导入抗逆基因的转双抗虫基因741杨的获得

Regeneration System and Genetic Transformation with AtNDPK2 Gene of Transgenic Hybrid Poplar 741 Carrying Two Insect-Resistant Genes

为培育更多抗非生物胁迫的转基因杨树,以转双抗虫基因741杨(P.alba×(P.davidaiana×P.sirnonii)×P.tomentosa)为材料,建立了叶片诱导的转双抗虫基因741杨的高频再生体系,并采用根癌农杆菌介导法,探索了影响其遗传转化效率的主要因子。结果表明:最佳不定芽诱导培养基为MS+1.0 mg/L 6-BA+0.1 mg/L IBA;继代培养基为MS+0.5 mg/L 6-BA+0.05 mg/L IBA,最佳生根培养基为1/2MS+0.05 mg/L IBA。对影响转化因子的研究结果表明,共培养时间为3 d时,不定芽诱导率最高;共培养培养基加入乙酰丁香酮、pH值调到5.2时,诱导潮霉素抗性芽效果最显著;最佳潮霉素筛选质量浓度为3 mg/L;将抗逆基因AtNDPK2通过农杆菌导入到转基因741杨中,经PCR检测,在抗性植株DNA中扩增出与目的基因大小相同的片断,初步确认目的基因已经整合到转基因741毛白杨基因组中。

A study was conducted to establish a high frequency leaf-induced regeneration system of transgenic hybrid poplar 741 （ P. alba x （ P. davidaiana x P. sirnonii） x P. tomentosa ） carrying two insect-resistant genes in order to cultivate transgenic poplar with resistance to abiotic stresses. The main factors influencing the transformation efficiency of the transgenie hybrid poplar 741 carrying two insect-resistant genes were analyzed by Agrobacterium tumefaciens mediation. Results showed that the optimal culture medium for adventitious bud regeneration was MS supplemented with 1.0 mg/L 6-BA and 0. 1 mg/L IBA; the subculture medium was MS containing 0.5 mg/L 6-BA and 0.05 mg/L IBA; the optimal medium for rooting was 1/2MS supplemented with 0.05 mg/L IBA. The highest induction rate of adventitious bud was obtained after 3 days of co-culture. A remarkable effect on induction of hygromycin-resistant shoots was observed in the co-culture medium with 200 mg/L acetosyringone when pH of Agrobacterium culture medium was adjusted to 5.2. The critical concentration of hygromycin for adventitious bud regeneration was 3 mg/L. PCR detection showed that the DNA fragments of similar size to the target gene were amplified in the poplar 741 transformed with AtNDPK2 gene by A. tumefaciens mediation, indicating that the target gene expressed in the genome of poplar.