Identification And Transgenic Application Of Long Non-coding RNAs In Silk Gland Of Silkworm Bombyx Mori

Silkworm is the model of Lepidoptera, silk gland is a special organ from which the silk proteinsse can be synthezed. Currently, studies of the silk gland were focus on the terms of coding genes and microRNAs(miRNAs), studies of long non-coding(lncRNA) in silkworm were rare.To mechanisms of lncRNAs during the development of silk gland, total RNAs of the silk gland of the dormant fourth instar larvea and the fifth instar larvea of silkworm were used, and two RNA libraries of silk gland were constructed with the methods of transcriptome and lnc RNA sequencing. The results were as following: Two RNA libraries of silk gland of the dormant fourth instar larvea and the fifth instar larvea were constructed, and 16071 novel RNAs were identified in the silk gland of the dormant fourth instar larvea, which included 9670 novel lnc RNAs and 6401 novel mRNAs. Furthermore, 13,561 novel RNAs were identified in the silk gland of the fifth instar larvea, which included 7507 novel lncRNAs and 6054 novel mRNAs. Of the novel lncRNAs, 556 located in the antisense strand of coding genes, 1755 located up-or-downstream of coding genes. In addition, 585 coding genes were upregulated in the silk gland of the dormant fourth instar larvea to the fifth instar larvea, but 1079 coding genes were downregulated.2. To further study the function of novel lncRNAs, the expression of 27 novel lncRNAs and their neighboring genes in the different tissues of silk gland of the 4th instar larvea and the 5th instar larvea were analyzed. Results showed that lncR11880, lncR17454, lncR25865 accumulated more in the silk gland of the 4th instar larvea but little in the silk gland of the 5th instar larvea as well as their upstream coding genes. lncR6576 and its downstream gene were all accumulated more in the posterior silk gland of the fifth instar. lncR5831 had more expression in the silk gland of the 4th instar larvea but little in the silk gland of the 5th instar larvea as well as its downstream olfactory receptor genes CSP12. lncR1436 expressed the middle silk gland of the 5th instar larvea, and its downstream gene paralytic peptide had reverse expression pattern to lncR1436 with more expression in the silk gland of the 4th instar larvea. Five lncRNAs lncR12412, lncR17829, lncR14013, lncR19478, lncR6141 which origin from the antisense strand of coding genes had consistent expression pattern with their sense-antisense gene pairs. lncR2036 origined from the upstream of P25 gene had consistent expression pattern with P25 gene, while lncR2034 which origined from the antisense downstream of P25 showed reverse expression pattern with P25. lncR18335 which origin from the downstream of Ser3 gene had reverse expression pattern with Ser3, and accumulated more in the silk gland of the 4th instar larvea. But lncR18325 which comes uptream of Ser3 had consistent expression pattern with Ser3 with more accumulation in the middle silk gland of the 5th instar larvea. lncR2453 located downstream of Ser1, and accumulated more in the silk gland of the 4th instar larvea, but with reverse expression pattern with Ser1 in the middle silk gland of the 5th instar larvea.3. lncR2034 and the promoter of fibroin P25 were co-transfected to the HEK293 T cells, results showed that the activity of P25 promoter were inhibited with a percentage of 64% after lncR2034 was overexpressed, which indicated that lncR2034 can significantly inhibited the promoter activity of P25, which can be used in the transgenetic application of silk gland in the future.