根癌农杆菌介导深绿木霉（Trichoderma atroviride）T23转化体系的建立以及高效降解敌敌畏突变株筛选与应用研究
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摘要
本研究成功建立了高效的农杆菌介导转化深绿木霉(Trichoderma atroviride)T23转化体系,转化效率可达50个转化子/107个孢子。从110株稳定遗传的突变株中筛选到7株敌敌畏降解率显著变化的突变子。其中两株(AMT-12和AMT-28)敌敌畏降解率较出发菌T23提高10%以上,生物学分析发现其产孢能力明显下降,但其对敌敌畏的耐受性与T23基本一致。另外,还有5株突变子AMT-18、AMT-25、AMT-44、AMT-64和AMT-92的降解率较T23降低70%,但产孢能力保持不变,它们对敌敌畏的耐受能力是T23的4倍(800μg/mL)。Southern杂交检测证实AMT-12和AMT-28是T-DNA单拷贝插入突变子,而另外5株降解率下降的突变子为T-DNA多拷贝插入。我们克隆了突变株AMT-12和AMT-28的侧翼序列,其长度分别为1845bp和1730bp。经BLAST分析发现序列与深绿木霉测序菌株IMI206040分别具有89%和88%的相似性。但是,这2个侧翼序列所在的基因与降解敌敌畏的关系还需深入研究。
为了深入研究木霉在处理敌敌畏废水中的作用,试验以突变株AMT-28为例,采用海藻酸钙滴珠法建立了其孢子固定化技术体系,并探讨了深绿木霉菌生物降解敌敌畏的可能机制。在纯培养条件下,以海藻酸钙固定107个孢子(107孢子/100mL的3%海藻酸钠悬液)降解敌敌畏的效果最好。与固定化菌丝和非固定化菌丝相比,固定化孢子对敌敌畏的降解率有显著性提高。在重复利用实验中,固定化孢子和固定化菌丝小球在5个连续批次测试中显示了良好的降解能力,且随着批次增加,固定化孢子对敌敌畏的降解率有一定程度的提高。固定化菌丝小球在储存一个月后,其降解能力大大下降,而固定化孢子非但没有下降,还有某种程度的提高。为了监测菌丝体对敌敌畏的降解情况,实验中采用了HPLC法分析了突变株AMT-28的发酵液,确认AMT-28能够在7天内完全降解敌敌畏。同时,利用缺陷培养基纯培养发现,AMT-28能够在无外加碳源或者磷源的情况下降解敌敌畏,氮元素对AMT-28降解敌敌畏的影响很大。解吸附实验证实敌敌畏的减少是由于木霉的生物降解而非吸附作用。酶定位研究结果提示深绿木霉菌T23中存在诱导型胞内敌敌畏降解酶。实验末期降解液的pH与敌敌畏添加初始浓度成正相关,由此推测木霉降解敌敌畏是通过生物矿化作用完成的。
A protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of biocontrol fungus Trichoderma atroviride strain T23 was developed to construct mutants with improved dichlorvos-degradation ability. A transformation frequency of 5×10-6 was achieved. Among 110 genetically stable T-DNA transformants of T. atroviride T23, two transformants, AMT-12 and AMT-28, confirmed by Southern blot analysis to have single-copy inserts of T-DNA, showed an increase in dichlorvos-degradation ability of more than 10% compared to that of the wild type, exhibited similar tolerance to the pesticide, but lower spore formation ability. Five transformants exhibited a reduction in degradation of more than 70%, exhibited wild-type spore formation, and tolerated up to 800μg/mL of dichlorvos. The left-flanking sequence of the insertion site in AMT-12 and AMT-28 were cloned as a 1845-bp and 1730-bp fragments and shown to have 89% and 88% identity to the DNA from T. atroviride IMI 206040, respectively; however, the involvement of these DNA fragments in dichlorvos degradation remains still to be determined. This study can promote both a more efficient isolation of DNA sequence flanking T-DNA integration site in T. atroviride mutants and a more rational utilization of these transformants in dichlorvos degradation.
An immobilizing conidia approach was successfully established to study the degradation ability of dichlorvos in AMT-28, and the biodegradation mechanism of DDVP in this fungus was also investigated. The beads immobilized 10'conidia per 100 mL of Na-alginate solution exhibited the highest degradation rate compared to that of 105 and 109 conidia under the experimental conditions. The immobilized AMT-28 conidia showed improved degradation abilities than immobilized or free mycelia. The beads immobilized cells (conidia and mycelia) of AMT-28 kept good storage stability and reutilization capacity, the degradation abilities of them did not decrease, but there were somewhat degree of increase in five bathes of samples through one-month determination test. The dichlorvos in Burk medium with mycelia of AMT-28 was confirmed to be completely removed using HPLC analysis. The dichlorvos degradation in auxotrophic Burk media (designated as N-, P- and C-, respectively) varied with different nitrogen, phosphorus and carbon sources, and it was found to be dramatically affected by nitrogen sources. Dichlorvos could be adopted as a sole carbon or phosphorus source of AMT-28. Meanwhile, The results revealed that the major reason for dichlorvos removal in AMT-28 should be attributed to the fungal biodegradation and there was no detectable biosorption in this study. Inducible intracellular degrading enzyme of dichlorvos could be promoted by a small amount of dichlorvos at the initial stage of this organophosphorus compound stress. Overall, the dichlorvos degradation in AMT-28 was likely to be a kind of Biomineralization process.
为了深入研究木霉在处理敌敌畏废水中的作用,试验以突变株AMT-28为例,采用海藻酸钙滴珠法建立了其孢子固定化技术体系,并探讨了深绿木霉菌生物降解敌敌畏的可能机制。在纯培养条件下,以海藻酸钙固定107个孢子(107孢子/100mL的3%海藻酸钠悬液)降解敌敌畏的效果最好。与固定化菌丝和非固定化菌丝相比,固定化孢子对敌敌畏的降解率有显著性提高。在重复利用实验中,固定化孢子和固定化菌丝小球在5个连续批次测试中显示了良好的降解能力,且随着批次增加,固定化孢子对敌敌畏的降解率有一定程度的提高。固定化菌丝小球在储存一个月后,其降解能力大大下降,而固定化孢子非但没有下降,还有某种程度的提高。为了监测菌丝体对敌敌畏的降解情况,实验中采用了HPLC法分析了突变株AMT-28的发酵液,确认AMT-28能够在7天内完全降解敌敌畏。同时,利用缺陷培养基纯培养发现,AMT-28能够在无外加碳源或者磷源的情况下降解敌敌畏,氮元素对AMT-28降解敌敌畏的影响很大。解吸附实验证实敌敌畏的减少是由于木霉的生物降解而非吸附作用。酶定位研究结果提示深绿木霉菌T23中存在诱导型胞内敌敌畏降解酶。实验末期降解液的pH与敌敌畏添加初始浓度成正相关,由此推测木霉降解敌敌畏是通过生物矿化作用完成的。
A protocol for efficient Agrobacterium tumefaciens-mediated transformation (ATMT) of biocontrol fungus Trichoderma atroviride strain T23 was developed to construct mutants with improved dichlorvos-degradation ability. A transformation frequency of 5×10-6 was achieved. Among 110 genetically stable T-DNA transformants of T. atroviride T23, two transformants, AMT-12 and AMT-28, confirmed by Southern blot analysis to have single-copy inserts of T-DNA, showed an increase in dichlorvos-degradation ability of more than 10% compared to that of the wild type, exhibited similar tolerance to the pesticide, but lower spore formation ability. Five transformants exhibited a reduction in degradation of more than 70%, exhibited wild-type spore formation, and tolerated up to 800μg/mL of dichlorvos. The left-flanking sequence of the insertion site in AMT-12 and AMT-28 were cloned as a 1845-bp and 1730-bp fragments and shown to have 89% and 88% identity to the DNA from T. atroviride IMI 206040, respectively; however, the involvement of these DNA fragments in dichlorvos degradation remains still to be determined. This study can promote both a more efficient isolation of DNA sequence flanking T-DNA integration site in T. atroviride mutants and a more rational utilization of these transformants in dichlorvos degradation.
An immobilizing conidia approach was successfully established to study the degradation ability of dichlorvos in AMT-28, and the biodegradation mechanism of DDVP in this fungus was also investigated. The beads immobilized 10'conidia per 100 mL of Na-alginate solution exhibited the highest degradation rate compared to that of 105 and 109 conidia under the experimental conditions. The immobilized AMT-28 conidia showed improved degradation abilities than immobilized or free mycelia. The beads immobilized cells (conidia and mycelia) of AMT-28 kept good storage stability and reutilization capacity, the degradation abilities of them did not decrease, but there were somewhat degree of increase in five bathes of samples through one-month determination test. The dichlorvos in Burk medium with mycelia of AMT-28 was confirmed to be completely removed using HPLC analysis. The dichlorvos degradation in auxotrophic Burk media (designated as N-, P- and C-, respectively) varied with different nitrogen, phosphorus and carbon sources, and it was found to be dramatically affected by nitrogen sources. Dichlorvos could be adopted as a sole carbon or phosphorus source of AMT-28. Meanwhile, The results revealed that the major reason for dichlorvos removal in AMT-28 should be attributed to the fungal biodegradation and there was no detectable biosorption in this study. Inducible intracellular degrading enzyme of dichlorvos could be promoted by a small amount of dichlorvos at the initial stage of this organophosphorus compound stress. Overall, the dichlorvos degradation in AMT-28 was likely to be a kind of Biomineralization process.
引文
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