利用原生质体融合构建竞争结瘤能力强且耐酸性固氮菌

Using Protoplast Fusion to Construct a Nitrogen Fixing Bacteria with Strong Competitive Nodules and Acid-resistance

为获得固氮、竞争结瘤能力强且耐酸的功能菌株,促进新型多功能微生物肥料的研究与开发,以前期分离、鉴定获得的竞争结瘤能力强的大豆根瘤菌菌株YA-1和耐酸性菌株BQ-2为材料,按照功能互补的原则进行原生质体融合,针对融合子采用全氮比色法测定其固氮能力,利用BOX-PCR生物学技术评价其竞争结瘤能力,以GGE双标图的数学模型绘制出融合子和p H环境图标,进行融合子耐酸性状和稳定性的评价分析。结果表明:两供试菌株原生质体易于融合,连续传代10次后,共获得稳定融合子5株。固氮活性测定结果显示融合子YB-3生物固氮量最高,较出发菌株YA-1和BQ-2有显著的提高;融合子在竞争结瘤能力方面对两出发菌株进行了平衡,接种融合子YB-3菌株图谱与结瘤菌株图谱一致性较高,其占瘤率最高为95%,菌株竞争结瘤能力强于土著根瘤菌。GGE双标图分析结果表明,菌株耐酸性顺序为YB-2> YB-3> BQ-2> YB-4> YA-1> YB-5> YB-1,稳定性顺序为YB-5> YB-3> YB-1> YA-1> YB-2> YB-4> BQ-2,耐酸性强且稳定性较好的融合子菌株为YB-3。综合比较而言,获得了一株表现出双亲优良性状的集高固氮、竞争结瘤能力强且耐酸性的新型固氮菌YB-3。

In order to obtain the functional strains with strong nitrogen fixing, competitive nodules and acid resistance, promo- ting research and development of new multifunctional microbial fertilizer, this study used the YA-1 strain isolated and identifi- eated with strong competitive nodules and BQ-2 strain that have acid resistance as materials. Protoplast fusion was performed according to the principle of complementary, we determined the nitrogen fixation capacity of the fusion hybrid hy the total ni- trogen colorimetry, evaluated its ability to compete tumor formation by BOX-PCR evaluate, drew the fusion child and pH envi- ronment icon with the mathematical model of GGE biplot, evaluate the acid resistance and stability of the fusion seeds. Results showed that： The protoplast of the two strains was liable to fuse, and after 10 consecutive generations, 5 stable trains of fusion were obtained. The results of nitrogen fixation showed that the nitrogen fixation was the highest in the sub YB -3, which was significantly higher than that of the starting strain YA-1 and BQ-2. The fusion cells were balanced in terms of their ability to compete for nodule formation. The consistency between the bacterial map of the fusion strain YB-3 and the tumor-forming strain was higher, with the highest tumor rate 95%. The competition ability of the YB-3 strains was stronger than that of indigenous rhizobia. The result of GGE double plot analysis showed that the acid resistance sequence was YB-2 〉 YB-3 〉 BQ-2 〉 YB-4 〉 YA-1 〉 YB-5 〉 YB-1, and the stability sequence was YB-5 〉 YB-3 〉 YB-1 〉 YA-1 〉 YB-2 〉 YB-4 〉 BQ-2, and the fusion substrain with strong acid resistance and better stability was YB-3. In the comprehensive comparison,we obtained a new type of nitrogen fixing bacteria YB-3, with high nitrogen fixation, competitive nodule ability and acid resistance.