Studies On Molecular Cloning, Expression And Function Of BmPLA2 And BmRab3 In Silkworm, Bombyx Mori L

Studies on Molecular Cloning, Expression and Function of BmPLA2 and BmRab3 in Silkworm, Bombyx mori LPart-Ⅰ Molecular cloning, expression and, in vivo inhibition of BmPLA2 containing conserved domain WD40 causes depletion of fat tissue in silkworm B. mori L PLA2 is a superfamily of enzymes which participate in hydrolyzing arachidonic acid, and other polyunsaturated fatty acids, from sn-2 position to release free arachidonic acid and a lysophospholipid. So far studies on PLA2 in invertebrate organisms mostly rely on immune response inflammations through bacterial infection. PLA2 enzymes comprise the largest class of phospholipases and participate in lipid signaling molecules like arachidonic acid release in vivo. Previous studies have reported the activities of PLA2 in immune associated tissues like hemocytes and fat bodies. In the present study, we cloned namely, BmPLA2 from fat tissue of silkworm B. mori. This newly cloned gene has a total sequence of 1.031kb size with a 31.90 kDa protein. In silico result of BmPLA2 indicated that the protein has a putative WD40 conserved domain and found its phylogeny tree clustered with Danaus plexippus species. We observed BmPLA2 highest expression in fat body tissue and lowest in skin tissue amongst the studied tissues of 3rd day 5th instar larvae. We also reported differential expression of BmPLA2 in early larval instars besides its highest expression in female pupa than male pupa. Our RNAi-mediated gene silencing results showed highest reduction of BmPLA2 expression in post-24 hour followed by post-48 and post-72 hours. Further, we observed RNAi interfered BmPLA2 larvae and pupae characterized by pharate adult lethality and underdevelopment. We also show phenotypic characters of fat body cells in RNAi-induced larvae further supported our findings. We conclude that a functional study on BmPLA2 could be a significant approach for future research.Part-Ⅱ BmRab3 with C-terminal hypervariability for GGT2 site expresses highest in MSG tissue of silkworm B. mori L.Almost two decades have passed since the yeast Yptl and Sec4 proteins including mammalian Rab (Ras-related protein found in brain) GTPases were first reported to be known as evolutionarily conserved, important membrane trafficking regulators. Rab GTPases are members of the biggest superfamily called Ras GTPases. So far, over 70 human Rab and Rab like members of the superfamily Ras have been reported. Sensibly, Rab GTPases are known for their molecular switches, cycling between their active and inactive states and in fact serving as scaffolds for integrating dual membrane trafficking and intracellular signaling. Despite their smaller size structurally, Rab GTPases are known to have multiple interaction sites and their interaction mechanistic function both functionally and structurally is important. Meanwhile, one of the members of Rab GTPases, Rab3 GTPases are known to play central role in vesicular trafficking, and expresses highest in brain and endocrine tissues besides their activities in adipocytes, peripheral tissues as well as exocrine glands. Interestingly, the silk gland of Lepidopteran insect such as silkworm B. mori has been an important tissue in research and translational medicine studies. With several unknown mechanism underlying the silk protein, and Rab3 mechanism alone, here for the first time we report the BmRab3 cloned from silk gland tissue having highest expression in MSG. In silico analysis of the cloned gene indicated a theoretical isoelectric point and molecular weight of 5.52 and 24.3 kDa. BmRab3 showed C-terminal hypervariability for GGT2 site but having two other putative GEF/GDI interaction sites. Multiple sequence alignment shows high similarity with Rab3 isoforms of other species. Phylogeny tree clustered BmRab3 between T. castanium and A. aegypti species. Expression of BmRab3 was analyzed by specific excision of MSG, PSG and, whole SG besides other tissues studied. Our result shows highest expression of BmRab3 in MSG than the PSG and the whole of silk gland tissues. Further, expression in whole body early instars larvae shows differential expression of BmRab3 followed by male pupa higher expression than female pupa. In vivo dsRNA interference of BmRab3 shows higher suppression of BmRab3 in post-24h than by post-72h in MSG tissue tissues. The resultant knockout witnessed underdevelopment of larvae and pharate pupae lethality.