摘要
重组克隆ＤＮＡ用ＫｐｎＩ／ＢａｍＨＩ双酶切得到马铃薯Ｙ病毒ＮＩｂ基因，将其插入质粒ｐＲＯＫ２相应切点中构成植物表达载体。用重组质粒ｐＲＯＫ２转化农杆菌（Ａｇｒｏｂａｃｔｅｒｉｕｍｔｕｍｅｆａ－ｃｉｅｎｓ）ＬＢＡ４４０４菌株，叶盘法将ＮＩｂ基因转入烤烟品种ＮＣ８９的染色体，获得抗卡那霉素的转化再生植株。经抗性筛选、ＰＣＲ检测、无性扩繁和大量重复抗病鉴定，结果表明，转化烟草植株ＤＮＡ中整合了外源目的基因，且表现抵抗２０μｇ／ｍｌＰＶＹＮ的侵染，ＥＬＩＳＡ分析认为抗性植株无病毒累积，初步筛选出对ＰＶＹＮ侵染具有较高抗性的转基因烟草植株。
The full length PVY N replicase gene(NIb gene)was cloned into plant expression vector pROK2 and the recombinanzed pROK2 was mobilized into Agrobacterium tumefaciens strain LBA 4404 by triparental mating.Leaf disks of Nicotiana tobacum cultivar NC89 were transformed with A.tumefaciens.Transgenic plants with resistance to the necrotic strain of PVY(PVY N) were obtained.Through screening plants for resistance to 20μg/me PVY N infection and polymerase chain reaction(PCR)detection,inoculation of 35 independent transgenic NC89 Ro plants identified 4 resistant plants,while inoculation of 50 controlled NC89 plants in none resistant,Indirect-ELISA also revealed that the 4 plants with resistance to 20μg/ml PVY N infection had no accumulation of PVY N.
出处
《中国烟草科学》
CSCD
1997年第3期23-26,共4页
Chinese Tobacco Science
基金
辽宁省烟草公司资助
