So sánh quá trình chuyển gen qua Agrobacterium của bốn giống lúa mạch sử dụng các gen báo cáo GFP và GUS

Plant Cell Reports - Tập 22 - Trang 397-402 - 2003
F. Murray1, R. Brettell1, P. Matthews1, D. Bishop1, J. Jacobsen1
1CSIRO Plant Industry, Canberra, Australia

Tóm tắt

Các thí nghiệm đã được thực hiện để sản xuất các cây lúa mạch chuyển gen thông qua việc nhiễm trùng các phôi chưa trưởng thành bằng Agrobacterium tumefaciens. Các callus đã được chuyển hóa được thu được bằng cách sử dụng tính kháng hygromycin như là một dấu hiệu lựa chọn và hoặc là protein huỳnh quang xanh (GFP) hoặc β-glucuronidase (GUS) như là một gen báo cáo. Tần suất chuyển hóa thực vật giảm đáng kể được ghi nhận với gen GFP so với GUS. Tuy nhiên, GFP chứng tỏ là một dấu hiệu báo cáo tuyệt vời cho các sự kiện chuyển hóa sớm và đã được sử dụng để so sánh bốn giống lúa mạch về hiệu quả trong hai giai đoạn của quá trình chuyển hóa: tạo ra callus lúa mạch đã được chuyển hóa ổn định và phục hồi cây con từ callus đã được chuyển hóa. Callus đã được chuyển hóa được tạo ra với tần suất cao (47–76%) ở cả bốn giống. Việc phục hồi cây con đã được chuyển hóa cũng được thực hiện cho cả bốn giống mặc dù tần suất này cao hơn nhiều đối với giống Golden Promise so với ba kiểu gen còn lại, khẳng định rằng kiểu gen là một yếu tố quan trọng trong khả năng phục hồi của lúa mạch. Nghiên cứu này đã chứng minh lần đầu tiên rằng chuyển hóa qua sự trung gian của Agrobacterium có thể được sử dụng để chuyển hóa các giống Sloop và Chebec của Úc.

Từ khóa

#Agrobacterium tumefaciens #chuyển gen lúa mạch #protein huỳnh quang xanh (GFP) #β-glucuronidase (GUS) #callus chuyển hóa #phục hồi cây con.

Tài liệu tham khảo

Ahlandsberg S, Sathish P, Sun C, Jansson C (1999) Green fluorescent protein as a reporter system in the transformation of barley cultivars. Physiol Plant 107:194–200 Aizawa H, Fukui Y, Yahara I (1997) Live dynamics of Dictyostelium cofilin suggests a role in remodelling actin latticework into bundles. J Cell Sci 110:2333–2344 Barakat A, Carels N, Bernardi G (1997) The distribution of genes in the genomes of Gramineae. Proc Natl Acad Sci USA 94:6857–6861 Barakat A, Gallois P, Raynal M, Mestre-Ortega D, Sallaud C, Guiderdoni E, Delseny M, Bernardi G (2000) The distribution of T-DNA in the genomes of transgenic Arabidopsis and rice. FEBS Lett 471:161–164 Barro F, Martin A, Lazzeri PA, Barceló P (1999) Medium optimisation for efficient somatic embryogenesis and plant regeneration from immature inflorescences and immature scutella of elite cultivars of wheat, barley and tritordeum. Euphytica 108:161–167 Bevan M, Barnes WM, Chilton M (1983) Structure and transcription of the nopaline synthase gene region of T-DNA. Nucleic Acids Res 11:369–385 Bregitzer P, Dahleen LS, Campbell RD (1998) Enhancement of plant regeneration from embryogenic callus of commercial barley cultivars. Plant Cell Rep 17:941–945 Carlson AR, Letarte J, Chen J, Kasha KJ (2001) Visual screening of microspore-derived transgenic barley (Hordeum vulgare L.) with green fluorescent protein. Plant Cell Rep 20:331–337 Chiu WL, Niwa Y, Zeng W, Hirano T, Kobayashi H, Sheen J (1996) Engineered GFP as a vital reporter for plants. Curr Biol 6:325–330 Cho MJ, Choi HW, Buchanan BB, Lemaux PG (1999) Inheritance of tissue-specific expression of barley hordein promoter-uidA fusions in transgenic barley plants. Theor Appl Genet 98:1253–1262 Christensen AH, Sharrock RA, Quail PH (1992) Maize polyubiquitin genes: genes, structures, thermal perturbation of expression and transcript splicing, and promoter activity following transfer to protoplasts by electroporation. Plant Mol Biol 18:675–689 Elliott AR, Campbell JA, Brettell RIS, Grof CPL (1998) Agrobacterium-mediated transformation of sugarcane using GFP as a screenable marker. Aust J Plant Physiol 25:739–743 Elliott AR, Campbell JA, Dugdale B, Brettell RIS, Grof CPL (1999) Green-fluorescent protein facilitates rapid in vivo detection of genetically transformed plant cells. Plant Cell Rep 18:707–714 Fang YD, Akula C, Altpeter F (2002) Agrobacterium-mediated barley (Hordeum vulgare L.) transformation using green fluorescent protein as a visual marker and sequence analysis of the T-DNA::barley genomic DNA junctions. J Plant Physiol 159:1131–1138 Garvin DF, Miller-Garvin JE, Viccars EA, Jacobsen JV, Brown AHD (1998) Identification of molecular markers linked to ant28–484, a mutation that eliminates proanthocyanidin production in barley seeds. Crop Sci 38:1250–1255 Haseloff J, Siemering KR (1998) The uses of GFP in plants. In: Chalfie M, Kain S (eds) Green fluorescent protein: strategies, applications and protocols. Wiley, New York, pp191–220 Hiei Y, Ohta S, Komari T, Kumashiro T (1994) Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J 6:271–282 Horvath H, Huang JT, Wong O, Kohl E, Okita T, Kannangara CG, von Wettstein D (2000) The production of recombinant proteins in transgenic barley grains. Proc Natl Acad Sci USA 97:1914–1919 Ishida Y, Saito H, Ohta S, Hiei Y, Komari T, Kumashiro T (1996) High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nat Biotechnol 14:745–750 Jefferson RA, Kavanagh TA, Bevan MW (1987) GUS fusions: β-glucuronidase as a sensitive and versatile gene fusion marker in higher plants. EMBO J 6:3901–3907 Jordan MC (2000) Green fluorescent protein as a visual marker for wheat transformation. Plant Cell Rep 19:1069–1075 Kaeppler HF, Menon GK, Skadsen RW, Nuutila AM, Carlson AR (2000) Transgenic oat plants via visual selection of cells expressing green fluorescent protein. Plant Cell Rep 19:661–666 Ke XY, McCormac AC, Harvey A, Lonsdale D, Chen DF, Elliott MC (2002) Manipulation of discriminatory T-DNA delivery by Agrobacterium into cells of immature embryos of barley and wheat. Euphytica 126:333–343 Kohli A, Gahakwa D, Vain P, Laurie DA, Christou P (1999) Transgene expression in rice engineered through particle bombardment: molecular factors controlling stable expression and transgene silencing. Planta 208:88–97 Kott LS, Howarth M, Peterson RL, Kasha KJ (1985) Light and electron microscopy of callus initiation from haploid barley embryos. Can J Bot 63:1801–1805 Lazo GR, Stein PA, Ludwig RA (1991) A DNA transformation-competent Arabidopsis genomic library in Agrobacterium. Biotechnology 9:963–967 Lührs R, Lörz H (1987) Plant regeneration in vitro from embryogenic cultures of spring- and winter-type barley (Hordeum vulgare L.) varieties. Theor Appl Genet 75:16–25 Matthews PR, Wang MB, Waterhouse PM, Thornton S, Fieg SJ, Gubler F, Jacobsen JV (2001) Marker gene elimination from transgenic barley, using co-transformation with adjacent ‘twinT-DNAs’ on a standard Agrobacterium transformation vector. Mol Breed 7:195–202 McCormac AC, Wu H, Bao M, Wang Y, Xu R, Elliott MC, Chen DF (1998) The use of visual marker genes as cell-specific reporters of Agrobacterium-mediated T-DNA delivery to wheat (Triticum aestivum L.) and barley (Hordeum vulgare L.) Euphytica 99:17–25 Ohta S, Mita S, Hattori T, Nakamura K (1990) Construction and expression in tobacco of a β-glucuronidase (GUS) reporter gene containing an intron within the coding sequence. Plant Cell Physiol 31:805–813 Patel M, Johnson JS, Brettell RIS, Jacobsen J, Xue GP (2000) Transgenic barley expressing a fungal xylanase gene in the endosperm of the developing grains. Mol Breed 6:113–123 Pesole G, Liuni S, Grillo G, Saccone C (1998) UTRdb: a specialized database of 5′- and 3′-untranslated regions of eukaryotic mRNAs. Nucleic Acids Res 26:192–195 Ryschka S, Ryschka U, Schulze J (1991) Anatomical studies on the development of somatic embryoids in wheat and barley explants. Biochem Physiol Pflanz 187:31–41 Tingay S, McElroy D, Kalla R, Fieg S, Wang M, Thornton S, Brettell R (1997) Agrobacterium tumefaciens-mediated barley transformation. Plant J 11:1369–1376. Trifonova A, Madsen S, Olesen A (2001) Agrobacterium-mediated transgene delivery and integration into barley under a range of in vitro culture conditions. Plant Sci 161:871–880 Van der Geest AHM, Petolino JF (1998) Expression of a modified green fluorescent protein gene in transgenic maize plants and progeny. Plant Cell Rep 17:760–764 Walmsley AM, Henry RJ, Birch RG (1995) Optimisation of tissue culture conditions for transformation studies using immature embryos of Australian barley cultivars. Aust J Bot 43:499–504 Wan Y, Lemaux PG (1994) Generation of large numbers of independently transformed fertile barley plants. Plant Physiol 104:37–48 Wang MB, Upadhyaya NM, Brettell RIS, Waterhouse PM (1997) Intron-mediated improvement of a selectable marker gene for plant transformation using Agrobacterium tumefaciens. J Gen Breed 51:325–334 Wang MB, Li Z, Matthews PR, Upadhyaya NM, Waterhouse PM (1998) Improved vectors for Agrobacterium tumefaciens-mediated transformation of monocot plants. Acta Hortic 461:401–407 Wang MB, Abbott DC, Upadhyaya NM, Jacobsen JV, Waterhouse PM (2001) Agrobacterium tumefaciens-mediated transformation of an elite Australian barley cultivar with virus resistance and reporter genes. Aust J Plant Physiol 28:149–156 Weir B, Gu X, Wang M, Upadhyaya N, Elliott AR, Brettell RIS (2001) Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as model system and green fluorescent protein as a visual marker. Aust J Plant Physiol 28:807–818