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采用农杆菌介导法,将透明颤菌血红蛋白Vitreoscilla hemoglobin(VHb)基因vgb导入银腺杨(Populus alba×P.glandulosa)。以经无菌培养的叶片为外植体,通过450个叶盘与根癌农杆菌(Agrobacterium tumefaciens)LBA4404共培养,将植物双元表达载体pPZV中vgb基因导入银腺杨基因组,经卡那霉素筛选后,共获得60株卡那霉素抗性植株。经PCR特异性扩增和Southern点杂交分析,证明其中52株再生植株基因组DNA中整合了vgb基因。RT-PCR分析结果表明,vgb基因在其中的44个株系中获得表达。在2年温室生长条件下,转基因无性系的外部形态与对照相比无显著差异。但在株高和地径上,有3株转基因无性系较对照植株有明显增加。
The Agrobacterium-mediated method was used to introduce Vitreoscilla hemoglobin (VHb) gene vgb into Populus alba × P.glandulosa. The vgb gene of plant binary expression vector pPZV was introduced into the genome of G. gossypii by co-culturing the Agrobacterium tumefaciens strain LBA4404 with 450 leaf discs using sterile leaf explants. After screening, a total of 60 Kanamycin resistant plants were obtained. PCR-specific amplification and Southern blot analysis showed that vgb gene was integrated into genomic DNA of 52 regenerated plants. RT-PCR analysis showed that vgb gene was expressed in 44 of them. Under the conditions of two years of greenhouse growth, there was no significant difference in the external morphology of the transgenic clones compared with the control. However, in the plant height and diameter, three transgenic clones had significantly increased compared with the control plants.