藓羽藻(Bryopsis hypnoides)核酮糖-1，5-二磷酸羧化酶／加氧酶的分离与活性测定

采用硫酸铵分部沉淀与凝胶过滤的方法，进行藓羽藻Rubisco的分离研究。结果表明，分离的藓羽藻Rubisco经SDS-聚丙烯酰胺凝胶电泳检测呈两条清晰条带，分别为Rubisco大亚基与小亚基；与菠菜相比，藓羽藻Rubisco大亚基分子量与菠菜基本相同，而小亚基较之稍大一些。藓羽藻Rubisco活力测定结果表明，Rubisco分离过程中用硫酸铵分部沉淀后活力降低许多，分离后活力有所上升，但仍比粗提液活力弱；在Rubisco活力测定过程中，藓羽藻Rubisco的活化温度与其它物种Rubisco活化的温度不同，在低温下活化效果较好。这些结果说明Rubisco的酶活力受硫酸铵的影响而且藓羽藻Rubisco相对陆地高等植物结构不稳定。     

Characterization of ribulose-1, 5-bisphosphate carboxylase/oxygenase and transcriptional analysis of its related genes in Saccharina japonica (Laminariales,Phaeophyta)

Saccharina japonica is a common macroalga in sublittoral communities of cold seawater environments,and consequently may have highly effi cient ribulose-1,5-bisphosphate carboxylase/oxygenase(Rubisco)activity for carbon assimilation.In our study,we cloned the full-length Rubisco gene from S.japonica(SJ-rbc).It contained an open reading frame for a large subunit gene(SJ-rbcL)of 1 467 bp,a small subunit gene(SJ-rbcS)of 420 bp,and a SJ-rbcL /S intergenic spacer of 269 bp.The deduced peptides of SJ-rbcL and SJ-rbcS were 488 and 139 amino acids with theoretical molecular weights and isoelectric points of 53.97 kDa,5.81 and 15.84 kDa,4.71,respectively.After induction with 1 mmol/L isopropyl-β-Dthiogalactopyranoside for 5 h and purifi cation by Ni 2+ affi nity chromatography,electrophoresis and western blot detection demonstrated successful expression of the 55 kDa SJ-rbcL protein.Real-time quantitative PCR showed that the mRNA levels of SJ-rbcL in gametophytes increased when transferred into normal growth conditions and exhibited diurnal variations: increased expression during the day but suppressed expression at night.This observation implied that Rubisco played a role in normal gametophytic growth and development.In juvenile sporophytes,mRNA levels of SJ-rbcL,carbonic anhydrase,Calvin-BensonBassham cycle-related enzyme,and chloroplast light-harvesting protein were remarkably increased under continuous light irradiance.Similarly,expression of these genes was up-regulated under blue light irradiance at 350 μmol/(m 2·s).Our results indicate that long-term white light and short-term blue light irradiance enhances juvenile sporophytic growth by synergistic effects of various photosynthetic elements.     

快速升温后极地甲藻Polarella glacialis的生理响应

Polarella glacialis最早发现于南极固定冰和北冰洋水层,最近在温带海域也发现其相关基因。专抗甲藻的Rubisco、PCNA抗体在该藻细胞内各检测到~53 kDa和~55 kDa的特异性条带。对该藻的梯度温度培养实验显示,4℃生长的P. glacialis细胞密度为1.1×105 cells/ml时仍能继续增殖,一天中藻细胞内Rubisco的表达量基本保持恒定,PCNA的表达量峰值与%S峰值相对应,当温度迅速上升至15、20℃时,藻株处于胁迫状态,细胞密度迅速减少,正常的细胞周期被干扰,大多数细胞分裂活动不活跃或停止分裂,Rubisco、PCNA的表达量大幅减少甚至消失,昼夜表达特征改变,其中20℃对藻株的胁迫作用更加显著。15℃培养下的P. glacialis细胞密度并不立即减少,细胞周期与两种指示蛋白表达特征显示仍有部分细胞完成分裂。本研究为该藻的相关基因在温带水域的出现提供了可能的解释,并推测在相对长期渐进的极地增温过程中,P. glacialis可能继续存在。     

Variation in Rubisco and other photosynthetic parameters in the life cycle of Haematococcus pluvialis

Cells of Haematococcus pluvialis Flot. et Will were collected in four different growth phases. We quantified the initial and total enzyme activity of ribulose-1,5-bisphosphate carboxylase (Rubisco) in crude extracts, and the relative expression of large-subunit ribulose-1,5-bisphosphate caboxylase / oxygenase (rbcL) mRNA. We measured the ratio of photosynthetic rate to respiration rate (P/R), maximal effective quantum yield of photosystem II (Fv/Fm), electron transport rate (ETR), actual photochemical efficiency of PSII in the light (ΦPSII), and non-photochemical quenching (NPQ). Green vegetative cells were found to be in the most active state, with a relatively higher P/R ratio. These cells also displayed the lowest NPQ and the highest Fv/Fm, ETR, and ΦPSII, indicating the most effective PSII. However, both Rubisco activity and rbcL mRNA expression were the lowest measured. In orange resting cysts with relatively lower P/R and NPQ, Rubisco activity and rbcL expression reached a peak, while Fv/Fm, ETR, and ΦPSII were the lowest measured. Taking into account the methods of astaxanthin induction used in industry, we suggest that Rubisco may participate in astaxanthin accumulation in H. pluvialis. A continuous and sufficient supply of a carbon source such as CO2 may therefore aid the large scale production of astaxanthin.     

晚中新世植被变更与光合作用演化

光合作用酶Rubisco出现于太古代,对于以后地质时期里大气的CO2降低并不适应。晚中新世大幅度扩展的C4植物,就是光合作用演化的一种途径,适应于CO2浓度较低的大气,也适应于温暖而季节性干旱的季风气候。C4与C3植物碳同位素的重大差异,又为利用古土壤和哺乳类牙齿珐琅质的δ13C分辨C3、C4植物在植被中的比例提供了条件。自从发现巴基斯坦晚中新世古土壤层δ13C突变以来的10余年,围绕着C4植物扩展究竟反映季风气候,CO2浓度下降,还是干旱化,国内外学术界展开了热烈的讨论,至今尚属未解之谜,但从中可以吸取研究地球系统演变的经验教训。     

蛋白核小球藻(Chlorella pyrenoidosa) Rubisco活化酶基因的克隆与表达分析

采用RACE技术克隆了单细胞绿藻蛋白核小球藻820Rubisco活化酶基因(rca)序列,并用荧光定量PCR方法研究了rca和Rubisco小亚基基因(rbcS)的表达规律。结果获得了1703bp的rcacDNA序列,包括66bp的5’非翻译区、1242bp的开放阅读框和395bp的3’非翻译区。序列比较和分析表明该蛋白核小球藻rca序列与其它绿藻的同源性高达78%—85%;偏好使用以G/C/T结尾的密码子;推测的RCA蛋白等电点和分子量分别为8.44和45.71kDa。荧光定量PCR结果表明随光照时间增加rca与rbcS转录表达逐渐降低;不同盐度处理下rbcS的mRNA量变化不大,而rca在37.5盐度下表达量为25盐度的2.69倍;1.0—3.0mmol/L水杨酸处理rca与rbcS表达均降低。