哈茨木霉厚垣孢子产生突变体的筛选及T-DNA标签序列的克隆

利用PD液体培养基从哈茨木霉T-DNA插入突变体库中筛选出在产孢性状上与野生型菌株明显不同突变子7株,其突变表型主要表现在分生孢子产生数量显著减少和菌丝上有大量厚垣孢子分化.利用TAIL-PCR方法对7株突变体的侧翼序列进行克隆,从5个突变体中获取了5条T-DNA侧翼序列.为木霉菌厚垣孢子产生相关全长基因的克隆和产孢机制的研究奠定了基础.

Study on Screening of Chlamydospore Formation Mutants from T-DNA Insertional Mutant Library of Trichoderma harzianum and Cloning of T-DNA Tagging Sequence

7 mutants with chlamydospore formation differences were selected from 1 192 transformants cultured in PD liquid medium.The conidia formation ability of all these 7 mutants was significantly lower than that of the wild type T. harzianum .The characterization of these 7 mutants was analyzed.The results showed that as for the mutants 918,1 137, 1 317 chlamydospore developed in the mycelium and failed off from mycelium when incubated time prolonged .However, as for the other 4 mutants 627,840,1 137,1 173 the chlamydospore couldn't failed off from mycelium when incubated time prolonged. Using TAIL-PCR to clone T-DNA right border sequences from spore formation mutants, we successfully obtained the fungal genomic DNA sequences flanking T-DNA right border from 5 transformants. The homologous sequences of 3 sequences were found by using Blastn and Blastx from NCBI GenBank. However, the homologous sequences of other 2 sequences were not found. They might be new genes related to spore formation which need to be further studied. This study is the base for the cloning of chlamydospore related gene and the mechanism research of chlamydospore formation.