Molecular cloning of the Robl gene from Bombyx mori and studies of its developmental and physicochemical regulation.
Dynein light chains function as motor acceptor to recruit cargos, which play vital roles in many cellular processes such as intracellular transport and mitosis. In this study, we cloned and expressed the dynein light chain LC7 gene BmRobl in silkworm. The full-length cDNA of the dynein light chain LC7 gene BmRobl is 757 bp and encoded 97 aa polypeptide. Its molecular weight was ~11 kDa confirmed by western blotting. The tissue and stage expression profile of BmRobl drafted by real time PCR revealed that presence of BmRobl transcript was examined in all tissue but prominent expression level was found in brain, wing disc, ovary and testis. In metamorphosis wing disc, BmRobl reached to peak during the prepupae stage compared with the larval and pupal stages. This indicated BmRobl might involve in wing discs development during metamorphosis. Besides, in vitro wing discs 20E cultivation was performed and BmRobl expression profile was detected. The results demonstrated that the BmRobl gene was significantly up-regulated with increase of 20E concentration; the mRNA level peaked at 2 μg/ml of 20E. However, the BmRobl expression nearly has no change cultivated by 20 μg/ml 20E compared with 0.02 μg/ml 20E. These indicated that BmRobl expression might directly or indirectly induced by 20E, besides, high concentration 20E was far too inducible, suggesting that low concentrations of ecdysteroid induce cell proliferation, whereas high concentrations inhibit cell proliferation. Moreover, the transport role of BmRobl was clarified by UV challenge and vanadate cultivation. Both the real time PCR and western blotting results showed that the BmRobl gene was degraded with increase in the concentration of sodium vanadate combined with elongation in the time of UV challenge. Interestingly, compared with the single treatment group and non-treatment group, the group treated by both sodium vanadate and UV have severe degradation. This indicated that UV and vanadate might down-regulate BmRobl synergetically. It was further speculated that BmRobl may function as a positive regulator of the dynein complex during cellular transport.
Institute of Sericulture & Apiculture, College of Animal Sciences, Zhejiang University, Hangzhou, People's Republic of China.
Hao
H
Wei
He
H
Xuling
Xu
X
Yusong
D000818:Animals; D012831:Bombyx / Q000235:genetics / Q000254:growth & development / Q000378:metabolism*; D002478:Cells, Cultured; D003001:Cloning, Molecular; D004398:Dyneins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D004441:Ecdysterone / Q000494:pharmacology; D040461:Gene Components; D015870:Gene Expression / Q000187:drug effects / Q000528:radiation effects; D020869:Gene Expression Profiling; D018507:Gene Expression Regulation, Developmental; D019476:Insect Proteins / Q000096:biosynthesis / Q000235:genetics* / Q000302:isolation & purification; D008675:Metamorphosis, Biological / Q000235:genetics; D008969:Molecular Sequence Data; D009928:Organ Specificity; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis / Q000235:genetics / Q000302:isolation & purification; D017422:Sequence Analysis, DNA; D014466:Ultraviolet Rays; D014638:Vanadates / Q000494:pharmacology; D014921:Wings, Animal / Q000254:growth & development / Q000378:metabolism
D019476:Insect Proteins; D011993:Recombinant Fusion Proteins; D014638:Vanadates; D004441:Ecdysterone; D004398:Dyneins