Co-expression of the tobacco anthranilate synthase β subunit with its feedback-insensitive α subunit as a selectable marker that also markedly increases the free tryptophan content
Tóm tắt
Anthranilate synthase (AS), a tetramer consisting of two α subunits and two β subunits, is the key control enzyme in the tryptophan (Trp) biosynthesis pathway. A naturally occurring α subunit of AS called ASA2 that is insensitive to Trp feedback inhibition was isolated from a tobacco suspension cell culture and has been extensively studied and used for both nuclear and plastid transformation. However, the obligate β subunit of AS had not been studied in tobacco. Therefore, the tobacco AS β subunit-encoding cDNA was cloned and its encoded protein was verified. Agrobacterium-mediated transformation of tobacco plants was performed, under the selection of the toxic Trp analogs, 7-methyltryptophan or α-methyltryptophan, with a construct containing both ASA2 and AS β subunit genes. Many transgenic plants overexpressing both subunits were identified and examined. Compared to the wild-type plants, the transgenic plants had higher levels of enzymatic activities for both holoenzyme and α subunit. The transgenic plants had 9 to 68 times the amount of free Trp as the wild-type plants, which was more pronounced than plants overexpressing ASA2 alone. This study demonstrates the potential of co-expressing AS α and β subunits as a robust plant transformation system as well as overcoming feedback inhibition to obtain high levels of Trp biosynthesis.
Tài liệu tham khảo
Anderson PC, Chomet PS, Griffor MC, Kriz AL (1997) Anthranilate synthase gene and its use thereof. World Intellectual Property Organization, 97/26366
Barone P, Widholm JM (2008) Use of 4-methylindole or 7-methyl-DL-tryptophan in a transformant selection system based on the feedback-insensitive anthranilate synthase α-subunit of tobacco (ASA2). Plant Cell Rep 27:509–517
Barone P, Zhang X-H, Widholm JM (2009) Tobacco plastid transformation using the feedback insensitive anthranilate synthase [α]-subunit of tobacco (ASA2) as a new selectable marker. J Exp Bot 60:3195–3202
Cho H-J, Brotherton JE, Song H-S, Widholm JM (2000) Increasing tryptophan synthesis in a forage legume Astragalus sinicus by expressing the tobacco feedback-insensitive anthranilate synthase (ASA2) gene. Plant Physiol 123:1069–1076
Cho H-J, Brotherton JE, Widholm JM (2004) Use of the tobacco feedback-insensitive anthranilate synthase gene (ASA2) as a selectable marker for legume hairy root transformation. Plant Cell Rep 23:104–113
Hong S-B, Peebles CAM, Shanks JV, San K-Y, Gibson SI (2006) Expression of the Arabidopsis feedback-insensitive anthranilate synthase holoenzyme and tryptophan decarboxylase genes in Catharanthus roseus hairy roots. J Biotechnol 122:28–38
Inaba Y, Brotherton JE, Ulanov A, Widholm JM (2007) Expression of a feedback insensitive anthranilate synthase gene from tobacco increases free tryptophan in soybean plants. Plant Cell Rep 26:1763–1771
Kreps JA, Town CD (1992) Isolation and characterization of a mutant of Arabidopsis thaliana resistant to alpha methyltryptophan. Plant Physiol 99:269–275
Li J, Last RL (1996) The Arabidopsis thaliana trp5 mutant has a feedback-resistant anthranilate synthase and elevated soluble tryptophan. Plant Physiol 110:51–59
Morollo AA, Eck MJ (2001) Structure of the cooperative allosteric anthranilate synthase from Salmonella typhimurium. Nat Struct 8:243–247
Murashige T, Skoog F (1962) A revised medium for rapid growth and bioassays with tobacco tissue cultures. Physiol Plant 15:473–497
Niyogi KK, Last RL, Fink GR, Keith B (1993) Suppressors of trp1 fluorescence identify a new arabidopsis gene, TRP4, encoding the anthranilate synthase beta subunit. Plant Cell 5:1011–1027
Poulsen C, Bongaerts RJM, Verpoorte R (1993) Purification and characterization of anthranilate synthase from Catharanthus roseus. Eur J Biochem 212:431–440
Radwanski ER, Last RL (1995) Tryptophan biosynthesis and metabolism: biochemical and molecular genetics. Plant Cell 7:921–934
Ranch JP, Rick S, Brotherton JE, Widholm JM (1983) Expression of 5-methyltryptophan resistance in plants regenerated from resistant cell lines of Datura innoxia. Plant Physiol 71:136–140
Song H-S, Brotherton JE, Gonzales RA, Widholm JM (1998) Tissue culture specific expression of a feedback-insensitive Nicotiana tabacum anthranilate synthase. Plant Physiol 117:533–543
Spraggon G, Kim C, Xuong N-H, Yee M-C, Yanofsky C, Mills SE (2001) The structures of anthranilate synthase of Serratia marcescens crystallized in the presence of (i) its substrates, chorismate and glutamine, and a product, glutamate, and (ii) its end-product inhibitor, L-tryptophan. Proc Natl Acad Sci 98:6021–6026
Tozawa Y, Hasegawa H, Terakawa T, Kyo Wakasa K (2001) Characterization of rice anthranilate synthase α-subunit genes OASA1 and OASA2. Tryptophan accumulation in transgenic rice expressing a feedback-insensitive mutant of OASA1. Plant Physiol 126:1493–1506
Tsai F-Y, Brotherton JE, Widholm JM (2005) Overexpression of the feedback-insensitive anthranilate synthase gene in tobacco causes tryptophan accumulation. Plant Cell Rep 23:548–556
Tsai F-Y, Zhang X-H, Ulanov A, Widholm JM (2010) The application of the yeast N-acetyltransferase MPR1 gene and the proline analogue L-azetidine-2-carboxylic acid as a selectable marker system for plant transformation. J Exp Bot 61:2561–2573
Wakasa K, Widholm JM (1987) A 5-methyltryptophan resistant rice mutant, MTR1, selected in tissue culture. Theor Appl Genet 74:49–54
Widholm JM (1972) Cultured Nicotiana tabacum cells with an altered anthranilate synthase which is less sensitive to feedback inhibition. Biochim Biophys Acta 261:52–58
Widholm JM (1973) Measurement of the five enzymes which convert chorismate to tryptophan in cultured Daucus carota cell extracts. Biochim Biophys Acta 320:217–226
Yamada T, Tozawa Y, Hasegawa H, Terakawa T, Ohkawa Y, Wakasa K (2004) Use of a feedback-insensitive a subunit of anthranilate synthase as a selectable marker for transformation of rice and potato. Mol Breed 14:363–373
Zhang X-H, Brotherton JE, Widholm JM, Portis AR (2001) Targeting a nuclear anthranilate synthase alpha-subunit gene to the tobacco plastid genome results in enhanced tryptophan biosynthesis. Return of a gene to its pre-endosymbiotic origin. Plant Physiol 127:131–141
Zhang X-H, Webb J, Huang Y-H, Lin L, Tang R-S, Liu A (2011) Hybrid Rubisco of tomato large subunits and tobacco small subunits is functional in tobacco plants. Plant Sci 180:480–488