摘要
以马铃薯Y病毒坏死株系(PVYN)的外壳蛋白(coat protein,CP)基因3′端50bp片段为hpRNA的茎,以pUC19不同长度的序列为hpRNA的环,构建了茎环比例分别为4∶1、2∶1、1∶1、1∶2、1∶4和1∶8的植物表达载体。利用农杆菌介导法转化烟草品种NC89,获得了多种转基因植株。室内抗病性检测发现:不同茎环比例的hpRNA介导的病毒抗性效率不同;茎环比例为4∶1、2∶1和1∶1时效率较高,抗性植株的比例达60%左右;随着环长度的逐渐增加,抗性植株的比例逐渐降低;当茎环比例为1∶8时,抗性植株的比例仅为9.52%。Southern blot分析结果表明:外源基因已整合于烟草的基因组中,且转基因植株的抗病性与转基因的拷贝数之间无明显的相关性。Northern blot分析结果表明:目的片段转录产物的积累量与植株的抗病性呈负相关,证明所获得的抗病性是RNA介导的。
Six binary vectors with 4 ∶1,2 ∶1,1 ∶1,1 ∶2,1 ∶4 and 1 ∶8 stem-loop proportion of hpRNA were constructed,respectively.The inverted repeat DNA sequence of hpRNA derived from 3＇ends 50 bp of PVY^N CP gene,and the spacer sequence of hpRNA derived from different part of pUC19.Recombinant binary vectors were then introduced into tobacco（NC89）plants via Agrobacterium tumefaciens-mediated transformation system.Transgenic plants transformed with different binary vectors were obtained respectively.Resistance tests indicated that hpRNA with different stem-loop proportion had different effect on inducing RNA-mediated gene silencing.When the proportion was 4 ∶1,2 ∶1 and 1 ∶1,hpRNA could effectively induce RNAi,about 60% transgenic plants were highly resistant to PVY^N infection;with the length of loop increased,the proportion of resistant transgenic plants became lower;when it was 1 ∶8,the proportion of resistant transgenic plants was only 9.52%.Southern blot analysis confirmed that the foreign gene had been integrated into the tobacco genome,and there were no relationship between resistance of transgenic plants and the transgene copy numbers.Northern blot results revealed an inverse correlation between transgenic transcript accumulation and virus resistance,it demonstrated that the resistance was RNA mediated.
出处
《植物病理学报》
CAS
CSCD
北大核心
2008年第5期468-477,共10页
Acta Phytopathologica Sinica
基金
国家自然科学基金资助项目(30471139)
