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【目的】将抗逆相关转录因子ABP9基因导入大豆(Glycine max L.)基因组,并对转化系统条件进行探索,为提高大豆遗传转化效率提供依据。【方法】以农杆菌介导的半种法大豆遗传转化系统为基础,对目的基因进行转化;以健康外植体获得率、抗性丛生芽获得率和抗性芽伸长比率为指标,对转化系统条件进行优化。【结果】共培养时保证充足的光照和氧气,外植体生长情况最好;在共培养或诱导丛生芽培养基中加入600mg·L-1的L-半胱氨酸可以使抗性丛生芽获得率由22.1%提高至32.6%;采用4种植物激素进行正交试验,诱导抗性芽伸长比率最高的激素配比为:6-BA为1.5mg·L-1,GA3为0.75mg·L-1,IAA为0.1mg·L-1,ZT为1.0mg·L-1。【结论】利用优化的方法进行遗传转化研究已获得转基因再生植株,经PCR和DNA测序等分子检测,证明目的基因ABP9已导入并整合到大豆基因组中;转化率为1.33%。
【Objective】 The objective of this study was to introduce the ABP9 gene, an anti-retrograde transcription factor, into the genome of soybean (Glycine max L.) and explore the conditions of the transformation system to provide a basis for improving the genetic transformation efficiency of soybean. 【Method】 Agrobacterium-mediated transformation system was used to transform the target gene. Based on the yield of healthy explants, the yield of resistant buds and the rate of elongation of resistant buds, Conversion system conditions to optimize. 【Result】 Adequate light and oxygen were obtained during co-culture, and the explants grew best. Adding 600 mg · L -1 L-cysteine in co-culture or inducing clump bud culture medium could make resistant cluster bud The yield was increased from 22.1% to 32.6%. Orthogonal test with four plant hormones showed that the ratio of hormones with the highest elongation rate of resistant buds was 1.5 mg · L -1 for 6-BA and 0.75 mg · L -1 for GA 3 L-1, IAA was 0.1 mg · L-1 and ZT was 1.0 mg · L-1. 【Conclusion】 The optimized method for genetic transformation has been obtained from transgenic plants. After PCR and DNA sequencing, the target gene ABP9 has been introduced and integrated into soybean genome. The transformation rate was 1.33%.