伯克氏固氮菌GXS16与甘蔗根系高效联合固氮的生理和分子基础研究

Some sugarcane germplasms are able to obtain high levels of nitrogen via N2-fixation by associated N2-fixing bacteria, but its therapeutic mechanism remains obscure. The roots are primary tissue for N2-fixation, and in practice, by most of the inoculation methods, endophytic diazotrophs will insert through the sugarcane roots and it will colonize inside the plant and fix the nitrogen. We have isolated a Burkholderia sp strain GXS16 from the wild Saccharum spontaneum L. plant, which is able to fix N2 in free-living states, but growth promoting abilities of GXS16 are different with the different sugarcane germplasms. Here, we plan to reveal physiological and molecular basis of high nitrogen fixation efficiency of GXS16 associated with roots of sugarcane, by using Guangxi lower nitrogen-fixing cultivar ROC22 and a Brazilian high nitrogen-fixing cultivar RB86-7515. In the first phase, we will introduce strain GXS16 into sugarcane plants by culturing micro propagated sugarcane plantlets and we will determine the rate of N2-fixation by using the 15N isotope dilution method in the pot. In the second phase, we will label strain GXS16 with constitutively expressed gfp or gfp regulated by nifH promoter, and use GFP fluorescence, immune-gold labeling and qRT-PCR technologies, along with confocal microscopy and transmission electron microscopy observation, to identify the root infected position, infected process, strain quantity or gene expression, in order to find out difference of bacterial localization patterns, organization and nifH gene expression frequencies of both sugarcane genotypes. We will also analyze the transcriptome sequences from the insertion to optimal colonization, and compare the differences between genotypes to find out the target genes for optimal nitrogen fixation ability. This study will provide valuable information for sugarcane genotype selection with the efficient nitrogen fixation ability or utilization of diazotrophs in order to improve the nitrogen fixation efficiency in the targeted sugarcane genotypes as well as new varieties.

某些甘蔗种质能与固氮菌进行高效联合固氮，但其机理尚不清楚。根系是甘蔗生物固氮的首要场所，不同接种方式下固氮菌主要通过根系进入甘蔗体内进行固氮。我们从野生割手密体内分离到伯克氏固氮细菌GXS16，初步鉴定了其对甘蔗根系促生的优异性和品种差异性。本项目研究GXS16与低固氮甘蔗品种ROC22和高固氮品种RB86-7515联合固氮效率差异的生理和分子基础。用GXS16接种生根期组培苗，15N同位素稀释法检测根系的联合固氮水平；用组成型表达gfp和nifH-gfp标记GXS16，结合GFP荧光、免疫胶体金、qRT-PCR、共聚焦显微镜和透射电镜技术，定位或定量指示菌株在根系内的定殖组织模式和固氮功能表达模式差异；通过转录组测序挖掘GXS16与不同基因型甘蔗根系实现联合固氮过程中基因表达种类和丰度变化差异，揭示固氮菌与甘蔗根系高效联合固氮的生理和分子基础，为实现甘蔗与固氮菌高效联合固氮提供基础信息。

不同甘蔗品种间的联合固氮效率差异显著，本项目研究伯克氏固氮菌GXS16与不同甘蔗品种联合固氮效率差异的生理和分子基础。取得结果如下：.1. GXS16对甘蔗品种B8的促生效应和联合固氮效率优于ROC22。接种后培养60 d，B8根系、地上部分和全株干重分别提高46.9%、33.2%和37.6%，ROC22只提高32.1%、26.2%和27.9%，B8不同部位氮含量分别提高32.7%、23.8%和24.9%，ROC22只提高19.5%、18.5%和18.7%，差异显著。B8从空气中获得氮的百分率也显著高于ROC22。.2. GXS16在B8和ROC22中的定殖模式大致相同。两品种与GXS16共培养12 h，单个或成群的GXS16可定殖在根成熟区的根毛、根冠或边缘细胞上；接种24 h，GXS16定殖数量显著增加，显示聚集的趋势；接种48 h，GXS16大量聚集包围根毛，在成熟区表面、侧根发生处、伸长区、根皮层细胞间隙形成生物薄膜。接种96 h，GXS16在根部的定殖模式与48 h时相同。.3. 接种GXS16对不同甘蔗品种根系生理生化作用的影响，结果表明根系中菌株数量随接种时间延长而增加，但B8根系菌株数量显著高于ROC22。接种后第1 d，B8中H2O2和MDA含量显著高于对照，整个处理期间Pro含量无显著变化，乙烯含量呈先升高后下降趋势。ROC22根系H2O2、MDA、Pro、乙烯含量在不同接种时期都显著高于对照。接种后第1、第3、第5 d，B8根系ABA含量分别比对照提高91.9%、43.9%和18.7%，与此相反，GA3含量比对照降低12.9%、28.5%和45.2%，ROC22根系ABA和GA3含量变化趋势与B8类似。接种GXS16后不同的时点，B8根系抗氧化酶中CAT、SOD、PPO、APX、GR活性显著提高，ROC22根系的抗氧化酶活性弱于品种B8。.4. 从接种后的第1、第3和第5 d的甘蔗品种B8根系分别筛选到2437、6678和4586个差异表达基因，其中不同处理期共有的差异表达基因601个，GO和KEGG显示差异表达基因代谢途径具有多样性。参与ABA、GA和乙烯代谢通路的差异表达基因分别有17、42和75个，与乙烯相关的差异表达基因数量及其表达量的多样性远高于其它两种植物激素，表明乙烯在调节B8甘蔗根系响应GXS16侵染定殖的进程中具有重要作用。