Hlips在保护蓝藻光系统II免受氨损伤中的机制研究

The amount of ammonia input has been increasing significantly with the human activities, resulting in the increase of lakes' trophic level all around the world. Meanwhile, the distribution of algal species in lakes have been changed significantly, and cyanobacterial blooms occur frequently. Previous studies indicated that high concentration of ammonia could inhibit photosystem II activity rapidly. Since different algal species showing wide range of ammonia sensitivity, ammonia is an important factor to influence the distribution of algal species in aquatic systems. Usually, cyanobacteria are dominant algal group in eutrophic lakes, and they are more resistant to ammonia inhibition than some other species. However, the mechanism of ammonia resistance in cyanobacteria is still unclear. In the previous study, the applicant found that transcript levels of high light inducible proteins (Hlips) in wild-type Synechocystis sp. PCC 6803 were significantly induced by ammonia treatment through transcriptome sequencing, and gene knocked-out mutants showed more sensitive to ammonia inhibition than wild-type Synechocystis sp. PCC 6803, which indicated Hlips played important role in ammonia resistance. On the basis of these results, this proposal will investigate the roles of different hlips genes play in ammonia resistance, by phenotype analysis on hlips genes combination knocked-out and complementary strains. By using BN-PAGE and mass spectrometry, different photosynthetic complexes will be isolated and identified among wild-type and hlips mutants treated with ammonia. With combination of chlorophyll fluorescence, immunoblotting, and protein-protein interaction analysis, functions of Hlips involved in PSII repair and protection will be elucidated. This study will reveal the mechanism of Hlips on the protection of PSII against ammonia damage, and provide theoretical support for understanding the inner mechanism of ammonia involved in the algae succession in aquatic systems.

人类活动的加剧极大程度地增加了氨氮排放，引起水体富营养化程度普遍升高，蓝藻水华频发。研究表明，高氨可抑制PSII活性，氨对不同藻类抑制效应上的差异，最终影响了水体中藻类类群的分布。蓝藻具有高氨耐受性，是富营养化高氨水体中优势藻类群，但其高氨耐受的分子机制还不清楚。申请人前期以集胞藻PCC 6803为研究材料，通过转录组测序筛选，发现其hlips基因家族受氨诱导上调表达显著，基因敲除后氨敏感性明显增强。本项目在此基础上，拟通过对各hlips基因的组合敲除及互补表型分析，确定不同Hlips在高氨耐受中的作用；结合蓝绿胶电泳及质谱鉴定，分析氨处理对野生型与突变株光合复合体的差异影响；通过叶绿素荧光、免疫印迹及蛋白互作分析，揭示Hlips在PSII修复与保护中的功能。本项目的顺利实施将阐明Hlips在保护蓝藻PSII免受氨损伤中的功能机制，为深入认识氨氮影响水体中藻类演替的内在机制提供理论支撑。

人类活动的加剧改变了全球环境，大量氨氮输入引起水体富营养化已是世界性问题。氨可抑制光合作用，蓝藻具有较强的氨毒害耐受性，常为富营养化水体中优势物种。蓝藻中具有一类受高光诱导表达的Hlips蛋白家族，在光合机构的形成及保护过程中发挥重要作用。本项目以模式蓝藻Synechocystis PCC 6803为研究材料，对其中四个hli基因（hliA/B/C/D）在氨毒害耐受中的功能进行研究。结果表明：氨处理后各基因在转录水平上均有不同程度增加，其中hliA/B两基因表达上升最为明显，且hliB转录本占hlips总转录本的90%以上。基因定向敲除获得各基因单敲除突变株，生长结果表明hliB-突变株在氨处理后生长较野生型藻株明显下降，表明该基因在细胞耐受氨毒害过程中发挥重要功能，这与基因表达水平结果一致。同时敲除两组同源基因后获得hliA-B-与hliC-D-双突变株，氨处理后两双突变藻株生长较各自单突变藻株出现叠加抑制现象，尤其是hliA-B-双突变株氨毒害敏感性明显增强，表明两组同源基因中功能冗余，且hliA与hliB功能更为重要。三敲除突变株藻株对氨毒害敏感性进一步增强，且仅保留hliB的三突变藻株较其它三突变藻株氨毒害耐受性更强，确证hliB的功能重要性。分离类囊体膜并结合免疫印迹分析，hliB-突变株类囊体中PsbO含量在氨处理后显著降低，表明HliB在维持放氧复合体（OEC）的稳定发挥重要功能。双敲除 hliA与hliB后，OEC稳定进一步削弱，而单敲除hliA/C/D，以及双敲除hliC/D均对氨处理后PsbO与类囊体膜的粘附没有影响。上述结果进一步表明HliB通过参与维持OEC的稳定，进而起到耐受氨毒害的作用。虽然hliA/B高度同源，但由于表达较低，HliA发挥作用较小；而双敲除 hliC/D后，蓝藻在氨处理后非光化学淬灭较hliA-B-双突变株明显下降，表明HliC/D参与光合机构的热耗散。酵母双杂交及Pull-down结果表明HliB可与FtsH2蛋白酶发生相互作用，因此HliB通过影响氨胁迫下受损D1蛋白的降解，进而影响放氧复合体的稳定。本研究阐明了Hlips家族成员在保护蓝藻光系统II免受氨毒害的差异功能，由于该基因家族在蓝藻中大量扩张，这也为蓝藻对逆境的高度适应性提供了解释。