RNAi KNOCKDOWN OF BmRab3 LED TO LARVA AND PUPA LETHALITY IN SILKWORM Bombyx mori L.

Abstract

Rab3 GTPases are known to play key a role in vesicular trafficking, and express highest in brain and endocrine tissues. In mammals, Rab3 GTPases are paralogs unlike in insect. In this study, we cloned Rab3 from the silk gland tissue of silkworm Bombyx mori, and identified it as BmRab3. Our in silico analysis indicated that BmRab3 is an isoform with a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa, respectively. Further, BmRab3 showed the C-terminal hypervariability for GGT2 site but having two other putative guanine nucleotide exchange factor/GDP dissociation inhibitor interaction sites. Multiple alignment sequence indicated high similarities of BmRab3 with Rab3 isoforms of other species. The phylogeny tree showed BmRab3 clustered between the species of Tribolium castaneum and Aedes aegypti. Meanwhile, the expression analysis of BmRab3 showed the highest expression in middle silk glands (MSGs) than all other tissues in the third day of fifth-instar larva. Simultaneously, we showed the differential expression of BmRab3 in the early instar larva development, followed by higher expression in male than female pupae. In vivo dsRNA interference of BmRab3 reduced the expression of BmRab3 by 75% compared to the control in the MSGs in the first day. But as the worm grew to the third day, the difference of BmRab3 between knockdown and control was only about 10%. The knockdown later witnessed underdevelopment of the larvae and pharate pupae lethality in the overall development of silkworm B. mori L.

Localization and functional analysis of the insect‐specific RabX4 in the brain of Bombyx mori

“…Lately, there have been some reports on the knockdown of important genes, such as transcription factors, phospholipase, and Rab3, using dsRNAi (Liang et al., ; Orville Singh et al., ; Singh et al. ; Yang et al., ; Zhang, Teng, Chen, & Li, ). In this study, RNAi‐knockdown of RabX4 resulted in decreased expression of bombyxin in the brain, indicating that RabX4 may function in the transport of bombyxin in the brain of B. mori .…”

“…; Li et al., ; Satoh, Tokunaga, Kawamura, & Ozaki, ; Singh et al. ; Tong et al., ; Wang, Liu, & Huang, ). Twenty‐nine Rab proteins have been identified in D. melanogaster (Pereira‐Leal & Seabra, ), in which a group of six Rab proteins, termed RabX1 to RabX6, have no homologs in humans.…”

“…In D. melanogaster and Helicoverpa armigera, Rab proteins regulate important cellular processes, such as synaptic functions, midgut remodeling, oogenesis, autophagy, and immunological system (Chan et al, 2011;Coutelis & Ephrussi, 2007;Hou et al 2011;Li et al, 2015;Satoh, Tokunaga, Kawamura, & Ozaki, 1997;Singh et al 2015;Tong et al, 2011;Wang, Liu, & Huang, 2012). Twenty-nine Rab proteins have been identified in D. melanogaster (Pereira-Leal & Seabra, 2001), in which a group of six Rab proteins, termed RabX1 to RabX6, have no homologs in humans.…”