand (Fig. 2).

'Fig. S2 RT-PCR validation of 20 randomly selected lncRNAs. Seventeen lncRNAs were successfully amplified and confirmed by sequencing. The PCR product in lane 15, BPHLNC-unc536, was not correctly amplified. Two lncRNAs, BPHLNC-unc525 and BPHLINC406, were not amplified and were not shown in the figure. Lane 1–18: BPHOGS10028742-AS-RA, BPHLINC074-RA, BPHOGS10028378-AS-RA, BPHOGS10006054-OT-RA, BPHLINC250-RA, BPHOGS10022296-OT-RA, BPHLNC-unc280-RA, BPHLINC164-RA, BPHOGS10026274-IT-RA, BPHOGS10006052-AS-RA, BPHOGS10017161-OT-RA, BPHOGS10027736-OT-RB, BPHOGS10003291-OT-RA, BPHLNC-unc005-RA, BPHLNC-unc536-RA, BPHOGS10000919-OT-RA, BPHOGS10035448-AS-RA, BPHOGS10030139-OT-RA, respectively. (TIFF 4040 kb)'

'Fig. 2 Strand-specific PCR of 17 randomly selected lncRNAs to determine the transcription orientation. BPHOGS10035448-AS-RA was not amplified with a correct band. So, 16 lncRNAs were successfully amplified and confirmed by sequencing (see Figure S2 for RT-PCR validation). The results indicated that 12 lncRNA transcribed from the antisense strand, and four from the sense strand. F: forward primer (sense); R: reverse primer (antisense); RT:reverse transcriptase'

Labs working on this Project

• Department of Entomology, College of Plant protection, Nanjing Agricultural University,Nanjing 210095, China.
• Department of City Construction, Shaoyang University, Shaoyang 422000, China.
• State Key Laboratory for Biocontrol/Institute of Entomology, Sun Yat Sen University,Guangzhou 510275, China.
• Ministry of Agriculture Key Lab of Agricultural Entomology, Institute of Insect Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou 310058, China.

Corresponding Author

• lifei18@zju.edu.cn