马铃薯双抗病毒转基因技术初步研究

Preliminary Research on Potato Dual-antiviral Transgenic Technology

针对马铃薯生产中存在的病毒型退化和转基因本身存在的生物安全性问题,把新近发展迅速的RNA干扰技术和无标记转基因技术引入植物抗病毒基因工程,培育生物安全型抗多病毒转基因马铃薯,探索一条多抗甚至多免疫的生物安全型转基因新途径。通过农杆菌介导,将抗马铃薯X病毒和Y病毒的RNA干扰型基因结构转入马铃薯大西洋、克新1号等新疆主裁栽培品种,通过PCR检测获得对X病毒和Y病毒具有双重高抗或免疫的无标记基因的高生物安全型转基因马铃薯植株。最终,通过多次筛选试验,确定了农杆菌OD600为0.3~0.5时为宜,但以0.5最佳,预培养2 d,侵染时间为10 min,共培养2~3 d的最佳转化条件。通过特异性引物PCR扩增,部分再生植株得到与阳性对照一致的目的条带,初步确定RNAi基因已整合到马铃薯基因组中。本研究获得的抗病毒无标记转基因马铃薯,其目的基因为RNA干扰型结构,转基因植株的抗性是RNA沉默的结果。由于所转基因的m RNA已经降解成小片段,转基因植株不存在标记基因,规避了目的基因和标记基因潜在的生物安全性问题。

According to the viral de generation and biosafety problems of transgene itself in potato production, RNA interference technology and marker free transgenic technology which have been developed rapidly were introduced into plant antiviral genetic engineering. Then, biological safety type anti virus transgenic potato was cultivated and a biosafety transgenic new pathway with multi-resistance or even multiple immunization was explored. Mediated by Agrobacterium tumefaciens, RNA interference gene structure of against potato virus V and virus Y was transferred into referee cultivated varieties in Xinjiang, including potato Atlantic and the new No. 1. After PCR detection, high biosafety transgenic potato plants without marker gene which had double high resistance or immunity to virus X and virus Y were obtained. Finally, through several times of screening test, it was determined that the Agrobacterium OD6oo was advisable between 0.3 and 0.5, with the optimal at 0.5. The best transformation condition was preculture for 2 d, infection for 10 min, and co-culture for 2-3 d. Through specific primer PCR amplification, part of the regeneration plant obtained the same target bands as the positive control group, preliminarily indicating that RNAi gene had been integrated into the potato genome. The target gene of the virus resistant marker flee transgenic potato was of RNA interference type structure, and the resistance of transgenic plants was the result of RNA silencing. Because the transgenic mRNA had been degraded into small fragments, there was no marker gene in transgenic plants, which avoided the potential biosafety problems of target genes and marker genes.