基于代谢与转录组分析的银沙槐内生菌共生抗干旱促种子萌发协同调控

Ammodendron argenteum (Pall.) Kuntze is one of the most important plants for desert ecosystems for it’s distinctively characteristic on drought-resistance. But it becomes endangered in recent decades for difficult germination by its critical value of drought tolerance with -0.12 Mpa. Studies have show that osmotic stress is the dominant inhibiting factor for germination. Our previous study indicated that inoculated with cultures of endophytic bacteria isolated from A. argenteum could enhance the germination rate by 0.8 to 1.5 times higher than control groups under drought stress with 25% polyethylene glycol (PEG) 6000. But the gemination delayed for 3-5 days. Inoculated with separated bacterial cells or supernatant that containg metabolites indicating that some isolates plays the role by cells but some with metabolites. Lugol's dyeing has show that the seed starch has disappeared seriesly during the germination with cells inculation under drought stress. Indicating the seeds’ metabolic or gene expression must be regulated. So, to understand the mechanical of the symbiosis, how they participate or regulate the host metabolism and/or gene express was the most important work to do. For the pourpose to understand its regulation network, the gradually changes of enzymes related to germination, drought resistance related metabolites, and osmotic related small molecules would be estimated by classed analysis and LC-MS or GC-MS. Then, the germinated seeds with different period would be grinded as medium and the strains would be cultured with as “in vitro” metabolism. The metabolites will be analyzed and the cultures also inoculated for seed germination. The gene expression would be assessmented by transcriptome and the key genes that involved in coordinated regulation would be selected and identified by real – time PCR. The innovative of this work was the symbiotic regulation network would be assessmented by metabolism and transcriptome and the symbiotic regulation net would be constructed. The results would be helpful for our understanding the mechanical of the environment adaptation of the desert plant and the application of microorganisms on desert ecosystems protection and restoration.

荒漠优良固沙植物银沙槐耐旱性极强但已渐濒危,其种子萌发耐旱临界值-0.12 Mpa使萌发困难是致濒原因之一，而渗透胁迫是萌发主导抑制因子。我们研究证实25%PEG6000干旱胁迫下接种内生菌培养物可使种子萌发率比对照提高0.8到1.5倍，但伴随萌发延迟3-5天；将培养物分离为细胞和上清液后接种，证实一些菌株以细胞发挥作用而一些则以代谢产物缓解胁迫；将接种细胞的种子不同萌发阶段切片以卢哥氏染色发现其中淀粉质逐步消失。为揭示内生菌参与种子抗旱萌发的调控机制，项目对以细胞为作用的共生萌发进程中相关酶、主要抗逆成分及小分子产物进行检测；以不同萌发阶段种子磨浆接种内生菌检测其体外代谢产物并回接种子进行萌发试验;以转录组分析和关键基因表达定量探索基因协同调控并筛选关键基因。创新性在于以共代谢与基因协同调控研究银沙槐共生抗旱以揭示生物互作规律。研究成果对了解共生适应机制及微生物应用于生态修复提供科学依据

荒漠优良固沙先锋植物银沙槐耐旱性极强,但近年已渐濒危,其种子萌发耐旱临界值0.12 Mpa使萌发困难是致濒因子之一。前期研究证实，在25% PEG 6000干旱胁迫下，接种分离自银沙槐的内生菌菌株AER314-4可提高种子萌发率8倍以上且是以菌株进入种子发挥作用的。为揭示内生菌参与种子抗旱萌发的调控机制，本研究以Illumina对三个处理组（对照组（CK）、干旱胁迫组（DS）、加菌试验组（DE））种子4个萌发阶段共9个样品进行转录组测序，获得了约80G基因表达信息，经de novo组装整合获得104764个Unigenes，GO注释显示其与抗逆相关的基因主要集中于“分子功能”；KEGG分析显示共生抗旱代谢通路主要集中于氨基酸代谢；由PlantTFDB数据库预测得到455条转录因子，分布于48个转录因子家族。干旱响应基因8246个，其中687个与渗透调节物质相关；干旱抑制基因650个；萌发关键基因11445个，其中仅136个为已知功能基因；AER314-4调控响应基因27个，但功能未知。基因表达调控表现为或使被干旱胁迫抑制的基因恢复表达，或使沉默基因激活，且主要调控途径是氨基酸的代谢。.同时提取该9个样品的全部代谢产物，以GC-MS进行了代谢组检测，结果显示，接种AER314-4使分子量小于200的代谢组分比例增加，其中醛类和醇类在萌发初期大量积累，降低种子内渗透势而促萌发。经对拟体内代谢分析，接种AER314-4促进了正亮氨酸、酪氨酸、缬氨酸、苯丙氨酸的生物合成，而抑制了乳酸和琥珀酸的合成。对脂肪酸代谢途径的分析显示，接种AER314-4激活了丙二酰-[载体蛋白]合成油酸的代谢途径，而抑制了棕榈酸的合成。.研究结果揭示了内生菌AER314-4通过基因表达和代谢调控缓解了银沙槐种子的抗干旱萌发，且该途径是以氨基酸相关基因表达与代谢调控为主。研究成果对揭示共生调控机理有重要价值,并为微生物应用于珍稀植物种群保护及荒漠生态系统恢复提供科学依据。