银沙槐内生菌抗干旱胁迫促种子萌发调控方式研究

Ammodendron argenteum (Pall.) Kuntze, one most important plant for desert ecosystem, has distinctively characteristic on drought-resistance. But it has becomes endangered in recent decades. One of the threatening factors is its critical value of drought tolerance with 0.12 Mpa on germination. Our previous study indicating that inoculation with cultures of endobacteria isolated from A. argenteum could enhance the germination rate by 11 times as high as control groups under drought stress of -1.2 Mpa by polyethylene glycol (PEG). Effects of the inoculation was more than pure water for stress releases. Most of the isolates could produce indoleacetic acid (IAA) and proline under laboratory culture but has no relationship with germination enhancing. The results also indicated that the effects of germination enhancing were delayed for a few days, presuming that there have some regulations on osmotic or metabolic of the seed. For the purpose to understand the pathways of symbiotic enhancing on germination and the regulation on seed metabolic, the cultures of the isolates would be inoculated to the surface and deep sterilized seeds. With gradient drought stress with PEG 6000, the cultures, cells resuspension with PBS, and the supernatants of the isolates that cultured with general laboratory medium would be used. Germination rates and seed composition with or without inoculation would be estimated. And then, the seeds with different germination period would be grind as medium and the strains would be cultures with it as "semi- in vivo" test. The metabolites will be analyzed and the cultures also inoculated for seed germination. The results will be helpful for our understanding the pathway of the endophyte on germination, the enhancing chemicals, and metabolically regulations. And the enhancing mechanism of the seed resistance for desert would be play a foundation for the application of microorganisms on desert ecosystems protection and restoration.

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，在梯度干旱胁迫下，接种分离自银沙槐的内生菌培养物可缓解甚至解除胁迫，种子萌发率最高比对照提高10倍以上，效果超过纯水;检测表明，多数分离株实验室培养可产生吲哚乙酸、脯氨酸等成分但却与促萌发无关，且高胁迫度下促萌发作用有延迟现象,推测与种子渗透或代谢调节有关。本项目在此基础上，以普通微生物培养基，或不同萌发阶段种子磨浆作为培养基接种银沙槐的内生菌，检测培养物抗干旱相关成分，再分别以培养物、菌体细胞或上清液接种经表面、深层灭菌种子,在梯度渗透胁迫下检测萌发率和种子成分变化;以"拟体内"代谢试验分析共生抗干旱方式和对种子代谢的调节,探索内生菌与种子相互作用途径,揭示共生调控机理,为微生物应用于珍稀植物种群保护及繁育提供科学依据。

具有重要固沙作用的荒漠植物银沙槐已被列入国家二级濒危植物名录。研究证实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。.本项目研究由荒漠植物银沙槐根部分离内生细菌37株并完成鉴定，同时鉴定1株被命名为Alcaligenes endophyticus的微生物新种并有效发表。.以25% PEG 6000干旱胁迫下接种内生菌试验其对对种子萌发的影响，结果显示有18株可显著缓解干旱胁迫，种子萌发率最高可提高11倍，同时发现，内生菌缓解干旱促种子萌发会延迟3-5天，显示内生菌发挥作用的方式具有特殊性。.将内生菌培养物分离为细胞和上清液进行促进种子萌发实验，发现部分菌株是以细胞、部分是以上清液，还有部分是以混合培养物缓解干旱。对种子不同萌发阶段剖面淀粉染色发现，干旱胁迫下淀粉不能被分解而无法萌发，而接种以细胞为作用方式的菌株AER314-4后淀粉迟于对照组分解且种子萌发，与前述萌发延迟现象吻合，证实内生菌参与或调节了种子物质代谢或基因表达。.对内生菌菌株体外培养物的吲哚乙酸、脯氨酸等主要抗干旱成分检测显示，大多数菌株均可产生这两类成分但与缓解干旱无关，进一步证实内生菌是以其它成分或在种子内部以调控方式参与了抗逆。.为了解内生菌的代谢机制，将银沙槐种子研磨、灭菌后作为培养基体外培养AER314-4，再以培养物的上清液作为添加剂进行种子萌发实验，发现以种子为培养基的上清液与以牛肉膏蛋白胨为培养基的上清液在缓解干旱胁迫促种子萌发功能上无显著区别，再次证实菌株的共生抗干旱是在种子内部发生的。.依据种子萌发进程中淀粉含量的变化，取相同淀粉含量阶段的9个样品进行了GC-MS分析，在检测到的64个成分中，干旱胁迫组均为大分子组分，而接种内生菌AER314-4后分子量小于200的组分增加3.3倍，证实是内生菌进入种子调节内部代谢所致。.结论：银沙槐内生菌具有共生抗干旱作用，其作用机制是调控了种子内部代谢或基因表达。