Burns C, Leach K M, Elliott T J, Challen M P, Foster G D, Bailey A
School of Biological Sciences, University of Bristol, Bristol BS8 1UG, UK.
Mol Biotechnol. 2006 Feb;32(2):129-38. doi: 10.1385/mb:32:2:129.
There is interest in establishing genetic modification technologies for the cultivated mushroom Agaricus bisporus, both for improved crop characteristics and for molecular pharming. For these methods to be successful, it is necessary to establish a set of transformation systems that include robust and reliable vectors for gene manipulation. In this article, we report the evaluation of a series of promoters for driving expression of the Escherichia coli hph gene encoding hygromycin phosphotransferase. This was achieved using the Aspergillus nidulans gpdA and the A. bisporus gpdII and trp2 promoters. The Coprinus cinereus beta-tubulin promoter gave contrasting results depending on the size of promoter used, with a 393-bp region being effective, whereas the longer 453-bp fragment failed to yield any hygromycin-resistant transformants. The C. cinereus trp1 and the A. bisporus lcc1 promoters both failed to yield transformants. We also show that transformation efficiency may be improved by careful selection of both appropriate Agrobacterium strains, with AGL-1 yielding more than LBA1126 and by the choice of the binary vectors used to mobilize the DNA, with pCAMBIA vectors appearing to be more efficient than either pBIN19- or pGREEN-based systems.
人们对为栽培蘑菇双孢蘑菇建立基因改造技术很感兴趣，这既有助于改善作物特性，也有助于分子制药。为使这些方法取得成功，有必要建立一套转化系统，其中包括用于基因操作的强大且可靠的载体。在本文中，我们报告了对一系列用于驱动编码潮霉素磷酸转移酶的大肠杆菌hph基因表达的启动子的评估。这是通过使用构巢曲霉gpdA以及双孢蘑菇gpdII和trp2启动子来实现的。灰盖鬼伞β-微管蛋白启动子根据所使用启动子的大小给出了不同的结果，一个393 bp的区域有效，而较长的453 bp片段未能产生任何潮霉素抗性转化体。灰盖鬼伞trp1和双孢蘑菇lcc1启动子均未能产生转化体。我们还表明，通过仔细选择合适的农杆菌菌株（AGL-1产生的转化体比LBA1126多）以及选择用于转移DNA的二元载体（pCAMBIA载体似乎比基于pBIN19或pGREEN的系统更有效），可以提高转化效率。
