抗氧化剂对农杆菌介导的大豆下胚轴GUS基因瞬时表达的影响

doi:10.17521/cjpe.2006.0044

摘要/Abstract

摘要：

大豆(Glycine max)下胚轴作为大豆遗传转化的外植体材料,能快速高频再生不定芽。然而,在遗传转化过程中褐化影响基因转化效率。在该研究中,我们用含有GUS染色基因和hpt II(Hygromycin phosphotransferase II)筛选基因的农杆菌(Agrobacterium tumefaciens) LBA4404侵染大豆下胚轴,并用组织化学定位法测定了GUS基因的瞬时表达,以确定大豆的优化基因转化条件。结果显示,在共培养基中加入硫代硫酸钠、L_半胱氨酸以及二硫苏糖醇等抗氧化剂,可以有效地抑制大豆下胚轴在组培过程中褐化的发生,并大幅度提高农杆菌在下胚轴的瞬时表达率。这些结果说明抗氧化剂可以降低这种影响并有效提高基因转化效率。

关键词: 大豆, 下胚轴, 农杆菌, GUS基因, 瞬时表达

Abstract:

Soybean hypocotyls have advantages of higher regeneration frequency and faster formation of adventitious shoots, and often be used in soybean transformation mediated by Agrobacterium tumefaciens. But the browning and necrosis of hypocotyls during co_cultivation greatly affects the efficiency of the transformation.The effects of antioxidants on the transient expression efficiency of GUS (β_glucuronidase) gene in soybean hypocotyls mediated byAgrobacterium tumefaciens were studied by adding antioxidants (thiosulfate, L_cysteine, and dithiothreitol) in the co_cultivation medium,and the transient expression efficiency of the GUS gene was measured by histochemically method. The results showed that the antioxidant in the co_cultivation medium increased the transient expression efficiency up to 23.4% in hypocotyls. Further investigation demonstrated that higher transient expression efficiency was associated with concomitant decreases in the levels of hypocotyl browning. The stable expression of GUS gene in hypocotyls was found in the cambium cells, which was formed as deep blue zone on the hypocotyls. Our results also showed that concentration of Agrobacterium tumefaciens and co_cultivation time were the important factors that affected the transient expression efficiency of GUS gene in hypocotyls. In our experiments, the highest transient expression efficiency of GUS gene obtained by inoculating hypocotyls in Agrobacterium tumefaciens (OD₆₀₀ 0.6) and co_cultivating for 3 days in the darkness. All results suggested that antioxidants could efficiently improve transient expression efficiency of GUS gene in soybean hypocotyls.

Key words: Soybean, Hypocotyls, Agrobacterium tumefaciens, GUS gene, Transient expression

汲逢源, 王戈亮, 许亦农. 抗氧化剂对农杆菌介导的大豆下胚轴GUS基因瞬时表达的影响. 植物生态学报, 2006, 30(2): 330-334. DOI: 10.17521/cjpe.2006.0044

JI Feng_Yuan, WANG Ge_Liang, XU Yi_Nong. THE EFFECTS OF ANTIOXIDANTS ON THE TRANSIENT EXPRESSION OF GUS GENE IN SOYBEAN HYPOCOTYLS MEDIATED BY AGROBACTERIUM TUMEFACIENS. Chinese Journal of Plant Ecology, 2006, 30(2): 330-334. DOI: 10.17521/cjpe.2006.0044

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链接本文: https://www.plant-ecology.com/CN/10.17521/cjpe.2006.0044

https://www.plant-ecology.com/CN/Y2006/V30/I2/330

图/表 5

图1 GUS基因在大豆下胚轴中的瞬时表达

Fig.1 Transient expression efficiency of GUS gene in soybean hypocotyls

图2 抗氧化剂对大豆下胚轴褐化的影响及GUS基因在下胚轴中的表达 a: 没有添加抗氧化剂时,在大豆下胚轴伤口处出现褐化和坏死 Browning and necrosis of the cut without antioxidant in CCM b: 添加抗氧化剂后,下胚轴没有褐化现象发生 There was almost no browning in the cut with antioxidant in CCM c. 添加抗氧化剂后,GUS基因在下胚轴中瞬时表达的位置 Location of GUS gene expression in soybean hypocotyls

Fig.2 The effects of antioxidants on the browning, necrosis of hypocotyls and the location of GUS expression in soybean hypocotyls

图3 不同共培养时间对大豆下胚轴GUS基因瞬时表达率的影响

Fig.3 The effects of co_cultivation time on transient expression efficiency of GUS gene in soybean hypocotyls

图4 不同农杆菌稀释浓度下大豆下胚轴的GUS基因瞬时表达率

Fig.4 The effects of re_suspend liquid medium dilute consistence on GUS gene transient expression efficiency in soybean hypocotyls

图5 抗氧化剂对不同品种大豆下胚轴GUS基因瞬时表达率的影响 HN41:‘黑农41号’‘Heinong41’ KF6:‘科丰6号’‘Kefeng6’ KX3: ‘科新3号’‘Kexin 3’ ZS18:‘早熟18号’‘Zaoshu18’

Fig.5 The effects of different soybean species on soybean hypocotyls GUS gene transient expression efficiency

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