具ACC脱氨酶的内生固氮菌提高甘蔗抗旱性的生理及分子基础研究

To increase the nitrogen and water utilization efficiency and reduce the farming costs are the keys to ensure the sustainable development of sugarcane industry in China. The ACC deaminase containing endophytic diazotrophs as the precious resources with the enormous potential of growth promotion and stresses tolerance improvement when inoculated with sugarcane haven’t been totally developed yet. Here, we select the nitrogen-fixing bacteria Burkholderia sp strain GXS16 which with high growth promotion and ACC deaminase ability, planning to investigate the interaction between the strain and roots of sugarcane variety ROC16 under water stress condition. We will construct an acdS mutant strain and demonstrate whether the ACC deaminase plays a role in assistance to sugarcane cultivars to tolerate drought stress by moderate the endogenous ethylene synthesis, we will label strain GXS16 with constitutively expressed gfp, and use GFP fluorescence, 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 between stress treated and untreated sugarcane roots, and the relationship between this kind of difference and drought tolerance in sugarcane. Through evaluate the changes of physiological and biochemical parameters when inoculated with GXS16 under water stress, combined with the transcriptome data mining, we will find out the key genes which take part in stresses tolerance improvement pathway regulated by ACC deaminase of GXS16 strain. This study will provide bacterial resources and application evidence to increase infertility- and drought-tolerance for sugarcane cultivars in China.

具有ACC脱氨酶的内生固氮菌是尚未开发的可能帮助甘蔗减肥增产、耐旱耐瘠的宝贵资源，通过接种此类甘蔗内生固氮菌有可能同时达到增产增质和增强抗逆性的目的。本项目拟选用对甘蔗生长具有优异促生作用并具备高ACC脱氨酶活性的固氮Burkholderia sp细菌GXS16，以水分胁迫下GXS16与不耐旱甘蔗品种ROC16根系的互作为主要研究内容，构建GXS16的ACC脱氨酶突变体，验证GXS16通过ACC脱氨酶调节内源乙烯含量来提高甘蔗抗旱性；用GFP荧光及qRT-PCR技术监测菌株侵染及定殖进程得到其在甘蔗根系的定殖组织模式并分析与甘蔗抗旱性关系；测定水分胁迫下接种GXS16对甘蔗相关生理生化代谢的影响，结合转录组测序挖掘乙烯从合成到信号转导及下游响应基因的表达变化，找出ACC脱氨酶调控乙烯介导的固氮菌提高甘蔗抗旱性的关键基因，分析基因间代谢通路关系，为利用内生固氮菌提高甘蔗抗旱水平提供基础信息。

接种具有ACC脱氨酶活性的内生固氮菌可能同时实现甘蔗生产增产增质和增强抗逆性的目的。本项目研究具ACC脱氨酶活性的伯克氏固氮细菌GXS16促进甘蔗生长及提高抗旱性的机理，取得结果如下：. 1. 菌株鉴定表明GXS16具有高固氮酶和ACC脱氨酶活性；具备合成IAA、分泌嗜铁素、溶磷作用；对烟草和洋葱鳞茎没有致病作用；菌株可在根冠、根毛、根分生组织、叶片中定殖，数量随侵染时间增加；接种GXS16能促进甘蔗生长，干重和氮含量分别提高32.6%和23.4%，接种60天后固氮百分率为6.87%。. 2. 通过插入失活构建GXS16的ACC脱氨酶突变体菌株acd S。水分及盐胁迫下，接种acd S的甘蔗株高、根长、根鲜重、根干重显著低于野生株型，表明acd S菌株丧失分泌ACC脱氨酶的能力，不能缓解过量乙烯对甘蔗生长的抑制。此外，水分胁迫下，接种GXS16比接种突变体株acd S具有更快速的渗透调节物质（proline）积累能力，更优良的植物激素（ABA、SA、IAA、GA3）代谢水平，及更强的抗氧化防护系统（CAT、SOD、APX、GR）活性，因此比接种突变体株acd S显示出更强的耐受水分胁迫能力。. 3. 菌量qRT-PCR结果显示，水分胁迫加剧了菌株在甘蔗根系的定殖，并随胁迫时间不断增加。接种GXS16加水分胁迫的差异表达基因数量远高于水分胁迫处理。从不同处理样品中筛选到乙烯生物合成及信号转导相关差异表达基因共201个。其中乙烯生物合成关键酶ACS、ACO基因46个，乙烯信号传导途径包括乙烯受体（ETR）、信号转导（CTR1、SIMKK、MPK6、EIN2）、关键转录因子（EIN3、EBF、ERF）基因共155 个，上述基因整体以下调表达为主，可能与水分胁迫下甘蔗降低乙烯生物合成，以减轻过量乙烯对植株生长的抑制有关。