[1]MALEKI Samaneh Sadat,MOHAMMADI Kourosh,季孔庶*.根癌农杆菌介导遗传转化烟草‘NC89'效率的影响因素[J].南京林业大学学报(自然科学版),2018,42(01):027-34.[doi:10.3969/j.issn.1000-2006.201705030 ]
 Samaneh Sadat MALEKI,Kourosh MOHAMMADI,Kongshu JI*.Influencing factors of genetic transformation efficiency in tobacco (Nicotiana tabacum ‘NC89')using Agrobacterium tumefacience[J].Journal of Nanjing Forestry University(Natural Science Edition),2018,42(01):027-34.[doi:10.3969/j.issn.1000-2006.201705030 ]
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根癌农杆菌介导遗传转化烟草‘NC89'效率的影响因素
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《南京林业大学学报(自然科学版)》[ISSN:1000-2006/CN:32-1161/S]

卷:
42
期数:
2018年01期
页码:
027-34
栏目:
研究论文
出版日期:
2018-01-31

文章信息/Info

Title:
Influencing factors of genetic transformation efficiency in tobacco (Nicotiana tabacum ‘NC89')using Agrobacterium tumefacience
文章编号:
1000-2006(2018)01-0027-08
作者:
MALEKI Samaneh Sadat MOHAMMADI Kourosh 季孔庶*
南方现代林业协同创新中心,南京林业大学林木遗传与生物技术省部共建教育部重点实验室,江苏 南京 210037
Author(s):
Samaneh Sadat MALEKI Kourosh MOHAMMADI Kongshu JI*
Co-Innovation Center for the Sustainable Forestry in Southern China, Key Laboratory of Forest Genetics & Biotechnology, Ministry of Education, Nanjing Forestry University, Nanjing 210037, China
关键词:
遗传转化 烟草 农杆菌介导 高效
Keywords:
Keywords:genetic transformation tobacco Agrobacterium-mediated high efficiency
分类号:
Q37; S722
DOI:
10.3969/j.issn.1000-2006.201705030
文献标志码:
A
摘要:
【目的】通过农杆菌介导将PBI121:CslA1基因转入烟草‘NC89',研究遗传转化的影响因素,旨在提高转化效率,优化相关技术。【方法】 通过培养基的筛选在MS培养基 +6-BA(2.0 mg/L)+NAA(1.0 mg/L)获得了大量的幼苗,以其作为转基因受体材料,通过控制农杆菌浓度、预培养时间、农杆菌侵染时间、共培养时间等条件,进行含卡那选择性培养基的筛选,获得转基因壮苗。【结果】 最佳的转基因植株培养方法为:共培养2 d以后,农杆菌的浓度D600为0.7,农杆菌诱导20 min,培养基的卡那质量浓度为100 mg/mL; 暗培养4 d易于提高再生率,更换选择性培养基后暗培养7 d,更容易缩短愈伤诱导的时间。用RT-PCR和 Southern 杂交进一步验证和分析了转基因的结果。【结论】 通过优化转基因的各环节条件,实现烟草的过量表达,提高了转基因的效率。研究结果可为农杆菌介导转化法在烟草及其相关植物的遗传转化应用提供参考。
Abstract:
【Objective】 In this study, we aim to report the production of transgenic tobacco ‘NC89' using Agrobacterium tumefaciens harboring the PBI121:CslA1 binary vector and the optimization of some transformation parameters to enhance transformation efficiency. 【Method】 By optimizing the Murashige and Skoog medium enriched with 6-BA(2.0 mg/L)and NAA(0.1 mg/L), we got a large number of seeding for subsequent usage and achieved the maximum number of shoots per explant. To find out the most effective condition for tobacco transformation, we examined some parameters, including the Agrobacterium concentration, pre-culture duration, Agrobacterium infection duration, co-culture period, treatment of selective media containing kanamycin and dark incubation in selective duration. Transgenic seedlings was obtained by controlling some transformation parameters including the Agrobacterium concentration, pre-culture duration, Agrobacterium transformation duration, co-culture period, to complete selection with media containing kanamycin. 【Result】 The highest transformation efficiency was obtained with two days of explant pre-culture, Agrobacterium concentration of D600 being 0.7, Agrobacterium inoculation for 20 min. The appropriate concentration of kanamycin at the concentration of 100 mg/L was applied to screen transformed cells, and dark incubation for four days during co-cultivation increased the regeneration rate. Incubation for seven days in darkness after changing the selective medium shortened the callus induction duration. Putative transformants were verified by RT-PCR and Southern blot hybridization. 【Conclusion】 The present study achieved the overexpression of transgenes in tobacco and increased the transformation efficiency through optimizing conditions of the several key steps in gene transformation. This Agrobacterium-mediated transformation protocol could help a lot for the genetic application of A. tumefaciens mediated transformation in Nicotiana tabacum and related species.

参考文献/References:

[1] GANAPATHI T R,SUPRASANNA P,RAO P S, et al. Tobacco(Nicotiana tabacum L.): a model system for tissue culture interventions and genetic engineering[J]. Indian Journal of Biotechnology,2004(3):171-184.
[2] VASIL V,HILDEBRANDT A C. Differentiation of tobacco plants from single,isolated cells in microcultures[J]. Science,1965,150(3698): 889-892. DOI:10.1126/science.150.3698.889.
[3] HORSCH R B,FRY J E,HOFFMAN N L,et al. A simple and general method for transferring genes into plants[J]. Science,1985,227(4691): 1229-1231. DOI:10.1126/science.227.4691.1229.
[4] FRALEY R T,ROGERS S G,HORSCH R B,et al. Genetic transformation in higher plants[J]. Critical Reviews in Plant Sciences,1986,4(1): 1-46. DOI:10.1080/07352688609382217.
[5] AN G. High efficiency transformation of cultured tobacco cells[J]. Plant Physiol,1985,79(2): 568-570. DOI:10.1104/pp.79.2.568.
[6] GALLOIS P,MARINHO P. Leaf disk transformation using Agrobacterium tumefaciens-expression of heterologous genes in tobacco[J]. Methods Mol Biol,1995,49: 39-48. DOI:10.1385/0-89603-321-X:39.
[7] TZFIRA T,LI J,LACROIX B,et al. Agrobacterium T-DNA integration: molecules and models[J]. Trends Genet,2004,20(8): 375-383. DOI:10.1016/j.tig.2004.06.004.
[8] GELVIN S B.Agrobacterium-mediated plant transformation: the biology behind the “Gene-Jockeying” tool [J]. Microbiology and Molecular Biology Reviews,2003,67(1): 16-37. DOI:10.1128/mmbr.67.1.16-37.2003.
[9] VELUTHAMBI K,GUPTA A K,SHARMA A. The current status of plant transformation technologies [J]. Current Science,2003,84(3):368-380.
[10] KOMARI T,ISHIDA Y,HIEI Y. Plant transformation technology:Agrobacterium-mediated transformation[G]// Handbook of Plant Biotechnology,2004(1):223-261. DOI:10.1002/0470869143.kc014.
[11] SHOU H, FRAME B R, WHITHAM S A,et al. Assessment of transgenic maize events produced by particle bombardment or Agrobacterium-mediated transformation [J]. Molecular Breeding, 2004, 13(2):201-208.
[12] GOUBET F, BARTON, C J, MORTIMER J C, et al. Cell wall glucomannan in Arabidopsis is synthesised by CSLA glycosyltransferases, and influences the progression of embryogenesis[J]. The Plant Journal, 2009, 60, 527-538. DOI: 10.1111/j.1365-313X.2009.03977.x.
[13] HOLSTERS M,DE WAELE D,DEPICKER A,et al. Transfection and transformation of Agrobacterium tumefaciens[J]. Mol Gen Genet,1978,163(2): 181-187. DOI:10.1007/bf00267408.
[14] HORSCH R B,FRY J,HOFFMANN N,et al. Leaf disc transformation[C]// GELVIN S B, SCHIPEROORT R A. Plant molecular biology manual, Netherlauds: Kluwer Academic Publishers, 1988:63-71.
[15] DOYLE J J,DICKSON E E. Preservation of plant samples for DNA restriction endonuclease analysis[J]. Taxon,1987,36(4): 715. DOI:10.2307/1221122.
[16] POREBSKI S,BAILEY L G,BAUM B R. Modification of a CTAB DNA extraction protocol for plants containing high polysaccharide and polyphenol components[J]. Plant Molecular Biology Reporter,1997,15(1): 8-15. DOI:10.1007/bf02772108.
[17] STOLARZ A,MACEWICZ J,LÖRZ H. Direct somatic embryogenesis and plant regeneration from leaf explants of Nicotiana tabacum L.[J]. Journal of Plant Physiology,1991,137(3): 347-357. DOI:10.1016/s0176-1617(11)80144-6.
[18] SKOOG F,MILLER C O. Chemical regulation of growth and organ formation in plant tissues cultured in vitro[J]. Symp Soc Exp Biol,1957,11: 118-130.
[19] SONG G Q,HONDA H,YAMAGUCHI K I. Efficient Agrobacterium tumefaciens-mediated transformation of sweet potato(Ipomoea batatas (L.)Lam.)from stem explants using a two-step kanamycin-hygromycin selection method[J]. In Vitro Cellular & Developmental Biology-Plant,2004,40(4): 359-365. DOI:10.1079/ivp2004539.
[20] HAN SN, O H P R, KIM H S, et al. Effects of antibiotics on suppression of Agrobacterium tumefaciens and plant regeneration from wheat embryo [J]. Crop Science and Biotechnology, 2007, 10(2): 92-98.
[21] KONDO T,HASEGAWA H,SUZUKI M. Transformation and regeneration of garlic(Allium sativum L.)by Agrobacterium-mediated gene transfer[J]. Plant Cell Reports,2000,19(10): 989-993. DOI:10.1007/s002990000222.
[22] ZHANG K,LETHAM D S,JOHN P C. Cytokinin controls the cell cycle at mitosis by stimulating the tyrosine dephosphorylation and activation of p34cdc2-like H1 histone kinase[J]. Planta,1996,200(1): 2-12. DOI:10.1007/bf00196642.
[23] KIEBER J J,SCHALLER G E. Cytokinins[J]. The Arabidopsis Book,2014,12: e0168. DOI:10.1199/tab.0168.
[24] DAVIES P J. The plant hormones: their nature,occurrence,and functions [G]// DAVIES P. Plant hormones. Netherlands: Springer,2010: 1-15. DOI:10.1007/978-1-4020-2686-7_1.
[25] KIM M J,KIM J K,KIM H J,et al. Genetic modification of the soybean to enhance the β-carotene content through seed-specific expression[J]. PLoS One,2012,7(10): e48287. DOI:10.1371/journal.pone.0048287.
[26] ISHIDA J K,YOSHIDA S,ITO M,et al. Agrobacterium rhizogenes-mediated transformation of the parasitic plant Phtheirospermum japonicum[J]. PLoS One,2011,6(10): e25802. DOI:10.1371/journal.pone.0025802.
[27] HAN J L,WANG H,YE H C,et al. High efficiency of genetic transformation and regeneration of Artemisia annua L. via Agrobacterium tumefaciens-mediated procedure[J]. Plant Science,2005,168(1): 73-80. DOI:10.1016/j.plantsci.2004.07.020.

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备注/Memo

备注/Memo:
基金项目:“十三五”国家重点研发计划(2017YFD0600304); 江苏高校优势学科建设工程资助项目(PAPD) 第一作者:MALEKI Samaneh Sadat(smgenetics.njfu@yahoo.com),博士生。*通信作者:季孔庶( kgenetics.njfu@yahoo.com),教授。
更新日期/Last Update: 2018-03-30