联合固氮菌GXGL-4A调控黄瓜根际氮循环及促生机理研究

In addition to biological nitrogen fixation, the nitrogen-fixing (NF) bacteria usually promote plant growth. Recently, the genetic modification and utilization of NF bacteria from non-leguminous plants aimed at improving the nitrogen fixation capacity and plant promoting effect has become a hot spot. The nitrogen content and morphological transformation rule, and the enzyme activities in the cucumber rhizosphere soil will be investigated by the inoculation of an associative NF bacterial strain Kosakonia radicincitans GXGL-4A on the rhizosphere of cucumber seedlings in this study. Meanwhile, a metagenomic analysis should be performed to monitor the composition of rhizosphere microbial community and the variation of colony abundance. After inoculation, the expression levels of nitrogen metabolism-related genes of GXGL-4A at different stages (early and late) will be compared by metatranscriptomic sequencing, and the key genes regulating nitrogen cycling in rhizosphere could be identified. A Tn5 insertional mutant library of GXGL-4A will be established to screen the mutants with a statistically significant absence or a significant improvement in plant growth-promoting effects on cucumber seedlings. In order to identify the functional genes closely associated with plant growth-promoting effect and the growth-promoting active substances, and further exploring the promotion mechanism of GXGL-4A on plant growth, the comparisons of differential transcriptome and mass spectrometry analysis of the growth promoting components in fermentation broth between the target mutants and the wild type strain GXGL-4A will be carried out. All these results are expected to lay a theoretical foundation for the study of the interaction between nitrogen-fixing bacteria and plants and the development of microbial fertilizer in the forthcoming future.

除生物固氮功能外，固氮菌通常还有促进植物生长的作用。近年来对非豆科植物来源的联合固氮菌进行固氮和促生功能的改造和利用成为热点。本研究以联合固氮菌Kosakonia radicincitans GXGL-4A为对象，接种黄瓜苗根际，考察其对根际土中氮素含量和形态转化及土壤酶活的影响；同时，采用宏基因组技术对根际微生物群落结构组成和丰度变化进行监测；利用宏转录组技术检测固氮菌施用后不同时期（早期和后期）土壤中GXGL-4A菌株的氮代谢相关基因的表达差异，明确调控根际氮循环的关键基因。建立GXGL-4A的Tn5转座突变株库，筛选对黄瓜苗促生作用统计学上显著缺失和显著提高的突变株，并与野生株GXGL-4A间进行差异转录组分析和发酵液促生成分的质谱分析比较，鉴定出与促生效应密切相关的功能基因和促生物质，解析该菌对植物的促生机制，为今后研究固氮菌与植物间的互作及微生物肥料开发奠定理论基础。

联合固氮菌GXGL-4A不仅能生物固氮，而且对黄瓜等植物有显著的促生作用。探究其施用对植物根际氮循环的影响及对植物的促生机理，可为深入理解固氮菌-植物间的互作及微生物肥料的开发奠定坚实的理论基础。项目组在完成固氮菌GXGL-4A全基因组测序基础上，采用链特异性转录组技术检测了该菌在限氮、富氮培养条件下氮代谢相关基因的转录差异，解析了固氮菌对氮素水平的响应规律；通过高渗培养的方法建立了固氮菌GXGL-4A高效转座突变技术体系，并构建了1个含有5000株以上的突变株库；采用CAS平板检测法筛选出2株高产嗜铁素突变株M81和M107，并克隆了插入位点基因trX和amt，灌根实验证实突变株对黄瓜幼苗有显著的促生作用。此外，筛选出1株丧失嗜铁素合成能力的突变株M246-2，确认突变基因为编码GMP合酶的guaA基因。通过构建缺失突变株及回补实验证明guaA基因正调控固氮菌GXGL-4A的嗜铁素合成；利用同源重组方法创制了铵载体基因amtB缺失突变株，发现其在无氮培养条件下泌铵能力和对黄瓜幼苗的促生作用较野生株均极显著提高。嗜铁素生物合成和泌铵能力在固氮菌对黄瓜的促生机制中占有重要地位。施用固氮菌可调控黄瓜根际土中总氮、硝态氮及氨态氮的含量，并通过显著改变土壤亚硝酸还原酶（S-NiR）、土壤过氧化物酶（S-POD）、土壤脱氢酶（S-DHA）和土壤蔗糖酶（S-SC）等的活性来影响黄瓜根际土中的碳氮循环利用。接种固氮菌可引起黄瓜根部部分硝酸盐转运蛋白基因nrt转录水平显著提高，促进黄瓜根中苯丙烷的合成，根系分泌物中有机酸种类增加，并检测到特有的与生长素合成有关的4-甲基吲哚及与植物抗菌有关的桉叶油素、2-正戊基呋喃、吡啶和1-烯丙基-1,2,4-三氮唑，这些差异成分有利于促进黄瓜植株的生长。16S多样性测序表明施用固氮菌可显著增加黄瓜根际土中菌群的多样性和丰度。