改造的马铃薯Y病毒复制酶基因介导高度抗病性

High Virus-resistance of Transgenic Tobacco Plants Mediated by Expression of Modified NIb Gene of Potato Virus Y

提取马铃薯Y病毒中国分离株(PVY-C)的mRNA作为模板,随机六聚脱氧核苷酸和寡聚dT为引物合成了单链cDNA。通过聚合酶链式反应(PCR)获得了PVY-C的核内含体b(NIb)全长cDNA克隆。在对其进行全序列分析的基础上,构建了PVY-C NIb基因全长,5＇端缺失381个碱基和NIb反义RNA三种不同形式高等植物表达载体。在土壤农杆菌LBA4404的介导下,转化烟草生产品种NC89,获得了所有三种表达载体的转基因植株。通过分子生物学检测和抗性分析发现不同形式的NIb基因序列的转基因植株对马铃薯Y病毒表现不同程度的抗性。其中,以5＇端缺失的NIb的基因转化植株表现最好,从总共20个这类转化株系中筛选到4个株系至少在100μg/ml PVY-C接种浓度下,表现完全的抗病效果。从总共39个全长NIb基因转化株系中,仅有一个株系,在100μg/ml PVY-C的攻毒接种下具有完全的抗病性。所有33个NIb基因反义RNA的转化植株中,无一株系表现完全的抗病效果,但是有部分株系能不同程度地延缓或减轻发病程度,并有部分植株在发病后50d左右有恢复健康的趋势。虽然能够在上述3种形式的NIb基因序列的转基因植物中检测到相应的RNA的转录产物,但是均未能检测到其相应的蛋白表达产物。

The entire nuclear inclusion b (Nib) gene of potato Y virus, Chinese isolate (PVY-C) was synthesized by polymerase chain reaction (PCR) using ss-cDNA of its genonic RNA as template. After sequence analyses to confirm the correctness of the NIb gene, three plant expression vectors harbing the full length, 381nt deleted from the 5' terminal coding region and the antisense of NIb gene were constructed, respectively. Nicotiana tobacum NC89 plants were transformed with Agrobacterium tumefaciens LBA4404 earring above vectors, respectively, and transgenic plants were obtained. The results of virus challenge test showed that transgenic plants derived from different forms of Nib gene displayed different degrees of PVY resistance. Among them the transgenic plants expressing 5' terminal deleted NIb gene are the best. Four lines out of total 20 in this transgenic plant group showed complete resistance at an inoculum doses of up to 100ug/ml PVY, the highest concentation tested. Just one line out of 39 transgenic lines of full length gene showed complete resistance to PVY-C at the same doses. None of lines from total trangenic lines of 33 antisense gene was resistant to PVY-C completely. But some of them could delay the symptom development and reduce the disease severity. Some of them could recover to normal 50 days post-inoculation. Although the specific RNA transcripts of all of the three forms of Nib gene could be found in transgenic plants, no specific proteins of the gene could be detected in them.