Baek J M, Kenerley C M
Department of Plant Pathology and Microbiology, Texas A&M University, College Station 77843, USA.
Fungal Genet Biol. 1998 Feb;23(1):34-44. doi: 10.1006/fgbi.1997.1025.
The arg2 gene which encodes the small subunit of carbamoyl phosphate synthetase for Trichoderma virens has been cloned and used to develop a homologous transformation system. A genomic clone containing the arg2 gene was isolated from a cosmid library of T. virens based on complementation of an arginine auxotrophic mutant of this fungus. The predicted amino acid sequence of the arg2 gene shows 56-82% identity with homologous polypeptides from other fungi. It also contains an upstream open reading frame which encodes 24 amino acids. As is observed with other gene sequences encoding this polypeptide in filamentous fungi, the N-terminus of the predicted polypeptide showed characteristic features of a mitochondrial signal sequence. The arg2 gene was used for genetic transformation of T. virens in frequencies of up to 370 transformants/microgram of DNA. Heat-shock treatment of T. virens protoplasts increased the transformation frequency by fivefold, but more than 85% of the transformants were abortive. Both single-copy, homologous integration events and ectopic, non-homologous integration events were detected by Southern analyses of genomic DNA from transformed strains.
编码绿色木霉氨甲酰磷酸合成酶小亚基的arg2基因已被克隆，并用于构建一个同源转化系统。基于该真菌精氨酸营养缺陷型突变体的互补作用，从绿色木霉的黏粒文库中分离出一个包含arg2基因的基因组克隆。arg2基因预测的氨基酸序列与其他真菌的同源多肽具有56%-82%的同一性。它还包含一个编码24个氨基酸的上游开放阅读框。正如在丝状真菌中其他编码该多肽的基因序列所观察到的那样，预测多肽的N端显示出线粒体信号序列的特征。arg2基因用于绿色木霉的遗传转化，转化频率高达每微克DNA 370个转化体。对绿色木霉原生质体进行热休克处理可使转化频率提高五倍，但超过85%的转化体是无效的。通过对转化菌株基因组DNA的Southern分析，检测到单拷贝同源整合事件和异位非同源整合事件。
