Hayes R J, Coutts R H, Buck K W
Department of Pure and Applied Biology, Imperial College of Science, Technology and Medicine, London, UK.
Nucleic Acids Res. 1989 Apr 11;17(7):2391-403. doi: 10.1093/nar/17.7.2391.
Bacterial beta-glucuronidase (gus) and neomycin phosphotransferase (neo) genes were introduced into coat protein replacement vectors based on DNA A of tomato golden mosaic virus (TGMV). Recombinant gus and neo vectors up to 1.1 kbp larger than DNA A were shown to replicate stably in transgenic plants containing partial dimers (master copies) of the vectors integrated into their chromosomal DNA in the absence of DNA B. Beta-glucuronidase and neomycin phosphotransferase activities in independently transformed plants were proportional to the copy number of the double-stranded forms of the vector. Deletion analysis has shown that an essential part of the TGMV coat protein promoter, including a TATA box, lies within 76 nt upstream of the initiation codon of the gene. An increase in expression of a neo gene was obtained by replacing this 76 nt sequence by an 800 nt sequence containing a cauliflower mosaic virus 35S RNA promoter with no effect on the ability of the vector to replicate or on its stability in transgenic plants. Systemic infection of plants by agroinoculation with TGMV vectors larger than DNA A in the presence of DNA B resulted in deletions in the vector DNA in some, but not all, plants. Possible reasons for vector instability in systemically infected plants, and vector stability in transgenic plants containing master copies of the vector, are discussed.
细菌β-葡萄糖醛酸酶（gus）和新霉素磷酸转移酶（neo）基因被导入基于番茄金色花叶病毒（TGMV）DNA A构建的外壳蛋白置换载体中。结果表明，比DNA A大1.1 kbp的重组gus和neo载体在不含DNA B且其染色体DNA中整合有载体部分二聚体（主拷贝）的转基因植物中能稳定复制。独立转化植物中的β-葡萄糖醛酸酶和新霉素磷酸转移酶活性与载体双链形式的拷贝数成正比。缺失分析表明，TGMV外壳蛋白启动子的一个重要部分，包括一个TATA框，位于该基因起始密码子上游76 nt范围内。通过用含有花椰菜花叶病毒35S RNA启动子的800 nt序列替换这76 nt序列，获得了neo基因表达的增加，且对载体的复制能力或其在转基因植物中的稳定性没有影响。在存在DNA B的情况下，用大于DNA A的TGMV载体进行农杆菌接种对植物进行系统感染，结果在部分（但不是全部）植物中导致载体DNA缺失。本文讨论了系统感染植物中载体不稳定以及含有载体主拷贝的转基因植物中载体稳定的可能原因。
