兰州大学机构库 >生命科学学院
UV-B 作用下小球藻的一氧化氮信号产生机制,传递途径和对氮素代谢的影响
Alternative TitleFormation mechanism of NO induced by UV-B radiation,transduction pathway and effect on N metabolism
陈坤
Thesis Advisor王勋陵
2002-05-12
Degree Grantor兰州大学
Place of Conferral兰州
Degree Name博士
Keyword小球藻 一氧化氮 信号转导 UV-B辐射
Abstract本文以小球藻为实验材料,研究在UV-B辐射下小球藻的一氧化氮信号的产生机制,转导途径,和对氮素代谢的影响,其主要内容如下: 1. UV-B作用下小球藻的一氧化氮信号产生机制的研究。 NO半衰期5-50秒,,能通过生物膜快速扩散,NO作为信号分子在单细胞生物体中能够释放在细胞外,在实验中利用氧合血红蛋白在水相捕捉NO的方法,测定当时水相中的NO的浓度。采用不同的UV-B辐射强度,来确定一氧化氮的释放量与UV-B辐射强度的关系,结果发现NO的释放量与UV-B的辐射强度成正相关。 为了研究UV-B作用下小球藻释放NO的底物,在培养液中加入了NOS的竞争性抑制剂L-NNA,结果发现L-NNA并不降低NO的释放量。同样在氮饥饿(24小时)的培养液中添加NR的抑制剂NaN3不影响一氧化氮的释放,说明NO的底物不是L-Arg和NO3- ,不是NOS和NR参与了NO的形成过程。为了更进一步研究NO的形成机理,在实验中利用外源的活性氧产生系统导致了NO产生,验证了 NO的形成来自活性氧作用。为了探究NO的底物,在培养液中添加20种氨基酸,发现每一种氨基酸都能促进NO 的形成,推测 NO来自氨基酸的氨基或者是氨基酸中间代谢产物。在离体的条件下和外源活性氧参与下,发现只有本丙氨酸作为底物导致NO的释放,其它氨基酸没有释放NO。 同时测定了PAL的活性在UV-B作用下大幅度提高。 2. UV-B辐射下的一氧化氮信号的传导途径中的研究。本文的实验中主要研究NO与活性氧之间以及APx,5’-AMP,PAL的信号的关系。UV-B辐射引起了小球藻的CAT ,SOD, POD, APx,5’-AMP,PAL活性上升。为了确定UV-B辐射引起了以上酶活性的变化是不是NO由引起的,在实验中加入NO吞噬剂NAC,NAC降低了CAT,SOD,POD,Apx,5’-AMP,PAL的活性,添加外源的GSH降低了CAT,SOD, POD, APx,5’-AMP,PAL的活性的上升。更进一步的研究在无UV-B辐射时添加SNP进一步提高了CAT ,SOD, POD, APx,5’-AMP,PAL的活性,可以认为UV-B辐射影响上述酶活性的变化是NO作用的结果。在UV-B辐射下,在培养液中加入SNP(0.5mmol/L)对小球藻的叶绿素具有保护作用。 3. UV-B辐射下的NO信号对氮素代谢的作用。UV-B辐射引起了小球藻的NR(离体和体内),NiR,GS, GOT活性下降。为了确定NO信号在小球藻细胞中对氮素代谢的影响,利用NAC,GSH和外源的 NO作用于小球藻,NAC降低了UV-B辐射对NR(离体和体内),NiR,GS, GOT的活性影响,GSH与NAC具有相同的效果。用外源的NO减低了NR(离体和体内),NiR,GS, GOT的活性,低浓度NO(0.1mmol/LSNP)的对NR(离体和体内),NiR,GS, GOT的活性影响不大,但是高浓度NO(0.5mmol/LSNP和1mmol/LSNP)的对NR(离体和体内),NiR,GS, GOT的活性有明显的降低作用。结果推定UV-B辐射下小球藻的氮素代谢受到抑制是由于NO作用的结果。
Other AbstractIn the paper, formation mechanism of NO induced UV-B radiation, transduction pathway and effect on N metabolism in Chlorella pyrenodosia, the main contents are on below: 1 Formation mechanism of NO induced UV-B radiation in Chlorella pyrenodosia have been studied. Half life of NO is 5-50 seconds. It rapidly crosses biological membranes and triggers various different process in a short period of time. NO was quantified by oxyhaemoglobin in liquid phase. It was investigated that the UV-B-induced NO formed in the green alga Chlorella pyrenodosa suspensions. Nw-nitro-Larginine (L-NNA), an inhibitor of NO synthase (NOS), had not any inhibitory effect on the rate of NO. N-Acetyl-L-cysteine (NAC), a scavenger of NO, blocked NO generation. Cultured in nutrient solution and 1mmol/L NaN3 with or without nitrate (NO3-, nitrate-starved 24hours) suspensions, the Chlorella pyrenodosa similarly generated production of NO and NO2-. The production of NO was accorded with UV-B intersity. Meanwhile, addition of exogenous sodium nitroprusside (SNP, NO generator), NO and NO2- that released in the alga suspensions were measured. NO2- could be generated from NO. Furthermore whether the reactive oxygen species (ROS) involved in NO emissions processes response to UV-B radiation in the green alga was investigated. Exogenous addition of antioxidants such as dithiothreitol (DTT), glutathione (GSH; reduced form) ascorbic acide (AsA; reduced form) blocked NO emission by UV-B. Exogenous reactive oxygen, singlet oxygen (1O2) and hydroxyl radicals (OH-) stimulated NO formation, but hydrogen peroxide (H2O2) have not the effect stimulated NO formation. The activities chance of superoxide dismutase (SOD) and catalase (CAT) were measured under UV-B expose also. Together these results, it was suggest that UV-B radiation leads to the generation of NO probably via-via ROS. The results indicate that NO3- was not substrate of NO and NO2- and also indicate that NO induced by UV-B radiance not through L-Arg-dependent NOS and NO3-. It is PAL that emitted NO in vivo with ROS and Phe as substrate. 2. Transduction pathway of NO induced by UV-B radiation. The acivities of CAT, SOD, POD, APx, 5’-AMP,PAL were increased by UV-B radiation. Exogenous addition of NAC,GSH reduced the activities of CAT, SOD, POD, APx, 5’-AMP,PAL. Exogenously addition of SNP stimituted activities of CAT, SOD, POD, APx, 5’-AMP. SNP protect Chl against UV-B irradiation. 3 Effect on N metabolism of NO induced by UV-B ra...
URL查看原文
Language中文
Document Type学位论文
Identifierhttps://ir.lzu.edu.cn/handle/262010/221832
Collection生命科学学院
Recommended Citation
GB/T 7714
陈坤. UV-B 作用下小球藻的一氧化氮信号产生机制,传递途径和对氮素代谢的影响[D]. 兰州. 兰州大学,2002.
Files in This Item:
There are no files associated with this item.
Related Services
Recommend this item
Bookmark
Usage statistics
Export to Endnote
Altmetrics Score
Google Scholar
Similar articles in Google Scholar
[陈坤]'s Articles
Baidu academic
Similar articles in Baidu academic
[陈坤]'s Articles
Bing Scholar
Similar articles in Bing Scholar
[陈坤]'s Articles
Terms of Use
No data!
Social Bookmark/Share
No comment.
Items in the repository are protected by copyright, with all rights reserved, unless otherwise indicated.