固氮细菌DX120E的gfp标记及对不同甘蔗品种的接种效应

摘要

[Objective]The infection and colonization of nitrogen-fixing bacteria DX120E in sugarcane seedlings and the effects on the growth and physiology in different sugarcane varieties were studied to provide reference for further ana-lysis of the interaction mechanism between nitrogen-fixing bacteria and sugarcane.[Method]Sugarcane varieties ROC22, GT21 and B8 were used as test materials. After DX120E was labeled with gfp gene,sugarcane was inoculated by root-soaking method,and infection and colonization of DX120E in sugarcane were observed. The plant height,chlorophyll content and fresh weight of sugarcane were investigated at 60,90 and 120 d after inoculation,and the key enzyme activi-ties of nitrogen metabolism were measured.[Result]Stably expressed DX120E-gfp zygote strains was obtained. On day 7 after inoculation,nifH gene expression was detected in the roots and leaves of three sugarcane varieties,and the fragment size was approximately 360 bp. The results of fluorescence microscopy showed that on day 7 after inoculation,nitrogen-fixing bacteria was colonized in the root hair zone,the neonatal lateral roots,the root fracture wounds and the cross-sec- tions of the leaves of sugarcane. Pot experiment showed that compared with the control of inoculation with no nitrogen-fixing bacteria,the sugarcane inoculated with nitrogen-fixing bacteria DX120E could effectively promote the growth of the three varieties. And 120 d after inoculation,the plant heights of ROC22,B8 and GT21 respectively increased by 23%,20% and 14%;the fresh weight of aboveground increased by 25%,31% and 11% respectively;the fresh weight of under-ground part increased by 40%,34% and 30% respectively. The activity of glutamine synthetase and nitrate reductase in leaves of the three sugarcane varieties was also higher than that of the control.[Conclusion]By using the root-soaking method to inoculate sugarcane,the nitrogen-fixing bacteria strain DX120E can colonize the roots and transfer to the leaves of sugarcane. Using this method to treat sugarcane has obvious promoting effects on sugarcane,but there are some differences between different varieties.%[目的]研究固氮菌株DX120E在甘蔗幼苗中的侵染定殖及其对不同甘蔗品种生长生理的影响,为进一步分析该固氮菌与甘蔗的相互作用机制提供参考依据.[方法]以3个甘蔗品种ROC22、B8和GT21为试验材料,将DX120E用gfp基因标记后,以浸根法接种甘蔗,观察其在甘蔗中的侵染定殖情况,分别在接菌后60、90和120 d,调查甘蔗的株高、叶绿素含量及鲜重,并测定氮代谢关键酶活性指标.[结果]获得了稳定表达的DX120E-gfp接合子菌株;接种后第7 d ,在3个甘蔗品种的根部和叶片中均检测出nifH基因表达,片段大小约为360 bp.荧光显微镜观察结果表明,接种后第7 d,固氮菌定殖在甘蔗的根毛区、新生侧根处、根断裂伤口处及叶片横切处.盆栽试验结果表明,接种固氮菌DX120E对3个甘蔗品种有明显促生作用,与不接菌对照相比,接菌后120 d,ROC22、B8和GT21的株高分别增加23%、20%和14%,地上部分鲜重分别增加25%、31%和11%,地下部分鲜重分别增加40%、34%和30%.且3个甘蔗品种叶片的谷氨酰胺合成酶和硝酸还原酶活性总体上高于对照.[结论]采用菌液浸根法接种甘蔗,固氮菌DX120E能在甘蔗根部定殖并向地上部分转移定殖.采用该方法处理甘蔗,对甘蔗有明显的促生效应,但不同品种间存在一定差异.