银沙槐内生菌共生抗旱促种子萌发蛋白质代谢与调控

Symbiosis with microbe is an important way for desert plants to environment adaptation. And the biological characteristics of plants are regulated by protein expression. In our previous study, 37 strains of endophytic bacteria were isolated from Ammodendron argenteum (Pall.), one of the most important desert plants especially with drought-resistance ability. Under drought stress with 25% polyethylene glycol (PEG) 6000, the cultures of the isolates enhanced the germination rate by 37%. Inoculated with separated cells or supernatant that contain metabolites indicated that some isolates plays the role of stress mitigating by cells, but the germination delayed for 3-5 days. The activity of amylase was inhibited under drought stress in the process of seed germination but been activated by the bacteria. The starch was hydrolyzed and the soluble sugar was increased after inoculation. The results of transcriptome sequencing analysis indicated that 282 Unigenes were related to starch metabolism. And the composition of the protein was also changed by the inoculation on preliminary analysis. For the purpose to understand the osmotic adjustment and regulation mechanism of protein on the process of symbiosis, osmotic adjustment and protein hydrolysis would be analyzed by free amino acid and protease activity estimation. Protein differences and key genes of drought-resistance and symbiotic responses would be selected and estimated by proteomics. The expression of the genes would be assessment by Real-time PCR. The network of protein regulation of symbiosis would be constructed based on the study. The results would be helpful for our understanding the biological mechanism of the symbiosis with drought-resistant.

共生是荒漠植物适应环境的重要方式，而微生物对植物生物学特性的调控作用最终都以蛋白质的形式表现。前期由荒漠固沙植物银沙槐分离到37株菌，其中菌株AER314-4通过进入/接触种子来缓解25% PEG 6000模拟的干旱胁迫，从而提高种子萌发率37%，但萌发会有3-5天的延迟；通过淀粉酶的检测证实，接种内生菌可激活因干旱胁迫而被抑制的淀粉酶，降解淀粉增加可溶性糖；转录组测序分析表明，1697个Unigene参与大分子物质代谢，与淀粉代谢相关基因有282条；蛋白质初步分析显示，蛋白质组成会因接种内生菌而发生变化。为了解共生抗干旱过程中的蛋白质作用与调控机制，本项目以银沙槐种子和内生菌为材料，以蛋白质代谢和游离氨基酸测定分析共生时的渗透调节；以蛋白质组学分析差异表达蛋白并验证其表达模式；构建共生条件下的蛋白调控网络，为揭示银沙槐共生抗逆的生物学机制提供科学依据。

课题组前期从荒漠植物银砂槐根部分离的一株芽孢杆菌AER314-4（Bacillus axarquiensis），在25% PEG6000 模拟干旱胁迫下能有效促进种子萌发。为揭示AER314-4菌株抗干旱促种子萌发蛋白质代谢调控机制，本研究通过4D-DIA蛋白质组测序技术，对水萌发（CK）、干旱胁迫（DS）、干旱胁迫加菌（DE）三个处理下种子萌发早期、中期和晚期样品中蛋白质表达差异进行分析；通过检测种子中可溶性蛋白质、游离氨基酸含量，丙二醛（MDA）、脯氨酸（Pro)含量及超氧化物歧化酶（SOD）、过氧化物酶（POD）活性以分析AER314-4菌株调节种子能量代谢及抗氧化水平。3个处理组27份种子样品中共鉴定出7874个蛋白质组数，覆盖总蛋白质组数据库的83.2%。其中，抗氧化类、脂类和糖类代谢相关蛋白响应AER314-4菌株调节参与种子抗旱萌发，主要包括Cu-Zn超氧化物歧化酶、苯丙氨酸氨裂解酶、脂氧合酶，呋喃果糖苷酶等。几丁质酶、铜锌超氧化物歧化酶、谷胱甘肽S-转移酶等在干旱胁迫的种子中持续上调表达，为干旱响应蛋白。通过转录组与蛋白质组关联分析获得加菌抗旱响应蛋白4个，分别涉及糖、脂肪及抗氧化代谢功能。种子中可溶性蛋白及主要抗氧化酶含量变化与蛋白质组学结果较一致。综上，本研究初步探明菌株AER314-4通过上调种子中糖、脂肪及抗氧化代谢相关蛋白参与抗旱促种子萌发调控，为揭示荒漠植物与微生物共生抗逆机制提供重要参考价值，值得深入研究。