Biotechnology Bulletin ›› 2023, Vol. 39 ›› Issue (6): 171-180.doi: 10.13560/j.cnki.biotech.bull.1985.2022-1341

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Construction and Functional Study of RagA Transgenic Drosophila

MENG Guo-qiang(), GUAN Jian-wen, NIU Chun-mei, ZHOU Ying, SHEN Su-lin, WEI You-heng()   

  1. College of Bioscience and Biotechnology, Yangzhou University, Yangzhou 225000
  • Received:2022-11-01 Online:2023-06-26 Published:2023-07-07
  • Contact: WEI You-heng E-mail:m17854338552@163.com;yhwei@yzu.edu.cn

Abstract:

Rag GTPase is GTP-binding proteins belonging to a member of the Ras family and localizes on lysosomes. Drosophila RagA protein, a homologue of mammalian RagA/RagB protein, forms a complex with RagC to regulate TORC1 activity. Here we found that knockdown of RagA caused Drosophila development to stagnate at the pupal stage. To explore the function of RagA on Drosophila development, we constructed the overexpressing vector pUASp-RagA-wt with RagA RNA interfering target sites changed. On this basis, the GTP-binding RagA-overexpressed plasmid pUASp-RagA-Q61L and the GDP-binding RagA-overexpressed plasmid pUASp-RagA-T16N were constructed by point mutation. The pUASp-RagA-wt, pUASp-RagA-Q61L and pUASp-RagA-T16N plasmids were screened by microinject and screentransgene lines. The effects of different forms of RagA on the TORC1 activity were evaluated by the method of somatic cell cloning, RagA regulated the TORC1 activity, and it was in the activated state after binding with GTP, and inactivated after binding with GDP. To determine the effects of RagA activity on the Drosophila development under GTP/GDP state, the UASp transgene lines were crossed with Tub-Gal4/TM6 and the eclosion rate of offspring was counted. The results showed that Drosophila with RagA knockdown failed to develop into adults due to obstacles in the pupal stage. The overexpression of wild-type RagA completely avoided the death of Drosophila caused by RagA knockdown, confirming that depletion of RagA was the cause of developmental defects in RagA RNAi. The overexpression of GTP-bound RagA-Q61L partially reduced the lethal effect of Drosophila with RagA knockdown, while the overexpression of GDP-bound RagA-T16N could not decrease the lethal effect of Drosophila with RagA knockdown at all. Our work suggests that RagA plays an important role in the growth and development, and the binding of RagA and GTP/GDP needs to be in a dynamic equilibrium. Binding of RagA only to GTP or GDP would make TORC1 activity too high or too low, thus affecting fruit fly growth and development.

Key words: RagA, microinjection, Drosophila