农杆菌介导小麦非组培转化方法的探讨
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摘要
根癌农杆菌是广泛应用于植物转基因的载体系统，是转化双子叶植物最常规的方法，近些年利用农杆菌介导单子叶植物的转化发展较快，在水稻、玉米上已经有比较成熟的转化方法，对于小麦来说，虽然在1997年就获得了突破，但经过近七年的发展，农杆菌介导法依然没有成为小麦基因转化的主导方法。原因之一是由于小麦组织培养难度高、品种依赖性强，其二是受体材料单一、方法单一。
本实验选取了小麦生长发育过程的两个时期：种子萌发期和开花期，系统的研究了利用农杆菌非组培转化小麦的方法。通过实验发现，在小麦穗中部第一小穗发育到授粉完成到成小半粒的时期进行花器浸沾接种，可以得到很高的GUS瞬时表达效率。在利用农杆菌转化小麦萌芽的实验中我们发现，难以转化的主要原因是由于农杆菌无法渗透至生长点部位、vir基因表达效率低。本实验通过在接种菌液中加入表面活性剂和真空渗透的方法促进了受体材料对农杆菌的吸收，并且在接种菌液中加入AS诱导vir基因的表达，并且在生长点部位检测到了GUS的瞬时表达。
通过实验，花器浸沾处理小麦的花器，染色频率可达到61％，对萌芽处理中生长点的染色频率也可达到22％，对染色材料的切片观察，也可以看到组织内层细胞的GUS染色现象。
本实验还尝试了一种新的利用潮霉素筛选小麦转化体的方法，通过筛选在692株经种子萌芽处理后再生的植株中筛选到6株明显呈阳性的植株。但对植株后来产生的新叶进行PCR鉴定时却没有扩增出特异性条带。
Agrobacterium tumefaciens has been widely utilized as a vector for plant genetic transformation, particularly on dicotyledonous plants. The last few years, the transformation of Monocotyledonous plants mediated with Agrobacterium has made good progress. There have been some routine method in transforming the rice and corn, in contrast, the wheat transformation are developing slowly, although the researcher has made breakthrough in using Agrobacterium to transform wheat since 1997. One of the main reason is the tissue culture for the wheat is very difficult and with the restriction to the different genotype, another one, the window of the reception material is so narrow that make there is no enough material for a long-time research.
We select two stage in the time of wheat growing, that is the stage of seedling and the stage of flowering, to develop the method to transform wheat without tissue culture. We use the floral dip method to deal with the flower of wheat and get the result that when the first spikelet in the middle of the whole ear begin pollination and until it become a half granule, we can get high efficiency of the GUS activity if we perform the inoculation in this course. The method that deal with the seedling is the first attempt in wheat transformation, and the difficulty is mainly because the Agrobacterium can not transfer into the inner of the seedling and the vir gene can not express efficiently, to resolve the problem we use the surfactants and vacuum infiltration to help the Agrobacterium to transfer into the inner tissue, and add AS to help vir gene express.
The frequency of GUS activity in the method of floral dip reach 61%, deal with the seedling we get the frequency of 22% for the GUS activity In the apex meristem of the seedling, slice the stained material and we observe the inner tissue also get blue.
We also develop a new screen system in using hygromycin to screen the transformant of wheat, in the method we can easily observe the difference, and is also easy to perform, using this method we have screen 6 plants, while it's regretful that we haven't detect the integration of T-DNA when perform the PCR in the later-grown leaf of the 6 selected plant.
本实验选取了小麦生长发育过程的两个时期：种子萌发期和开花期，系统的研究了利用农杆菌非组培转化小麦的方法。通过实验发现，在小麦穗中部第一小穗发育到授粉完成到成小半粒的时期进行花器浸沾接种，可以得到很高的GUS瞬时表达效率。在利用农杆菌转化小麦萌芽的实验中我们发现，难以转化的主要原因是由于农杆菌无法渗透至生长点部位、vir基因表达效率低。本实验通过在接种菌液中加入表面活性剂和真空渗透的方法促进了受体材料对农杆菌的吸收，并且在接种菌液中加入AS诱导vir基因的表达，并且在生长点部位检测到了GUS的瞬时表达。
通过实验，花器浸沾处理小麦的花器，染色频率可达到61％，对萌芽处理中生长点的染色频率也可达到22％，对染色材料的切片观察，也可以看到组织内层细胞的GUS染色现象。
本实验还尝试了一种新的利用潮霉素筛选小麦转化体的方法，通过筛选在692株经种子萌芽处理后再生的植株中筛选到6株明显呈阳性的植株。但对植株后来产生的新叶进行PCR鉴定时却没有扩增出特异性条带。
Agrobacterium tumefaciens has been widely utilized as a vector for plant genetic transformation, particularly on dicotyledonous plants. The last few years, the transformation of Monocotyledonous plants mediated with Agrobacterium has made good progress. There have been some routine method in transforming the rice and corn, in contrast, the wheat transformation are developing slowly, although the researcher has made breakthrough in using Agrobacterium to transform wheat since 1997. One of the main reason is the tissue culture for the wheat is very difficult and with the restriction to the different genotype, another one, the window of the reception material is so narrow that make there is no enough material for a long-time research.
We select two stage in the time of wheat growing, that is the stage of seedling and the stage of flowering, to develop the method to transform wheat without tissue culture. We use the floral dip method to deal with the flower of wheat and get the result that when the first spikelet in the middle of the whole ear begin pollination and until it become a half granule, we can get high efficiency of the GUS activity if we perform the inoculation in this course. The method that deal with the seedling is the first attempt in wheat transformation, and the difficulty is mainly because the Agrobacterium can not transfer into the inner of the seedling and the vir gene can not express efficiently, to resolve the problem we use the surfactants and vacuum infiltration to help the Agrobacterium to transfer into the inner tissue, and add AS to help vir gene express.
The frequency of GUS activity in the method of floral dip reach 61%, deal with the seedling we get the frequency of 22% for the GUS activity In the apex meristem of the seedling, slice the stained material and we observe the inner tissue also get blue.
We also develop a new screen system in using hygromycin to screen the transformant of wheat, in the method we can easily observe the difference, and is also easy to perform, using this method we have screen 6 plants, while it's regretful that we haven't detect the integration of T-DNA when perform the PCR in the later-grown leaf of the 6 selected plant.
引文
1．安贤惠．(1998)拟南芥及其研究进展．西北农业学报，7，92-94．
2．付永彩，成卓敏．(1999)农杆菌介导的禾本科作物遗传转新进展．农业生物技术学报，10(3)增刊，1-5．
3．贺晨霞，夏光敏．(1999)农杆菌介导单子叶植物基因转化研究进展．植物学报 16，567-573．
4．李子银，胡会庆．(1998) 农杆菌介导的植物遗传转化进展．生物工程进展，18，22-26．
5．王关林，方宏筠．(1998)《植物基因工程原理与技术》．科学出版社
6．肖兴国，张爱民，聂秀玲(2000)转基因小麦的进展与展望。农业生物技术学报，2000，8(2)：111—116
7．许东晖，李宝健，刘煜，黄光纾，古练权．(1996)对农杆菌vir区基因具诱导作用的水稻信号分子的分离和确定．中国科学C辑，26，535-541．
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16. Cheng, X.Y., Sardana, R., Kaplan, H. & Altosaar, L. (1998)Agrobacterium-transformerl rice expressing synthetic crylAb and crylAc genes are highly toxic to striped stem borer and yellow stem borer. Proc. Natl Acad. Sci. USA, 95, 2767-2772.
17. Clough, S.J. & Bent, A.F.(1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 16, 735-743.
18. Deng, W., Chen, L., Liang, X. et el., VirE1 is a specific molecular chaperone for the exported single-stranded- DNA-binding protein VirE2 in Agrobacterium, Mol. Microbiol., 1999, 31(6): 1795.
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2．付永彩，成卓敏．(1999)农杆菌介导的禾本科作物遗传转新进展．农业生物技术学报，10(3)增刊，1-5．
3．贺晨霞，夏光敏．(1999)农杆菌介导单子叶植物基因转化研究进展．植物学报 16，567-573．
4．李子银，胡会庆．(1998) 农杆菌介导的植物遗传转化进展．生物工程进展，18，22-26．
5．王关林，方宏筠．(1998)《植物基因工程原理与技术》．科学出版社
6．肖兴国，张爱民，聂秀玲(2000)转基因小麦的进展与展望。农业生物技术学报，2000，8(2)：111—116
7．许东晖，李宝健，刘煜，黄光纾，古练权．(1996)对农杆菌vir区基因具诱导作用的水稻信号分子的分离和确定．中国科学C辑，26，535-541．
8. A. Pellegrineschi·R.M. Brim· L.Velazquez(2002), The effect of pretreatment with mild heat and drought stresses on the explant and biolistic transformation frequency of three durum wheat cultivars, Plant Cell Rep (2002) 20:955-960
9. Alicja Ziemienowicz etc, Import of Agrobacterium T-DNA into Plant Nuclei: Two Distinct Functions of VirD2 and VirE2 Proteins, The Plant Cell, Vol. 13,369-383, February 2001
10. Allen, G. C., G. E. Hall, S. Michalowski, W. Newman, S. Spiker, A. K. Weissinger, and W. F. Thompson. 1996. High-level transgene expression in plant cells: effects of a strong scaffold attachment region from tobacco. Plant Cell 8:899-913.
11. Aoyama, T. A., and N.-H. Chua. 1997. A glucocorticoid-mediated transcriptional induction system for transgenic plants. Plant J. 11:605-612.
12. Ashok Kumar Sahrawat, Dirk Becker, Stephanie Lutticke, Horst Lrz, Genetic improvement of wheat via alien gene transfer, an assessment, Plant science 165(2003): 1147-1168
13. Ben Miflin, Crop improvement in the 21th Century, 1999
14. Brian Weir, Xu Gu, Mingbo Wang, Narayana Upadhyaya, Adrian R.Elliott and Richard I. s. Brettell. (2001)Agrobacterium tumefaciens-mediated transformation of wheat using suspension cells as a model system and green fluorescent protein as a visual marker. Aust .J. Plant Physiol. 28,807-818.
15. Cheng, M., Fry, J.E., Pang, S., Zhou, H., Hironka, C.M., Duncan, D.R., Conner, T.W. & Wan, Y. (1997) Genetic Transformation of Wheat Mediated by Agrobacterium tumefaciens. Plant Physiol. 115, 971-980.
16. Cheng, X.Y., Sardana, R., Kaplan, H. & Altosaar, L. (1998)Agrobacterium-transformerl rice expressing synthetic crylAb and crylAc genes are highly toxic to striped stem borer and yellow stem borer. Proc. Natl Acad. Sci. USA, 95, 2767-2772.
17. Clough, S.J. & Bent, A.F.(1998) Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. The Plant Journal, 16, 735-743.
18. Deng, W., Chen, L., Liang, X. et el., VirE1 is a specific molecular chaperone for the exported single-stranded- DNA-binding protein VirE2 in Agrobacterium, Mol. Microbiol., 1999, 31(6): 1795.
19. E. Zuther etc Complex nested promoters control tissue-specific expression of acetyI-CoA carboxylase genes in wheat, 2003
20. Enriquez, O. G. A., Vazquez, P. R. I., Prieto, S. D. L. et al., Herbicide-resistant sugarcane (Saccharum officinarum L.) plants by Agrobacterium-mediated transformation, Planta, 1998, 206(1): 20.
21. F.barro (1998),The influence of auxins on transformation of wheat and tritordeum and analysis of transgene integration patterns in transformants, Theor Appl Genet (1998)97:684-695
22. Feldmann, K.A. & Marks, M.D. (1987) Agrobacterium-mediated transformation of germinating seeds of Arabidopsis thaliana: a non-tissue culture approach. Mol. Gen. Genet. 208, 1-9.
23. G. Leckband. H. Transformation and expression of a stilbene synthase gene of Vitis vinifera L. in barley and wheat for increased fungal resistance. Theor Appl Genet (1998)96:1004-1012
24. G.L. Sharp, J.M. Martin, S.P. Lanning, N.K. Blake, C.W. Brey, E. Sivamani, R. Qu, L.E. Talbert, Field evaluation of transgenic and classical sources of wheat streak mosaic virus resistance, Crop science 42(2002)105-110
25. H.K. Khanna· G. E. Daggard, Agrobacterium tumefaciens-mediated transformation of wheat using a superbinary vector and a polyamine-supplemented regeneration medium, Plant Cell Rep (2003) 21:429--436
26. H. Wu·C. Sparks·B. Amoah·H.D. Jones, Factors influencing successful Agrobacterium-mediated genetic transformation of wheat, Plant Cell Rep (2003) 21:659-668
27. 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. The Plant Jounal, 6, 271-292.
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