Dev Kamal, Maheshwari Ramesh
Department of Biochemistry, Indian Institute of Science, Bangalore 560012, India.
Curr Microbiol. 2003 Sep;47(3):220-5. doi: 10.1007/s00284-002-3991-3.
We transformed wild-type Neurospora crassa with hph gene encoding hygromycin phosphotransferase to obtain hygromycin-resistant (HygR) transformants and studied their behavior in the vegetative and sexual phases of growth. During vegetative growth in the absence of hygromycin, the hph gene was stable for at least three successive transfers with conidia. On the other hand, the behavior of the transformants in the sexual phase was different. The segregation of hph gene in the meiotic progeny was in accordance with the Mendelian ratio as inferred from PCR analysis. However, in spite of inheriting the hph gene, a proportion of the meiotic progeny failed to grow in the presence of hygromycin. This suggested that the hph gene is silenced in some progeny. The silencing effect was not confined to hph gene expression, since one-half of the meiotic progeny also showed poor conidiation. Genomic Southern analysis indicated deletions/rearrangements of the transgene in the progeny. A heterokaryon between silenced and non-silenced strains was able to grow on hygromycin-containing medium, showing that silencing was recessive. Silencing was reversed in homokaryotic nuclei extracted from such heterokaryon.
我们用编码潮霉素磷酸转移酶的hph基因转化野生型粗糙脉孢菌，以获得抗潮霉素（HygR）转化体，并研究它们在营养生长和有性生长阶段的行为。在无潮霉素条件下进行营养生长时，hph基因在至少连续三代分生孢子转接中保持稳定。另一方面，转化体在有性阶段的行为有所不同。从PCR分析推断，减数分裂后代中hph基因的分离符合孟德尔比例。然而，尽管继承了hph基因，但一部分减数分裂后代在潮霉素存在的情况下无法生长。这表明hph基因在一些后代中被沉默。沉默效应不仅限于hph基因表达，因为一半的减数分裂后代也表现出分生孢子形成不良。基因组Southern分析表明后代中存在转基因的缺失/重排。沉默菌株和非沉默菌株之间的异核体能够在含潮霉素的培养基上生长，表明沉默是隐性的。从这种异核体中提取的同核体细胞核中的沉默被逆转。
