利用原生质体融合技术选育淀粉发酵产DHA的新型裂殖壶菌

Screening a New Type of Schizochxtrium Strain Producing DHA Using Starch by Protoplast Fusion Technology

以生产DHA的裂殖壶菌(Schizochxtrium)B4D1和黑曲霉(Aspergillus niger)CGMCC 3.316为出发菌株,利用原生质体融合技术选育可以利用淀粉发酵生产DHA的新型裂殖壶菌。用裂解酶制得了两亲本的原生质体,通过研究两亲本培养时间、培养方法、酶解时间等条件对原生质体产量影响的基础上,以PEG介导进行了原生质体的融合,最终确定了原生质体融合最佳条件为40%的PEG6000,融合温度30℃,融合时间为10 min,在此条件下融合率可达1.9%。通过比较菌落外观、颜色、形态以及分离培养筛选获得了一株利用淀粉裂殖壶菌融合子。经过RAPD验证表明B4D1与CGMCC 3.316发生了重组,融合菌株表达了更多源于B4D1的遗传信息。

Schizochxtrium B4D1 and Aspergillus niger CGMCC 3.316 as original strains, a new strain of Schizochxtrium was selected using starch for producing DHA. The protoplasts from two parents strains were obtained respectively with Lysing enzyme. The study was investigated on the effect of culture time, culture method, enzymolysis time on the protoplasts preparation. Finally, the protoplasts fusion was done under the PEG mediated condition. The optimum condition of protoplast fusion was as follows：PEG6000 concentration of 40%, fusion temperature of 30℃, fusion time of 10 min. Furthermore, the fusion rate reached 1.9%at the optimum condition. Through comparing its colony characteristics, color, morphology and purified spore culture, a new strain has been selected using starch as carbon source. RAPD verification showed that the fusant having integrated genome of B4D1 and CGMCC 3.316 expressed more genetic information from B4D1.