Expression profile of two genes from Caenorhabditis elegans

Propaganda
56º Congresso Brasileiro de Genética
Resumos do 56º Congresso Brasileiro de Genética • 14 a 17 de setembro de 2010
Casa Grande Hotel Resort • Guarujá • SP • Brasil
www.sbg.org.br - ISBN 978-85-89109-06-2
343
Expression profile of two genes from Caenorhabditis
elegans treated with trehalose-sucrose solution
Evangelista, CCS¹; Guidelli, GV¹; Pereira, TC¹.
Lab. de Genética Molecular da Anidrobiose, Depto de Biologia, FFCLRP - USP
[email protected]
1
Keywords: gene expression, C. elegans, trehalose, anhydrobiosis, PCR
Introdution: Trehalose and sucrose are disaccharides capable of protecting biomolecules against extreme water
stress. Anhydrobiotic species accumulate such molecules during drought and thus survival almost total water
absence. “Anhydrobiotic engineering” is a recent research area that aims at conserving biological samples by using anhydrobiosis-related strategies, such as soaking in trehalose-sucrose solutions. However, genetic effects of
such treatments in vivo are not known. Objectives: to assess gene expression profiles of two genes in C. elegans
after trehalose-sucrose treatment for 24 hours. Additionally, to evaluate the efficiency of whole-worm trypan
blue staining aiming greater accuracy in viability studies. Methods: C. elegans worms were soaked in trehalosesucrose solutions with two different osmolarities (128 e 320 mOsm) for 24 hours; as negative control we used
worms soaked in M9. Total RNA was extracted and subjected to RT-PCR using primer pairs derived from P. superbus kinase sequences (an anhydrobiotic nematode). For staining test, live worms (soaked in M9) and dead
(75ºC for 15 minutes) were stained with trypan blue (0,4w/v) for one hour. Dye was removed by washing twice
with M9 and analysed via optical microscopy. Results: RT-PCR reactions yielded single amplicons for two primer
pairs. The first transcript showed no expression alterations in different treatments, while the second transcript
remained unaltered in 128 mOsm solution but was up-regulated in 320 mOsm. Trypan blue staining successfully
revealed that virtually all live worms were not stained, while nearly all dead worms were intensely stained. Conclusion: gene induction by soaking in 320 mOsm of trehalose-sucrose solution treatment for 24 hours reveals
the existence of molecular alterations. Since these primers are derived from kinase sequences, it is possible that
this up-regulation observed in C. elegans is related to signaling process during trehalose-sucrose treatment. Amplicon sequencing will allow us to determine its identity. Additionaly, successful whole-worm trypan blue staining will allow us accurately determining viability of C. elegans in future studies with trehalose-sucrose treatment.
Supported by: Fapesp, CNPq
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