标记基因的选择是影响植物遗传转化和转基因后代筛选成败的关键因素。hpt与bar作为两种常用的水稻转化筛选标记被广泛应用于水稻的转化。为比较两者在实际应用中的效果, 文章首先对比了在潮霉素和除草剂(Bialaphos)两种筛选剂下水稻遗传转化的情况。研究表明, hpt基因的转化筛选体系相对于bar基因在转化效率上提高近两倍, 转化周期提前至少10 d, 且插入基因拷贝数更低。随后, 文章分析了利用潮霉素浸种法在田间筛选转基因水稻的可行性, 研究显示当潮霉素浓度大于167 mg/L时, 可以对以水稻品种kitaake为亲本的转基因材料进行有效筛选, 达到常规除草剂的筛选效果。但与除草剂相比较, 潮霉素的田间筛选成本却处于劣势。文章研究和讨论了hpt和bar基因在遗传转化和后代田间筛选中的优缺点, 并提供了一种利用潮霉素浸种法筛选转基因后代阳性植株的手段, 为将来在水稻转基因研究工作中根据实际需求选择合适的遗传转化、筛选体系提供参考。
The decision of using selection marker is one of the key factors for success of plant genetic transformation and offspring screening. As two commonly used selection markers, hpt and bar genes are widely used in tissue culture-based rice transformation. To experimentally compare their performance, we investigated the efficiency of two transformation systems using Hygromycin and Bialaphos as the selection agents, respectively. The result indicated that the system using hpt gene as the selection marker saved 10 days and had double transformation efficiency and lower transgene copy number in comparison to the system using bar gene. Then, we assessed the feasibility of screening transgenic rice in the field by soaking the wild-type and transgenic seeds in a series of solutions containing step diluted hygromycin for two days. We targeted the suitable concentration for distinguishing the transgenic seeds from WT Kitaake seeds was 167 mg L-1. However, the cost of screening by hygromycin is still much higher than that of Basta in field test. Therefore, this study experimentally demonstrated the advantages and disadvantages of the hpt and bar gene as the selection markers and thus provided a reference for choose of an appropriate selection marker ac-cording to the practical applications.
[1] Miki B, McHugh S. Selectable marker genes in transgenic plants: applications, alternatives and biosafety. J Bio-technol, 2004, 107(3): 193-232.
[2] 黄大年, 朱冰, 杨炜, 薛锐, 肖晗, 田文忠, 李良材, 戴顺洪. 抗菌肽B基因导入水稻及转基因植株的鉴定. 中国科学(C辑), 1997, 27(1): 55-62.
[3] Oard JH, Linscomde SD, Braverman MP, Jodari F, Blouin DC, Leech M, Kohli A, Vain P, Cooley JC, Christou P. Development, field evaluation, and agronomic perform-ance of transgenic herbicide resistant rice. Mol Breed, 1996, 2(4): 359-368.
[4] Comai L, Stalker DM. Mechanism of action of herbicides and their molecular manipulation. Plant Mol Cell, 1986, 3(2): 167-195.
[5] Aybes NM, Pabk WD. Genetic transformation of rice. Crit Rev Plant Sci, 1994, 13(3): 219-239.
[6] 王慧中, 赵培洁, 颜美仙, 黄大年. Basta对水稻愈伤组织生长及分化的影响. 杭州师范学院学报, 2000, 15(3): 49-51.
[7] 周玲艳, 姜大刚, 吴豪, 庄楚雄. 潮霉素和PPT对水稻愈伤组织筛选效果的比较. 仲恺农业技术学院学报, 2003, 16(2): 10-15.
[8] Hiei Y, Ohta S, Komari T, Kumashiro T. Efficient trans-formation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J, 1994, 6(2): 271-282.
[9] Aldemita RR, Hodges TK. Agrobacterinm tumefaciens-mediated transformation of Japonica and indica rice varieties. Planta, 1996, 199(4): 612-617.
[10] Rashid H, Yoka S, Toriyama K, Hinata K. Transgenic plant production mediated by Agrobacterium in indica rice. Plant Cell Rep, 1996, 15(10): 727-730.
[11] 唐祚舜, 王象坤, 李良才, 田文忠. 基因枪法转基因水稻中hpt基因稳定遗传. 遗传学报, 2000, 27(1): 26-33.
[12] Wang MB, Waterhouse PM. A rapid and simple method of assaying plants transformed with hygromycin and PPT resistance genes. Plant Mol Biol Rep, 1997, 15(3): 209-215.
[13] 殷奎德, 张兴梅, 郑桂萍, 戴凌燕, 宁德利. 一种快速检测转基因水稻潮霉素抗性标记的方法. 黑龙江八一农垦大学学报, 2010, 22(1): 45-48.
[14] 王忠华, 舒庆尧, 贺华龙, 夏英武, 倪新强. Bt转基因水稻对抗生素反应的初步研究(简报). 植物生理学通讯, 2001, 37(2): 111-113.
[15] Toki S. Rapid and efficient Agrobacterium-mediated transformation in rice. Plant Mol Biol Rep, 1997, 15(1): 16-21.
[16] 王育花, 赵森, 陈芬, 肖国樱. 利用实时荧光定量PCR法检测转基因水稻外源基因拷贝数的研究. 生命科学研究, 2007, 11(4): 301-305.
[17] Saika H, Toki S. Mature seed-derived callus of the model indica rice variety Kasalath is highly competent in Agrobacterium-mediated transformation. Plant Cell Rep, 2010, 29(12): 1351-1364.
[18] Pipatpanukul T, Bunnag S, Theerakulpisut P, Kosittrakul M. Transformation of indica rice (Oryza sa-tiva L.) cv. RD6 mediated by Agrobacterium tumefaciens. Songklanakarin J Sci Technol, 2004, 26(1): 1-13.
[19] 钱海丰, 薛庆中. 两种筛选标记基因在水稻(Oryza sativa L.)遗传转化中的比较. 生物技术, 2004, 14(6): 12-14.
[20] 赵艳, 王慧中, 于彦春, 黄大年. 转基因植物中标记基因的安全性新策略. 遗传, 2003, 25(1): 119-122.
[21] 杨英军, 周鹏. 转基因植物中的标记基因研究新进展. 遗传, 2005, 27(3): 499-504.
[22] He ZQ, Fu YP, Si HM, Hu GC, Zhang SH, Yu YH, Sun ZX. Phosphomannose-isomerase (pmi) gene as a selectable marker for rice transformation via Agrobacterium. Plant Sci, 2004, 166(1): 17-22.
