Biologia plantarum 52:381-384, 2008 | DOI: 10.1007/s10535-008-0080-x

Transient RNAi based gene silencing of glutathione synthetase reduces glutathione content in Camellia sinensis (L.) O. Kuntze somatic embryos

P. Mohanpuria1, N. K. Rana1, S. K. Yadav1,*
1 Biotechnology Division, Institute of Himalayan Bioresource Technology (CSIR), Palampur, India

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.

Keywords: Agrobacterium; PCR; RNA interference
Subjects: Agrobacterium strains; Camellia sinensis; glutathione synthetase; polymerase chain reaction (PCR); RNA

Received: September 22, 2006; Accepted: March 25, 2007; Published: June 1, 2008Show citation

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Mohanpuria, P., Rana, N.K., & Yadav, S.K. (2008). Transient RNAi based gene silencing of glutathione synthetase reduces glutathione content in Camellia sinensis (L.) O. Kuntze somatic embryos. Biologia plantarum, 52(2), 381-384. doi: 10.1007/s10535-008-0080-x.
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    References

    1. Allen, R.S., Millgate, A.G., Chitty, J.A., Thisleton, J., Miller, J.A.C., Fist, A.J., Gerlach, W.L., Larkin, P.J.: RNAi-mediated replacement of morphine with the non-narcotic alkaloid reticuline in opium poppy.-Nat. Biotech. 22: 1559-1566, 2004. Go to original source...
    2. Baulcombe, D.: RNA silencing in plants.-Nature 431: 356-363, 2004. Go to original source...
    3. Chuang, C.F., Meyerowitz, E.M.: Specific and heritable genetic interference by double stranded RNA in Arabidopsis thaliana.-Proc. nat. Acad. Sci. USA 97: 4985-4990, 2000. Go to original source...
    4. Fukusaki, E.I., Kawasaki, K., Kajiyama, S., An, C.I., Suzuki, K., Tanaka, Y., Kobayashi, A.: Flower color modulations of Torenia hybrida by downregulation of chalcone synthase genes with RNA interference.-J. Biotechnol. 111: 229-240, 2004. Go to original source...
    5. Hamilton, A.J., Baulcombe, D.: A species of small antisense RNA in posttranscriptional gene silencing in plants.-Science 286: 950-952, 1999. Go to original source...
    6. Lee, S., Kim, J., Han, J.J., Han, M.J., An, G.: Functional analyses of the flowering time gene OsMADS50, the putative SUPPRESSOR OF OVEREXPRESSION OF CO 1 / AGAMOUS-LIKE 20 (SOC1 / AGL20) ortholog in rice.-Plant J. 38: 754-764, 2004. Go to original source...
    7. Lipardi, C., Wei, Q., Paterson, B.M.: RNAi as random degradative PCR. siRNA primers convert mRNA into dsRNAs that are degraded to generate new siRNAs.-Cell 107: 297-307, 2001. Go to original source...
    8. May, M.J., Vernoux, T., Leaver, C., Van Montagu, M., Inze, D.: Glutathione homeostasis in plants: implications for environmental sensing and plant development.-J. exp. Bot. 49: 649-667, 1998. Go to original source...
    9. Miki, D., Itoh, R., Shimamoto, K.: RNA silencing of single and multiple members in a gene family of rice.-Plant Physiol. 138: 1903-1913, 2005. Go to original source...
    10. Mondal, T.K., Bhattacharya, A., Ahuja, P.S., Chand, P.K.: Transgenic tea [Camellia sinensis (L.) O. Kuntze cv. Kangra Jat] plants obtained by Agrobacterium mediated transformation of somatic embryos.-Plant Cell Rep. 20: 712-720, 2001. Go to original source...
    11. Noctor, G., Gomez, L., Vanacker, H., Foyer, C.H.: Interactions between biosynthesis, compartmentation and transport in the control of glutathione homeostasis and signalling.-J. exp. Bot. 53: 1283-1304, 2002. Go to original source...
    12. Ogita, S., Uefuji, H., Morimoto, M., Sano, H.: Application of RNAi to confirm theobromine as the major intermediate for caffeine biosynthesis in coffee plants with potential for construction of decaffeinated varieties.-Plant mol. Biol. 54: 931-941, 2004. Go to original source...
    13. Page, D.R., Grossniklaus, U.: The art and design of genetic screens: Arabidopsis thaliana.-Nat. Rev. Genet. 3: 124-136, 2002. Go to original source...
    14. Ribas, A.F., Kobayashi, A.K., Pereira, L.F.P., Vieira, L.G.E.: Genetic transformation of Coffea canephora by particle bombardment.-Biol. Plant. 49: 493-497, 2005. Go to original source...
    15. Sijen, T., Fleeneor, J., Simmer, F., Thijssen, K.L., Parrish, S., Timmons, L., Plasterk, R.H., Fire, A.: On the role of RNA amplification in dsRNA-triggered gene silencing.-Cell 107: 465-476, 2001. Go to original source...
    16. 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. Go to original source...
    17. Smith, N.A., Singh, S.P., Wang, M.B., Stoutjesdijk, P.A., Green, A.G., Waterhouse, P.M.: Total silencing by intronspliced hairpin RNAs.-Nature 407: 319-320, 2000. Go to original source...
    18. Stam, M., DeBruin, R., Kenter, S., Van der Hoorn, R.A.L., Van Blokland, R., Mol, J.N.M., Kooter, J.M.: Post-transcriptional silencing of chalcone synthase in Petunia by inverted transgene repeats.-Plant J. 12: 63-82, 1997. Go to original source...
    19. Stoutjesdijk, P.A., Singh, S.P., Liu, Q., Hurlstone, C.J., Waterhouse, P.A., Green, A.G.: hp-RNA-mediated targeting of the Arabidopsis FAD2 gene gives highly efficient and stable silencing.-Plant Physiol. 129: 1723-1731, 2002. Go to original source...
    20. Tang, G., Reinhart, B.J., Bartel, D.P., Zamore, P.D.: A biochemical framework for RNA silencing in plants.-Gene Dev. 17: 49-63, 2003. Go to original source...
    21. 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. Go to original source...
    22. 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. Go to original source...

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