Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping
Strategies for development of functionally equivalent promoters with minimum sequence homology for transgene expression in plants: cis-elements in a novel DNA context versus domain swapping
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DOI：
10.1104/pp.103.020602
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发表时间：
2003-06-01
期刊：
影响因子：
7.4
通讯作者：
Burma, PK
中科院分区：
文献类型：
--
作者：
Bhullar, S;Chakravarthy, S;Burma, PK
花椰菜花叶病毒35 S（35 S）启动子已广泛用于双子叶植物中转基因的组成型表达。已知相同启动子的重复使用由于启动子同源性而诱导转基因失活。作为规避该问题的一种方式，我们测试了两种不同的策略用于开发功能等同但具有最小序列同源性的合成启动子。这样的启动子可以通过（a）在新的或合成的DNA片段中引入已知的顺式元件或（B）“结构域交换”来产生，其中一个启动子的结构域可以用来自其他异源启动子的功能等同结构域替换。我们评估了两种策略的启动子修饰使用域A（由最小的启动子和亚域A1）的35 S启动子作为模型。开发了一组经修饰的35 S启动子，使用β-葡萄糖醛酸酶作为报告基因将其强度与35 S启动子本身进行比较。在瞬时检测系统中对报告基因的表达分析表明，结构域交换导致启动子活性的显着下降。相比之下，通过将顺式元件置于新的DNA环境中开发的启动子显示出与35 S相当的表达水平。然后通过将最小启动子和亚结构域A1的核心序列置于不同的DNA序列中来设计两个启动子构建体Mod 2A 1 T和Mod 3A 1 T。用这两种构建体和作为对照的35 S在烟草（Nicotiana tabacum）中开发的转基因被用于评估启动子在初级转化体的不同组织中的活性。发现Mod 2A 1 T和Mod 3A 1 T在所有测试的组织中具有活性，其水平与35 S相当。此外，Mod 2A 1 T启动子在T-1代幼苗中的表达也与35 S启动子相似。本发明的策略打开了通过修饰顺式元件之间存在的序列以用于植物中的转基因表达来创建具有最小序列同源性并且表达水平与野生型原型相当的一组合成启动子的可能性。
The cauliflower mosaic virus 35S (35S) promoter has been extensively used for the constitutive expression of transgenes in dicotyledonous plants. The repetitive use of the same promoter is known to induce transgene inactivation due to promoter homology. As a way to circumvent this problem, we tested two different strategies for the development of synthetic Promoters that are functionally equivalent but have a minimum sequence homology. Such promoters can be generated by (a) introducing known cis-elements in a novel or synthetic stretch of DNA or (b) "domain swapping," wherein domains of one promoter can be replaced with functionally equivalent domains from other heterologous promoters. We evaluated the two strategies for promoter modifications using domain A (consisting of minimal promoter and subdomain A1) of the 35S promoter as a model. A set of modified 35S promoters were developed whose strength was compared with the 35S promoter per se using beta-glucuronidase as the reporter gene. Analysis of the expression of the reporter gene in transient assay system showed that domain swapping led to a significant fall in promoter activity. In contrast, promoters developed by placing cis-elements in a novel DNA context showed levels of expression comparable with that of the 35S. Two promoter constructs Mod2A1T and Mod3A1T were then designed by placing the core sequences of minimal promoter and subdomain A1 in divergent DNA sequences. Transgenics developed in tobacco (Nicotiana tabacum) with the two constructs and with 35S as control were used to assess the promoter activity in different tissues of primary transformants. Mod2A1T and Mod3A1T were found to be active in all of the tissues tested, at levels comparable with that of 35S. Further, the expression of the Mod2A1T promoter in the seedlings of the T-1 generation was also similar to that of the 35S promoter. The present strategy opens up the possibility of creating a set of synthetic promoters with minimum sequence homology and with expression levels comparable with the wild-type prototype by modifying sequences present between cis-elements for transgene expression in plants.