Transient RNAi based gene silencing of glutathione synthetase reduces glutathione content in Camellia sinensis (L.) O. Kuntze somatic embryos
- 259 Downloads
We report on gene silencing of glutathione synthetase (GSHS) that reduces reduced glutathione (GSH) content in somatic embryos of Camellia sinensis L. Using degenerate primers with cDNA of Camellia sinensis, a 457 bp GSHS gene fragment was cloned through polymerase chain reaction. This fragment was used in making ihpRNA. For this it was cloned in sense at AscI and SwaI and in anti-sense at Bam HI and XbaI restriction sites of pFGC5941 that has chalcone synthase (Chs) intron between SwaI and BamHI restriction sites. Resultant RNAi construct was used for C. sinensis somatic embryos transformation through Agrobacterium. After 11, 13 and 15 d of transformation, embryo GSHS transcript levels and GSH content decreased to a great extent which documented the feasibility of RNAi based gene silencing in C. sinensis.
Additional key wordsAgrobacterium PCR RNA interference
Camellia sinensis glutathione synthetase
double stranded RNA
small interfering RNA
Unable to display preview. Download preview PDF.
- Singh, K., Raizada, J., Bhardwaj, P., Ghawana, S., Rani, A., Singh, H., Kaul, K., Kumar, S.: 26S rRNA-based internal control gene primer pair for reverse transcription-polymerase chain reaction-based quantitative expression studies in diverse plant species.-Anal Biochem 335: 330–333, 2004.PubMedCrossRefGoogle Scholar
- Volohonsky, G., Tuby, C.N.Y.H., Porat, N., Rousseau, M.W., Visvikis, A., Leroy, P., Rashi, S., Steinberg, P., Stark, A.A.: A spectrophotometric assay of γ-glutamylcysteine synthetase and glutathione synthetase in crude extracts from tissues and cultured mammalian cells.-Chem.-Biol. Inter. 140: 49–65, 2002.CrossRefGoogle Scholar
- Wesley, S.V., Helliwell, C.A., Smith, N.A., Wang, M.B., Rouse, D.T., Liu, Q., Gooding, P.S., Singh, S.P., Abbott, D., Stoutjesdijk, P.A., Robinson, S.P., Gleave, A.P., Green, A.G., Waterhouse, P.M.: Construct design for efficient, effective and high-throughput gene silencing in plants.-Plant J. 27: 581–590, 2001.PubMedCrossRefGoogle Scholar