阿特拉津降解融合子的原生质体制备及其筛选

Preparation and Selection of Atrazine Degradation Fusants

以吉林省玉米带黑土中的优势真菌黑曲霉（A-02—10）为受体，阿特拉津降解菌青霉（P—02-07）的灭活原生质体为供体，对利用原生质体融合技术将降解基因导人土壤微生物优势种细胞，进而提高阿特拉津降解菌在土壤中定殖能力的可行性进行了初步研究。结果表明：（1）通过原生质体融合技术可显著提高阿特拉津降解菌在土壤中的定殖能力和降解效果，筛选出的融合子Al在自然土壤中的定殖数量可维持在6．4×10^6CFU·g^-1，使土壤中阿特拉津降解的半衰期由亲本（青霉P-02—07）的30d，缩短为10d。（2）供试的2个亲本具有相似的原生质体制备条件，即供试菌株的原生质体制备溶壁酶（Lywallzyme）浓度为2．0%-2．5％，最适酶解温度为30℃，酶解时间为5h，酶解液的pH值为6．0～6．5。此外，还讨论了单亲灭活原生质体融合技术在农药降解菌筛选中的独特技术优势——无需对菌株进行抗性标记，完全可以通过用农药配制的选择性培养基，依据菌落特征和农药溶解圈进行融合子的筛选，其中由农药配制而成的选择性培养基不仅可以用于降解融合子的定性筛选，而且还可以用于降解融合子在土壤中定殖数量的动态描述。原生质体融合技术仍是现阶段最为现实可行，最易广泛开展并收到实效的生物工程技术手段，通过原生质体融合进行基因体内重组构建具有降解优势基因工程菌的生物技术手段，应重新得到人们广泛关注。

Duo to its safety, high efficiency and lower cost, bioremediation technology has been regarded as an effective method to remediate the soils which polluted by pesticide and herbicide. But there are still some technological obstacles such as the relationship between the inoculums and indigenous microbes, soil type and other environmental factors ere, which hold-back to put it into practice. It is well known many bioremediation are failure due to the function microbial strains have not grown and multiplied in the polluted soils. The aim of this research is to investigate the possibility of making atrazine degradation genes recombination into soil dominated species, and then enhance the ability of the inoculums multiply and effect of atrazine degradation in soil. Single inactivate protoplasts technology was used, the parent strains for test were dominated species A spergillus niger（ A-02-10 ）in black soil and atrazine mineralization strain Penieillium sp（ P-02-07 ）, respectively. The proper conditions of protoplasts preparation were researched and a high efficiency fusant was selected. The results showed： （ 1 ）the two parents had the similar protoplast preparation conditions ： the enzyme concentration, enzymolysis temperature, enzymolysis time and pH value was 2.0%-2.5%, 30 ℃, 5 h, 6.0-6.5, respectively. （2）Making genetic recombination of atrazine degradation genes into soil dominated species by protoplast fusion could increase the ability of the atrazine degradation fusant multiply and effect of atrazine degradation in soil. The amounts of atrazine degradation fusant cells could keep on 6.4× 10^6 CFU· g^-1 in soil after inoculated 20 d; the half-decay period of atrazine in soil was decreased to 10 d from 30 d of the parent Penieillium sp P-02-07.