【作者】 汪本凡；

【导师】 唐欣昀；

【作者基本信息】 安徽农业大学 ， 微生物学， 2004， 硕士

【摘要】 本文系统研究了杜氏盐藻的生物学特性，主要内容包括杜氏盐藻无菌纯化、杜氏盐藻生理学特性、杜氏盐藻遗传学特性等三部分。采用抗生素结合离心技术、离心洗涤结合稀释技术以及盐藻耐盐特性等三种不同方法对杜氏盐藻进行无菌纯化，获得无菌杜氏盐藻藻株A1、A7、A13，A14和A21。经过不同方法检测，确证该5藻株属无菌藻株。利用无菌藻株A1研究了影响杜氏盐藻生长的营养因子，如盐度、维生素、激素、有机碳源、有机氮源、动植物抽提液等。结果表明：该盐藻藻株可适应0～292.5g·L-1NaCl浓度，NaCl浓度的突增会使杜氏盐藻的生长出现停滞，停滞期的长短与NaCl终浓度有关；添加150μg·L-1VB1、160μg·L-1VB6、0.5μg·L-1VB12，320mg·L-1VC及2.0μg·L-1VH可在一定程度上促进藻的生长；液体培养发现葡萄糖的促长效果比醋酸钾好，以10~15g·L-1浓度为最佳，而固体培养时则以10~30g·L-1效果最佳；尿素的促长效果要好于甘氨酸，以0.1g·L-1浓度为最佳； NAA、肌醇、6-BA 、KT和GA促进藻细胞生长的最佳浓度分别为3、55、4.0、0.1和0.2mg·L-1；20 ~ 40ml·L-1鱼汤可强烈地促进藻细胞分裂，建议在盐藻培养基中添加一定浓度的鱼汤作为盐藻完全培养基，以用于筛选营养缺陷型藻株；100ml·L-1土豆汁和100ml·L-1豆芽汁均可以在一定程度上促进藻落的生长；蛋白胨以4~6g·L-1效果最佳。选取四种促长效果最佳的营养因子进行正交，结果表明以58.5g·L-1NaCl、320mg·L-1VC、0.2g·L-1尿素和15g·L-1葡萄糖为最优组合。杜氏盐藻对抗生素、除草剂和代谢产物类似物敏感程度因抗生素、除草剂和代谢产物种类不同而有所差异。液体培养中Cm、Em和PPT对盐藻最低抑制浓度分别为120、28和5mg·L-1；固体培养Cm、Em、PPT和Act对盐藻最低抑制浓度60、16、3和0.4mg·L-1；固体培养中盐藻对Sm、Km、Amp和Nm均不敏感，甚至浓度达1000mg·L-1亦不能产生任何抑制作用。液体培养中刀豆氨酸、异烟肼、玲蓝氨酸和6-巯基嘌呤对盐藻的最低抑制浓度均超过200mg·L-1，而ρ-氟苯丙氨酸最低抑制浓度则为180mg·L-1。盐藻无完整的纤维质细胞壁，有一层糖蛋白包裹于外层。研究发现以5mmol·L-1 HEPES pH7.5为缓冲液和0.5mol·L-1山梨醇为稳渗剂，采用400mg·L-1的链霉蛋白酶，30℃，酶解1小时可以获得较高的原生质体形成率，且效果要略好于蛋白酶K、木瓜蛋白酶和胃蛋白酶。和其它一些微藻相比，杜氏盐藻对紫外线抵抗能力较强。紫外线致死剂量略大于<WP=7>60J·m-2。90%致死剂量为13.92 J·m-2，该剂量适于作为诱变剂量。采用不同方法对盐藻进行保种研究，结果表明盐藻适宜于液体、平板和斜面保种。更多还原

【Abstract】 In this thesis, the biological characteristic of Dunaliella salina, which included the axenic purification, physiology characteristics and genetics characteristics, was studied systematically. Three different methods including the combination of antibiotics and centrifugal technique, the combination of centrifugal washing technique and dilution method, and high concentration of salt were designed to proceed for purification of D. salina. Five axenic strains of D. salina were obtained, and axenic status was confirmed by different methods.The influence of different nutrition factors, the salt concentration, vitamins, hormones, organic carbon sources, organic nitrogen sources and extracts of animal and plant, on the growth of strain A1 of D. salina were investigated, The results showed that D. salina can adapt to 0~ 292.5g·L-1 NaCl, and abrupt increase of NaCl would induce lag phase of the growth of D. salina, and the length of the period has been related with concentration of NaCl. The growth of D. salina was promoted in some way by VB1, VB6, VB12, VC or VH, and the best concentrations were 150μg·L-1, 160μg·L-1, 0.5μg·L-1, 320mg·L-1and 2.0μg·L-1, respectively. The promoting effect of glucose was better than potassium acetate, and the most suitable concentrations in liquid and solid medium were 10~15g·L-1and 10~30g·L-1respectively. The promoting effect of urea was better than glycine, and the most suitable concentration in liquid medium was 0.1g·L-1. Inositol, NAA, 6-BA, KT and GA could also promote the growth of D. salina, and the best concentrations were 55, 3, 4, 0.1 and 0.2mg·L-1 respectively. 20 ~ 40ml·L-1 fish soup, 100ml·L-1 bean sprout juice, 100ml·L-1potato juice and 4~6g·L-1peptone could promote the division of D. salina. The Johnsons medium with fish soup can be regarded as a complete medium of D. salina and might be used to select the auxotroph strain of D. salina. The orthonogal test was designed to search the optimum concentration of major affecting factors of NaCl, VC, urea, and glucose. The result showed the optimum medium for mixotrophic growth of strain A1 was NaCl 58.5g·L-1, VC 320mg·L-1, urea 0.2g·L-1 and glucose 15g·L-1.The sensitive degree of D. salina to antibiotics, herbicide and metabolite analog was different. The minimum inhibited concentrations (MIC) of Cm, Em and PPT in liquid ferment amounted to 120, 28 and 5mg·L-1, respectively, while the MICs of Cm, Em, PPT and Act in solid medium were only 60, 16, 3 and 0.4mg·L-1, respectively. The MICs of Sm, <WP=9>Km, Amp, Nm were over 1000mg·L-1 in solid medium. The MICs of isoniazid, L-canavanine, L-azetidine-2-carboxylic acid and mercaptopurine for D. salina in liquid medium were over 200mg·L-1, while the MIC of ρ-Fluorophenylalanine was only 180mg·L-1.D. salina has no complete fibrous cell wall, but glycoprotein coat on the external lamella of the plasmalemma. The protoplasts can be obtained by pronase B in buffer of 5mmol·L-1 HEPES pH7.5 with 0.5mol·L-1 sorbitol as osmotics, and the effect of pronase B was little better than those of proteinase K, papain, and pepsin. The best enzymolysis parameters were as following: 400mg·L-1pronase B, 1h of isolation time, 30℃.The resistant ability of D. salina to the ultraviolet ray was stronger than other microalgaes. The irradiation at a dose over 60J·m-2 could cause death of D. salina completely. The 90% lethal dose was 13.92J·m-2, and the dose was suitable for the isolation of mutant of D. salina. The preservation of axenic strain of D. salina was investigated with different ways. The results showed the algae could be preserved in liquid medium, plate, and slant.更多还原