转Bt基因棉对AM真菌磷转运基因表达及对菌根吸收、转运磷的影响

The high-affinity phosphate (P) transporter of arbuscular mycorrhizal (AM) fungi can significantly improve the uptake of P by the host plants. The gene expression of P transporter is induced by the symbiotic processes with plant roots. However, the symbiotic relationship is sensitive to the plant gene structures and insecides. Transgenic Bacillus thuringiensis (Bt) plants not only have the different gene structures with the wide type, but also release insecticidal proteins to the soil, which has the effect on the symbiotic relationship of AM fungi with their hosts. Thus the effect on gene expression of P transporter is highly worthy of attention. This project plans to study the effect of transgenic Bt cotton and Bt insecticidal protein on the symbiotic character and P transporter gene expression of AM fungi. During the research, Ri T-DNA-transformed cotton roots inoculated by AM fungi spores will be grown in Petri plates with two compartments. Bt insecticidal protein will be added to the culture medium. The Bt and non-Bt cotton will be planted in pots, inoculated by AM fungi or not. Quantitative real-time PCR analyses, direct fluorescent in situ RT-PCR and confocal laser scanning microscopy will be used to examine phosphate transporter gene expression profiles in extraradical myceliun and mycorrhizal roots.33P and energy dispersive X-ray microanalysis will be used to examine the uptake and transport of P from extraradical mycelium to mycorrhizal roots in the mycorrhizal root systems. The effect of gene expression level on the uptake and transport of P in the AM symbiosis will be discussed. The results can elucidate the mechanism of how transgenic Bt cotton impact on the AM symbiosis efficiency. The research will be very important for defining of the impact of transgenic Bt cotton on the ecological functions of AM fungi. It will provide scientific information for assessing environmental risks of transgenic Bt plants.

AM真菌与植物共生形成菌根，AM真菌磷转运基因所编码的高亲和性磷转运蛋白显著促进植物对磷的吸收，磷转运基因的表达受AM真菌与植物共生过程诱导，共生过程与特性对植物基因型和杀虫剂敏感。Bt基因导入植物不仅改变植物基因组成，还引起杀虫蛋白在土壤中积累，影响AM真菌与植物共生特性，对磷转运基因表达的影响值得关注。本项目拟采用Ri T-DNA棉花毛状根-AM真菌隔室双重培养与盆栽Bt棉方法，应用实时荧光定量RT-PCR、荧光原位RT-PCR与激光共聚焦显微镜等技术，研究Bt棉/Bt蛋白对AM真菌共生特性及对磷转运基因在菌丝与根内共生体中表达的影响；应用同位素标记与能量散射X线谱分析系统，研究磷在菌丝、（菌）根中的吸收、转运特征，探明磷转运基因表达水平与菌根体系磷的吸收、转运的关系，揭示Bt基因导入影响棉花-AM真菌共生系统磷吸收的分子机制。研究结果对阐明Bt棉对AM真菌共生效应的影响具有重要意义。

近年来，转Bt基因棉在我国得到大面积推广，2015年种植面积达370万公顷，占棉花总种植面积的96%。在Bt棉生长期间，Bt蛋白可通过根系分泌物、花粉飘落及植物残体进入土壤生态系统，进入土壤的Bt蛋白很快与土壤粘土矿物及腐殖质等结合，在土壤中可保持几个月的生物活性。Bt棉对土壤微生物生长、群落结构及生理生态功能的影响备受关注。丛枝菌根（AM）真菌是土壤中广泛存在的一类功能性微生物，能与80%以上的陆生植物品种形成共生关系，促进宿主对水分及营养元素，特别是磷的吸收。AM真菌还可提高宿主植物抗性，促进土壤团聚体形成，在土壤生态系统中具有重要的作用。本项目以AM真菌为研究对象，盆栽和大田种植条件下，采用激光共聚焦显微镜技术，实时荧光定量PCR技术及生物信息学分析技术，探讨了Bt棉对AM真菌发育、共生特性与种群结构的影响，并进一步探讨了Bt棉对AM真菌功能基因表达及关键酶活性的影响，揭示Bt棉对AM真菌的影响机制。结果表明，Bt蛋白和Bt棉根际物质对AM真菌孢子萌发、菌丝分化有显著抑制作用；Bt棉降低了AM真菌的侵染性，根段侵染率与丛枝丰度下降，Bt棉根细胞内丛枝结构降解；Bt棉叶片腐熟物显著抑制AM真菌在紫云英根部定殖，根段侵染率及丛枝丰度下降，AM真菌关键酶活性降低，菌丝中多聚磷酸盐相对含量、紫云英地上地下部分磷吸收量显著下降；与常规棉相比，大田种植条件下Bt棉根中AM真菌种群结构不同，Bt棉改变了丰度较大的几个OTU的丰度；基因检测显示Bt棉降低了AM真菌共生相关基因GintMST2，磷转运蛋白基因GintPT及碱性磷酸酶基因GintALP表达，共生体中碱性磷酸酶、酸性磷酸酶及脲酶活性下降。本研究结果表明Bt棉抑制了AM真菌发育与共生活性，影响了AM真菌的种群结构，降低了菌根对磷的吸收，这可能与AM真菌功能基因表达及关键酶活性受抑制有关，该结果可为Bt作物环境风险评价提供理论参考。