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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 mRNA of PVY-C was extracted as a template, and single-stranded cDNA was synthesized by random hexadecyl deoxyribonucleotides and oligo dT primers. The full-length cDNA clone of nucleosome b (NIb) of PVY-C was obtained by polymerase chain reaction (PCR). Based on the full sequence analysis, three different forms of higher plant expression vectors were constructed, including full length of PVY-C NIb, deletion of 381 bases at 5 ’end and NIb antisense RNA. Under the control of Agrobacterium tumefaciens LBA4404, the tobacco cultivar NC89 was transformed to obtain transgenic plants of all three expression vectors. Transgenic plants with different forms of NIb gene sequences were found to show different degrees of resistance to potato virus Y by molecular biology tests and resistance analysis. Among them, the NIb gene-deleted plants which were deleted at the 5 ’end performed the best, and 4 strains were screened from a total of 20 such transformed lines for at least 100 μg / ml PVY-C inoculation concentration, showing complete resistance effect. Only one of a total of 39 full-length NIb genetically transformed lines showed complete resistance against challenge with 100 μg / ml PVY-C. None of the 33 NIb antisense RNA transformants showed complete disease resistance, but some of them could delay or reduce the degree of disease to some extent and some plants recovered after 50 days Healthy trend. Although the transcripts of the corresponding RNAs can be detected in the transgenic plants with the above three forms of NIb gene sequences, none of their corresponding protein expression products can be detected.