Cvitanich Cristina, Judelson Howard S
Department of Plant Pathology, University of California, Riverside, CA 92521, USA.
Curr Genet. 2003 Jan;42(4):228-35. doi: 10.1007/s00294-002-0354-3. Epub 2002 Dec 19.
Germinated asexual sporangia, zoospores, and mycelia of Phytophthora infestans were transformed to G418-resistance by microprojectile bombardment. After optimization, an average of 14 transformants/shot were obtained, using 10(6) germinated sporangia and gold particles coated with 1 microg of vector. Transformants displayed tandem or simple insertions of vector sequences within chromosomes. Most primary transformants were heterokaryons of transformed and wild-type nuclei, a state which generally persisted for generations, even with G418 selection. Transgenic homokaryons were easily obtained from primary transformants through G418 selection of zoospores. To facilitate the optimization of transformation, experiments were performed using a vector containing neomycin phosphotransferase (npt) and beta-glucuronidase (GUS) genes fused to oomycete transcriptional regulatory sequences. To indicate which orientations of transgenes would maximize their expression, head-to-head, head-to-tail, or tail-to-tail orientations of npt and GUS were compared. Each yielded similar rates of transformation and levels of GUS activity, indicating little transcriptional interference.
通过微粒轰击，将致病疫霉的无性孢子囊、游动孢子和菌丝体转化为对G418具有抗性。经过优化，使用10⁶个萌发的孢子囊和包被有1微克载体的金颗粒，平均每次轰击可获得14个转化体。转化体在染色体上显示出载体序列的串联或简单插入。大多数初级转化体是转化核与野生型核的异核体，即使在G418选择下，这种状态通常也会持续几代。通过对游动孢子进行G418选择，很容易从初级转化体中获得转基因同核体。为便于优化转化，使用了一种含有与卵菌转录调控序列融合的新霉素磷酸转移酶（npt）和β-葡萄糖醛酸酶（GUS）基因的载体进行实验。为了表明转基因的哪种方向能使表达最大化，比较了npt和GUS的头对头、头对尾或尾对尾方向。每种方向产生的转化率和GUS活性水平相似，表明转录干扰很小。
