雨生红球藻八氢番茄红素合成酶基因的克隆及表征

雨生红球藻是一种单细胞绿藻,在多种逆境胁迫条件下能够大量合成并迅速积累虾青素,其积累量最高可达细胞干重的4%,从而成为目前最理想的天然虾青素合成工具.八氢番茄红素合成酶(PSY)是虾青素合成途径中第一个限速酶.分离了八氢番茄红素合成酶基因(psy)的全长cDNA及基因组DNA.其全长cDNA包括1200个碱基,编码400个氨基酸,基因组DNA包括5个外显子,4个内含子.系统发育分析结果显示,绿藻的八氢番茄红素合成酶基因形成一个进化枝,它们与高等植物的psy亲缘关系比较近.通过GenomeWalking的方法,分离了psy基因约1kb的5′侧翼序列.将含有TATA-box和CAAT-box的297bp的序列与LacZ报告基因构成嵌合的表达载体,用基因枪法转化雨生红球藻.lacZ的瞬间表达检测结果表明,这段上游序列能够驱动lacZ表达,具有启动子活性.     

Carotenoid hydroxylase from Haematococcus pluvialis: cDNA sequence, regulation and functional complementation.

A cDNA homologous to beta-carotene hydroxylase from Arabidopsis thaliana was isolated from the green alga Haematococcus pluvialis. The predicted amino acid sequence for this enzyme shows homology to the three known plant beta-carotene hydroxylases from Arabidopsis thaliana and from Capsicum annuum (38% identity) and to prokaryote carotenoid hydroxylases (32-34% identities). Heterologous complementation using E. coli strains which were genetically engineered to produce carotenoids indicated that the H. pluvialis beta-carotene hydroxylase was able to catalyse not only the conversion of beta-carotene to zeaxanthin but also the conversion of canthaxanthin to astaxanthin. Furthermore, Northern blot analysis revealed increased beta-carotene hydroxylase mRNA steady state levels after induction of astaxanthin biosynthesis. In accordance with the latter results, it is proposed that the carotenoid hydroxylase characterized in the present publication is involved in the biosynthesis of astaxanthin during cyst cell formation of H. pluvialis.     

雨生红球藻β－胡萝卜素酮化酶（bkt）启动子功能分析

单细胞绿藻———雨生红球藻在逆境条件下积累大量的虾青素。β-胡萝卜素酮化酶(bkt)催化在β-胡萝卜素和玉米黄素的β-紫罗酮环C-4位引入酮基的反应,是虾青素合成过程中的关键酶。我们利用凝胶阻滞的方法研究雨生红球藻中bkt基因309bp(-617/-309)启动子区域的转录因子结合位点并发现在-396/-338的59bp探针存在特异的核蛋白结合位点。通过序列分析,发现此59bp区域并不包含TATA或者CAAT-box,而是存在对光、缺氧、p-香豆酸及激素反应的G-box。     

UV-B辐射对雨生红球藻光合特性和虾青素含量的影响及其响应

实验研究了不同强度的UV-B(280-320 nm)辐射对雨生红球藻(Haematococcus pluvialis)的光合活性、生物量、色素含量、活性氧(ROS)含量和抗氧化酶活性等的影响, 以探讨利用UV-B辐射诱导虾青素生物合成增强的可能性。结果发现, 经UV-B辐射处理后,雨生红球藻的光合活性降低、生物量增长被抑制。UV-B辐射对叶绿素影响不大, 但会改变细胞的类胡萝卜素和虾青素含量:0.1和0.3 W/m2强度的UV-B辐射使细胞中的这两种色素含量升高, 0.5 W/m2组的色素含量短暂升高后恢复到对照水平。中低强度的UV-B可以促进雨生红球藻单细胞虾青素含量的增加, 但由于其对细胞生长的抑制作用, 并不能使虾青素大量积累。随辐射时间延长, 细胞内ROS含量未明显增加,但抗氧化酶(过氧化氢酶和超氧化物歧化酶)活性下降, 雨生红球藻可能主要依靠虾青素来淬灭ROS。以上结果表明, UV-B辐射对雨生红球藻的主要生理生化过程有抑制作用, UV-B辐射既可以诱导虾青素的合成又会消耗一部分虾青素, 对虾青素含量的影响与其强度有关, 而利用虾青素来清除细胞内的ROS可能是雨生红球藻抵御这种不利环境条件的最重要的途径。       

Transformation of the green alga Haematococcus pluvialis with a phytoene desaturase for accelerated astaxanthin biosynthesis

Astaxanthin is a high-value carotenoid which is used as a pigmentation source in fish aquaculture. Additionally, a beneficial role of astaxanthin as a food supplement for humans has been suggested. The unicellular alga Haematococcus pluvialis is a suitable biological source for astaxanthin production. In the context of the strong biotechnological relevance of H. pluvialis, we developed a genetic transformation protocol for metabolic engineering of this green alga. First, the gene coding for the carotenoid biosynthesis enzyme phytoene desaturase was isolated from H. pluvialis and modified by site-directed mutagenesis, changing the leucine codon at position 504 to an arginine codon. In an in vitro assay, the modified phytoene desaturase was still active in conversion of phytoene to zeta-carotene and exhibited 43-fold-higher resistance to the bleaching herbicide norflurazon. Upon biolistic transformation using the modified phytoene desaturase gene as a reporter and selection with norflurazon, integration into the nuclear genome of H. pluvialis and phytoene desaturase gene and protein expression were demonstrated by Southern, Northern, and Western blotting, respectively, in 11 transformants. Some of the transformants had a higher carotenoid content in the green state, which correlated with increased nonphotochemical quenching. This measurement of chlorophyll fluorescence can be used as a screening procedure for stable transformants. Stress induction of astaxanthin biosynthesis by high light showed that there was accelerated accumulation of astaxanthin in one of the transformants compared to the accumulation in the wild type. Our results strongly indicate that the modified phytoene desaturase gene is a useful tool for genetic engineering of carotenoid biosynthesis in H. pluvialis.     

Optimization of biomass, total carotenoids and astaxanthin production in Haematococcus pluvialis Flotow strain Steptoe (Nevada, USA) under laboratory conditions

The microalga Haematococcus pluvialis Flotow is one of the natural sources of astaxanthin, a pigment widely used in salmon feed. This study was made to discover optimal conditions for biomass and astaxanthin production in H. pluvialis from Steptoe, Nevada (USA), cultured in batch mode. Growth was carried out under autotrophic (with NaNO3, NH4Cl and urea) and mixotrophic conditions (with 4, 8, 12 mM sodium acetate) under two photon flux densities (PFD) (35 and 85 mumol m-2 s-1). The carotenogenesis was induced by 1) addition of NaCl (0.2 and 0.8%), 2) N-deprivation and 3) high PFD (150 mumol m-2 s-1). Total carotenoids were estimated by spectrophotometry and total astaxanthin by HPLC. Ammonium chloride was the best N-source for growth (k = 0.7 div day-1, 228-258 mg l-1 and 2.0 x 10(5)-2.5 x 10(5) cells ml-1 at both PFD, respectively). With increasing acetate concentration, a slight increment in growth occurred only at 85 mumol m-2 s-1. Light was the best inductive carotenogenic factor, and the highest carotenoid production (4.9 mg l-1, 25.0 pg cell-1) was obtained in cultures pre-grown in nitrate at low light. The NaCl caused an increase in carotenoid content per cell at increasing salt concentrations, but resulted in a high cell mortality and did not produce any increment in carotenoid content per volume compared to cultures grown at 150 mumol m-2 s-1. The highest carotenoid content per cell (22 pg) and astaxanthin content per dry weight (10.3 mg g-1) (1% w/w) were obtained at 85 mumol m-2 s-1 with 0.8% NaCl.     

Haematococcus astaxanthin: applications for human health and nutrition

The carotenoid pigment astaxanthin has important applications in the nutraceutical, cosmetics, food and feed industries. Haematococcus pluvialis is the richest source of natural astaxanthin and is now cultivated at industrial scale. Astaxanthin is a strong coloring agent and a potent antioxidant - its strong antioxidant activity points to its potential to target several health conditions. This article covers the antioxidant, UV-light protection, anti-inflammatory and other properties of astaxanthin and its possible role in many human health problems. The research reviewed supports the assumption that protecting body tissues from oxidative damage with daily ingestion of natural astaxanthin might be a practical and beneficial strategy in health management.     

UV-B对雨生红球藻生长及虾青素积累的影响

以雨生红球藻(Haematococcus pluvialis)为材料,研究不同强度的UV-B对雨生红球藻生长、光合作用及虾青素积累的影响和其作用机理。设置5种紫外线强度,分别在正常光照培养条件下补充不同强度UVB(100—500 lx),标记为CK、U100、U200、U300、U400和U500六组。结果表明,经UV-B辐射后雨生红球藻细胞密度、PSⅡ最大光化学效率(F_v/F_m)、非光化学淬灭系数(NPQ)和叶绿素(Chl.a和Chl.b)含量等均呈现下降趋势,且与辐射强度相关。相反,虾青素含量在100—400 lx强度下随UV-B辐射强度的增加而升高。与对照相比,高强度UV-B辐射(U400)36h和72h后藻细胞虾青素含量分别提高了35.68%和56.23%,达到5.82和7.06 mg/L。qRT-PCR检测发现雨生红球藻虾青素合成关键酶基因(IPI、PSY、BCH和BKT)的表达量随紫外辐射强度和辐射时间的增加均有不同程度升高。UV-B辐射亦调控紫外光受体UVR8及其信号转导通路核心元件(COP1、SPA1、HYH和HY5)的基因表...     

Identification and characterization of a suppressor element in the 5'-flanking region of the mouse androgen receptor gene.

Androgens play an important role in the development and maintenance of male reproductive organs through the androgen receptor (AR). In order to study the mechanism of regulation of AR at the molecular level, a 1571 bp fragment in the 5'-flanking region of the mouse androgen receptor (mAR) gene was isolated and sequenced. Transfection of 5'-deletion constructs cloned into vectors containing the chloramphenicol acetyl transferase (CAT) gene indicated the presence of a promoter in the sequence -146 to +131. These experiments also suggested the presence of a suppressor element. Further characterization indicated that the suppressor is present between -486 to -351. It is functional in the context of the natural AR promoter and the heterologous thymidine kinase promoter. Transfection of a -546/ + 131 construct in which the putative suppressor element (-421 to -448) had been deleted caused increased basal CAT activity suggesting that the suppressor is limited to this 28 bp element in the 5'-flanking region of the mouse AR gene.