gna基因在大豆上的转化及转化体系的优化
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摘要
本研究通过对大豆组织培养消毒处理方法对种子萌发的影响，来研究不同消毒剂及不同处理方法对大豆种子消毒效果的影响，从而减少由于种子消毒不彻底造成实验材料的浪费并影响后期的研究工作。利用大豆不同品种的下胚轴、顶芽、子叶节、原位再生等几种外植体进行诱导植株再生的实验，并通过添加不同浓度的6-BA来观察诱导愈伤组织和出芽的情况，为下一步的转化实验建立一个良好的组培平台。最后对大豆转化体系的几个因素进行优化实验，并以原位再生体系为平台进行gna基因的转化。实验主要结果如下：
1.大豆组织培养最佳消毒处理方法为70％酒精预处理后饱和漂白粉溶液4℃处理1～2h；或用0.1％HgCl2溶液作消毒剂4℃条件下处理2h，能取得较好的消毒效果。
2.各种外植体诱导愈伤组织和出芽最适6-BA浓度不同，其中子叶节为1.0mg/L，顶芽为2.0mg/L，原位为1.0mg/L至2.0mg/L；下胚轴为2.0mg/L。原位在1.0mg/L 6-BA的基础上添加0.1mg/L IBA时出芽率比单独加6-BA要高。
3.大豆下胚轴、子叶、顶芽均较易诱导出愈伤组织，但这三种外植体出芽率较低，子叶节和原位再生出芽率较高，五种外植体出芽率依次为原位＞子叶节＞顶芽＞下胚轴＞子叶。
4.不同基因型的外植体在诱导愈伤组织和出芽上存在着显著差异，分析品种间差异表现发现，基因型稳定性在不同外植体诱导愈伤组织和出芽上较一致，稳定性影响诱导愈伤组织和出芽，其中品种东农42号表现好。表明应该注重不同基因型稳定性上的差异。
5.大豆品种对农杆菌的敏感性普遍较低，并且存在着较强的基因型依赖性。
6.外植体不同的预培养时间、感染时间和共培养时间对大豆的转化有 明显的影响，以预培养1d，感染10min后共培养3d为宜。
7.以再生频率较高的原位再生体系为平台进行gna基因的转化，通过卡那霉素进行抗性筛选后得到再生植株，随机选取15株进行PCR检测，9株呈现阳性反应。
To establish the most optimal methods of sterilization on soybean, variety Hefeng No.35 were taken as material to compare effects of seed germination with different disinfectants and different treating time at 28℃ or 4℃ in tissue culture.
The different explants, hypocotyl, terminal bud, cotyledonary node, seedling were studied to induce bud or callus with different concentrations of 6-BA and to establish a better system for transformation. Several factors in transformation system of soybean were studied and gna gene was transferred into soybean with seedling explants. The results show:
1. In soybean tissue culture , the most optimal methods of sterilization is one-two hour's treatment with saturate bleaching powder solution in 4℃ after 30 seconds' pretreatment with 70% ethanol or two hour's treatment with 0.1% aqueous mercuric chloride in 4℃.
2. The different explants need different concentration of 6-BA to induce callus and bud, cotyledonary node is 1.0mg/L, terminal bud 2.0mg/L, seedling 1.0mg/L to bud-inducing, and hypocotyls is 2.0mg/L to induce callus. The effect is better with 6-BA 1.0mg/L supplemented with IBA 0.2mg/L for seedling explant's bud-inducing.
3. Hypocotyl, cotyledon, terminal bud are all easy to induce callus, but the inducing rate of bud are lower than those of cotyledonary node and seedling. Regeneration rates of various explants were , in the order from high to low, seedling, cotyledonary node, terminal bud, hypocotyls, cotyledon.
4. There exists obvious difference among different genotypes . these results indicates that the stability is uniform between callus and bud inducing with different explants and it affects callus and bud inducing. Dongnong 42 is better than other tested genotypes on its stability. It shows we should pay attention to the difference on the stability of genotypes.
5. The sensitivity of soybean cultivars to Agrobacterium tumefaciens is commonly low and there is significant difference among genotypes.
6. The preculture, duration of infection and co-culture with Agrobacterium tumefaciens influences soybean transformation evidently. The best combination is preculture 1 day, infection 10 minutes and co-culture 3 days under the experimental conditions.
7. gna gene was transferred with regeneration system of soybean seedling, whose regeneration is higher than others. After selected by kanamycin, Among the 15 putative transgenic plants, nine gave positive PCR reaction.
1.大豆组织培养最佳消毒处理方法为70％酒精预处理后饱和漂白粉溶液4℃处理1～2h；或用0.1％HgCl2溶液作消毒剂4℃条件下处理2h，能取得较好的消毒效果。
2.各种外植体诱导愈伤组织和出芽最适6-BA浓度不同，其中子叶节为1.0mg/L，顶芽为2.0mg/L，原位为1.0mg/L至2.0mg/L；下胚轴为2.0mg/L。原位在1.0mg/L 6-BA的基础上添加0.1mg/L IBA时出芽率比单独加6-BA要高。
3.大豆下胚轴、子叶、顶芽均较易诱导出愈伤组织，但这三种外植体出芽率较低，子叶节和原位再生出芽率较高，五种外植体出芽率依次为原位＞子叶节＞顶芽＞下胚轴＞子叶。
4.不同基因型的外植体在诱导愈伤组织和出芽上存在着显著差异，分析品种间差异表现发现，基因型稳定性在不同外植体诱导愈伤组织和出芽上较一致，稳定性影响诱导愈伤组织和出芽，其中品种东农42号表现好。表明应该注重不同基因型稳定性上的差异。
5.大豆品种对农杆菌的敏感性普遍较低，并且存在着较强的基因型依赖性。
6.外植体不同的预培养时间、感染时间和共培养时间对大豆的转化有 明显的影响，以预培养1d，感染10min后共培养3d为宜。
7.以再生频率较高的原位再生体系为平台进行gna基因的转化，通过卡那霉素进行抗性筛选后得到再生植株，随机选取15株进行PCR检测，9株呈现阳性反应。
To establish the most optimal methods of sterilization on soybean, variety Hefeng No.35 were taken as material to compare effects of seed germination with different disinfectants and different treating time at 28℃ or 4℃ in tissue culture.
The different explants, hypocotyl, terminal bud, cotyledonary node, seedling were studied to induce bud or callus with different concentrations of 6-BA and to establish a better system for transformation. Several factors in transformation system of soybean were studied and gna gene was transferred into soybean with seedling explants. The results show:
1. In soybean tissue culture , the most optimal methods of sterilization is one-two hour's treatment with saturate bleaching powder solution in 4℃ after 30 seconds' pretreatment with 70% ethanol or two hour's treatment with 0.1% aqueous mercuric chloride in 4℃.
2. The different explants need different concentration of 6-BA to induce callus and bud, cotyledonary node is 1.0mg/L, terminal bud 2.0mg/L, seedling 1.0mg/L to bud-inducing, and hypocotyls is 2.0mg/L to induce callus. The effect is better with 6-BA 1.0mg/L supplemented with IBA 0.2mg/L for seedling explant's bud-inducing.
3. Hypocotyl, cotyledon, terminal bud are all easy to induce callus, but the inducing rate of bud are lower than those of cotyledonary node and seedling. Regeneration rates of various explants were , in the order from high to low, seedling, cotyledonary node, terminal bud, hypocotyls, cotyledon.
4. There exists obvious difference among different genotypes . these results indicates that the stability is uniform between callus and bud inducing with different explants and it affects callus and bud inducing. Dongnong 42 is better than other tested genotypes on its stability. It shows we should pay attention to the difference on the stability of genotypes.
5. The sensitivity of soybean cultivars to Agrobacterium tumefaciens is commonly low and there is significant difference among genotypes.
6. The preculture, duration of infection and co-culture with Agrobacterium tumefaciens influences soybean transformation evidently. The best combination is preculture 1 day, infection 10 minutes and co-culture 3 days under the experimental conditions.
7. gna gene was transferred with regeneration system of soybean seedling, whose regeneration is higher than others. After selected by kanamycin, Among the 15 putative transgenic plants, nine gave positive PCR reaction.
引文
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Barwale UB, HR Kerns and JM Widholm. 1986. Pant regeneration from callus cultures of several soybean genotypes via embryogenesis and organogenesis. Planta. 167: 473-481
2.
Bechtold, N., J. Ellis and G. Pelletier. 1993. In planta Agrobacterium mediated gene transfer by infiltration of adult Arabidopsis thaliana plants. C.R. Acad. Sci. Paris, Life Sci. 316:1194-1199
3.
Blades, R, M Moos and K Geider. 1987. Transformation of soybean protoplasts from permanent suspension cultures by cocultivation with cells of Agrobacterium tumefaciens. Plant Mol. Biol. 9:135-145
4.
Boulter D, Gatchouse A M R, Gatehouse T A, et al. 1993. Genetically engineered insect resisitance to the brown plant hopper(BPH) and other sucking insect.Proceedings of Sixth Annual Meeting of the International Program on Rice Biotechnology.Chiang Mai Thailand, 9
5.
Broadway R M, Duffey S S. J 1986. Insect Physiol. 32: 827-833
6.
Chee PP, KA Fober, JL Slightom. 1989. Transformation of soybean (Glycine max) by infecting germinating seeds with Agrobacterium tumefaciens. [J] Plant Physiol. (91):1212-1218
7.
Chen L, Y Dan, JM Tyler and NA Reichert. 1999. Transgenic soybean generated from hypocotyl explants. In Vitro Cell. Dev. Biol. 35:61A.
8.
Christianson ML, DA Warnick and PS Carlson. 1983. A morpho-genetically competent soybean suspension culture. Science. 222: 632-634
9.
Christou, P, JE Murphy and WF Swain. 1987. Stable transformation of soybean by electroporation and root formation from transformed callus. Proc. Natl. Acad. Sci. USA 84:3962-3966
10.
Clough, SJ and AF Bent. 1998. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16:735-743
11.
Dan, Y and NA Reichert. 1998. Organogenic regeneration of soybean from hypocotyl explants. In Vitro Cell. Dev. Biol. 34P:14-21
12.
Di R, V Purcell, GB Collins and S Ghabrial. 1996. Production of transgenic soybean lines expressing the bean pod mottle virus coat protein precursor gene. [J] Plant Cell Rep. (15):746-750
13.
Edward GA, Hepher A, Clerk SP, et al. 1991. Pea lectin is correctly processed, stable and active in leaves of transgenic potato plants. Plant Mol. Biol. 17:89-100
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