核基质结合区对马铃薯Y病毒全长非翻译CP基因介导抗病性的影响

Effect of matrix attachment regions on the RNA-mediated resistance indu ced by PVY^N-CP gene

为探索核基质结合区(m atrix attachm ent reg ions,MAR s)对RNA介导的病毒抗性的影响,我们将从烟草中克隆到的核基质结合区TM 2构建在包含马铃薯Y病毒全长非翻译CP基因的植物表达载体pRPVYCPN的表达盒的两侧,构建了植物表达载体pRTM 2CPNTM 2。采用农杆菌介导基因转化法,将表达载体pRPVYCPN和pRTM 2CPNTM 2转入烟草品种NC89中,分别获得了144株和344株转基因烟草。抗病性检测发现,核基质结合区的存在能明显提高RNA介导抗性的产生效率。在含MAR s转基因植株中,抗病植株的比率为15.1%,而不含核基质结合区的转基因植株的抗病比率则为8.3%。这一研究结果对抗病毒植物的分子育种和转基因表达调控有指导意义。

In order to investigate the effect of matrix attachment regions （MARs） on the RNA-mediated resistance to plant virus, matrix attachment region TM2 cloned from tobacco was constructed into the expression vector of pRPVYCPN which contains the untranslatable coat protein （CP） gene of Potato virus Y （ PVYN ）, resulting in the plant expression vector pRTM2CPNTM2. The expression vector pRPVYCPN was constructed based on the plasmid of pROKⅡ. Tobacco plants of cultivar NC89 were then transformed with either the expression vector pRPVYCPN or the vector pRTM2CPNTM2 via Agrobacterium-mediated transformation method. The transcription products of these two vectors were predicted to be full-length untranslatable PVY^N-CP RNA. The expression vector pROK Ⅱ was also used as control. The transformed tissues were then screened in the presence of 100 mg/L kanamycin. After tissue culture and screening, we obtained 144 and 344 transgenic plants transformed with pRPVYCPN and pRTM2CPNTM2, respectively. After challenge inoculation with PVYN, the resistance was assayed by symptom observation and ELISA detection. Upon inoculation, all the plants transformed with plasmid pROK I1 or the non-transgenic plants were susceptible to PVYN infection. However, 8.3 % of the transgenic plants transformed with pRPVYCPN without MARs were highly resistant to PVYN, while 15.1% of the transgenic plants transformed with pRTM2CPNTM2 were highly resistant to PVYN infection. Southern blot analysis and PCR detection confirmed that all of the transgenic plants contained the expected cDNA fragments. Northern blot analysis showed that the untranslatable transcripts of PVYN-CP gene were hardly detected or present in low levels in all the virus-resistant plants, whereas the CP RNAs in the virus-susceptible plants were accumulated to higher levels than that in the virus-resistant plants, which indicated that the resistance of the resistant plants was RNA-mediated. The results indicated that MAR sequence could considerably increase the ratio of transgenic plants which were highly resistant to virus infection, implying that this strategy could be useful in managing plants for more highly effective RNA-mediated resistance to virus infection.