【作者】 王娟；

【导师】 黄荣峰；

【作者基本信息】 中国农业科学院 ， 生物化学与分子生物学， 2010， 硕士

【摘要】 维生素C(VC)是普遍存在于植物和真核藻类体内的高丰度水溶性小分子抗氧化物质,在植物的生长发育、非生物胁迫和生物胁迫应答中具有重要的生物学功能。已有证据表明植物体内存在多条VC合成途径,包括L-半乳糖途径、古洛糖途径、糖醛酸途径和肌醇途径。其中L-半乳糖途径把VC的生物合成融入碳水化合物的主要代谢过程并与多糖合成及蛋白质糖基化联系起来,已被公认为植物中VC合成的最主要途径。研究表明,拟南芥GDP-甘露糖焦磷酸化酶(GMPase)是此途径中的一个关键酶,编码该酶的基因命名为VTC1。VTC1点突变体vtc1-1并没有改变VTC1的转录表达,只是降低了35%酶活以及70%VC含量(仅为野生型VC含量的25-30%),导致对臭氧、UV-B、SO2、高光强、盐、H2O2的敏感性。光是影响植物体内VC含量的一个重要环境因子,且VC在光保护中起重要作用。光合作用、光呼吸和其它氧化胁迫产生的活性氧(ROS)都可以通过VC清除。拟南芥L-半乳糖途径的几个重要酶均在转录水平上受到光调控,VTC1、GPP、GalLDH、VTC2在持续光照下基因表达升高,持续暗处理下基因表达降低。但是目前光调控VC合成的机制尤其是在蛋白水平上还不是很清楚。以AtVTC1为诱饵通过酵母双杂技术筛选拟南芥三天黄化苗的cDNA文库,得到三个与其互作的蛋白质因子。其中一个即为CSN5B,CSN5B是与“组成型光形态建成”密切相关的COP9信号复合体(COP9 signalosome,CSN)的一个亚基,CSN由8个亚基组成,依次为CSN1—8,此复合物可以通过泛素降解途径调节植物对光信号的应答,从而使植物体完成从光形态建成到暗生长的转换。CSN是通过对SCF型E3-泛素连接酶的去NEDD化来调控泛素降解系统的,CSN5对于整个复合体而言具有相对独立性,CSN5的单个亚基突变就能引起CSN突变体所有表型的出现,CSN5对植物的生长发育具有重要的调控作用。通过酵母双杂交、BiFC、pull-down和CoIP等实验验证了AtVTC1与AtCSN5B确实存在相互作用。为了进一步探讨CSN5B对VTC1的调控作用,我们从植物体VC含量和对氧化胁迫耐受性方面考察了它们之间的相互作用。CSN5B缺失突变体csn5b的生长发育与野生型并无明显差异,但检测到其VC含量比野生型要高20%左右,而过表达CSN5B后VC含量比野生型低;同时过表达CSN5B后其活性氧含量比野生型有所增加。从蛋白水平上看,拟南芥VTC1过表达植株与CSN5B过表达植株杂交后VTC1的蛋白含量比杂交前明显降低。以上结果表明,拟南芥中CSN5B通过调控VTC1的降解而影响植物体内的VC含量,并进而引起植物对氧化胁迫耐受性的变化。更多还原

【Abstract】 Vitamin C (VC), the most abundant water-soluble small molecular antioxidant in plants and eukaryotic algae, plays roles in plant development and response to abiotic and biotic stresses. It has been verified that there are several pathways of VC biosynthesis in plants, including L-galactose pathway, Gulose pathway, Uronic acid pathway, and myo-inositol pathway. The L-galactose pathway integrates VC biosynthesis to carbohydrate metabolism, amylase synthesis and protein glycosylation, which makes it to be the major route of VC synthesis in plants. VTC1 encodes one key enzyme GDP-D-Mannose pyrophosphorylase (GMPase). VC-reduced Arabidopsis thaliana mutant vtc1-1 habors a mis-sense mutation, resulting in the enzyme activity reduction by 35% compared with that of wild type without any changes of VTC1 transcripts. And the VC contents in the mutant decrease to 25-30% of that in wild type, consequently increasing the sensitivity to ozone, UV-B, SO2, high light, salt stress and H2O2.Light intensity is the major environmental factor affecting leaf VC contents, meanwhile VC plays a role in preventing plants from light-induced damage. The reactive oxygen species (ROS) produced in photosynthesis, photorespiration and oxidative stresses could be scavenged through VC transformation from reduced to oxidized status. The key enzymes of L-galactose pathway in A. thaliana are light-regulated in transcript level, and expression of VTC1、GPP、GalLDH、VTC2 increase in constant light-acclimated leaves and decrease under constant darkness. However, the mechanism that how light regulates VC synthesis, especially in post-translation level, is still not clear.In this work, we used the yeast two-hybrid system to isolate proteins able to interact with AtVTC1. By the system, we have isolated 3 putative proteins that might interact with AtVTC1, screened from a 3 day-old etiolated Arabidopsis seedling cDNA library. One of the proteins is CSN5B known as a subunit of COP9 signalosome (CSN), which is consist of 8 subunits named CSN1-8. The CSN complex plays a part in promoting an alternative developmental program (skotomorphogenesis to photomorphogenesis) through ubiquitin degradation pathway in response to a changing light environment. By monitoring the derubylation status of SCF E3 ubiquitin ligases, CSN participates in ubiquitin degradation system, CSN5 is relatively independent to the whole complex, the single mutated CSN5 would results in the phenotypes of all CSN mutants, and CSN5 has a crucial modulation in plant development.After identification of the interaction between AtVTC1 and AtCSN5B by way of yeast two hybrid, BiFC, pull-down and CoIP, we then detected plants VC content and tolerance to oxidative stress. The VC content in csn5b mutant, complete lack of the CSN5B transcripts, was 20% more than that in wild type, although seedlings did not show obvious different phenotypes between wild type and csn5b. The transgenic lines overexpressing CSN5B decreased VC content, compared to that of wild type, which resulted in the ROS in OECSN5B increased. And AtVTC1 protein quantities in OEVTC1/OECSN5B were apparently less than that in OEVTC1.The results above indicate that CSN5B plays an important role in modulation of plant VC synthesis and in influence of plant response to oxidative stress in A. thaliana, as a result of the degradation of VTC1 protein by CSN5B.更多还原