改良ATMT转化技术在深绿木霉基因敲除中的应用

Optimal Agrobacterium tumefaciens-Mediated Transformation Technology Applied in Gene Knockout for Trichoderma atroviride

木霉菌是环境中具有重要经济价值的丝状真菌之一。高效率的基因敲除技术是深入研究木霉菌功能的必要手段。研究改进了传统的农杆菌介导的遗传转化技术（ATMT）,成功构建深绿木霉T23中碳代谢抑制因子cre1基因敲除突变株。首先查找深绿木霉全基因组序列,比对并扩增cre1基因侧翼序列,以改造后的p1300qh质粒为骨架构建cre1敲除载体p C1300qh：cre1-up∷hyg∷cre1-down,转化到农杆菌AGL-1。通过优化ATMT转化中木霉菌分生孢子浓度,改良培养方式和延长诱导转化时间等参数,获得最佳转化条件：木霉菌分生孢子浓度为8×10~6,筛选培养基改为IM培养基,诱导转化时间延长,成功筛选到可能的转化子10个。最后,经鉴定有1个转化子为cre1敲除转化子,9个为T-DNA随机插入。因此,为深绿木霉菌基因功能研究提供了可借鉴的高效便捷的遗传转化方法。

Trichoderma. spp was one of most economic filament fungi in various environments. And the efficient gene knock out technologies are key ways to do intensive researches of gene functions in Trichoderma. spp. The aim is to optimize the Agrobacterium tumefaciens-mediated transformation （ATMT）, and successfully construct the crel gene knock out strain of Trichoderma atroviride. Firstly, the strain of T. atroviride 23 is adopted as original strain, analysis of genome sequence and confirmation DNA sequence of crel and its flanking sequence by combination of bioinformatics and molecular genetics operation strategy. Secondly, the flanking sequences of the upstream and downstream for crel are cloned and purified and then litigated into the plasmid pl300qh in sequence. Then, the knock-out vector pl3OOqhsilent-cre! is transform into Agrobacterium tumefaciens AGL-1. The spore concentration, culture mode and inducing period were optimizing. The optimal condition of ATMT is as follows ： concentration of conidia attains 8 × 10^6, IM medium the screening medium and increase of the period of induction. As a result, 8 tranformants which was much more than that by traditional methods were got. After verification, there were one crel gene knock out strain needed and seven tranformants of T-DNA random insertion. This contributes to an efficient, reliable and convenient method of ATMT for gene functional analysis in Trichoderma atroviride.