杜氏盐藻纯化及生物学特性研究
|
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
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。
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,
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.
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
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。
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,
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.
引文
[1]
Ettl H．Taxono mische Bemerkungen zuden phytononadina．Nova Hedvigia, 1983, 35: 731-736
[3]
Chardard R．Cryptogamie Algologie．1987, 8 (3 ) : 173~189
[3]
Pringsheim E. G.．Pure Culture of Algae．1949
[4]
汪志平，贾小明，等．不同形态钝顶螺旋藻藻丝体分离和纯化方法 浙江农业学报 1998，10（5）：275~277
[5]
唐欣昀，甘旭华，等．螺旋藻的无菌纯化．中国微生物学会五十周年会议摘要，2002
[6]
林伟．几种海洋微藻的无菌化培养．海洋科学，2000，24（10）：4~6
[7]
王高歌，张宝红，等．无菌钝顶螺旋藻单细胞的制备与再生．高技术通讯，2001，（4）
[8]
徐增富，刘晓勤，等．无菌短藻丝体钝顶螺旋藻藻株的获得．中山大学学报（自然科学版），1997，36（5）：123~126
[9]
Les protistes mesotrophes, C. R. Acad.．Sci. Paris, 1929, 185, 114
[10]
汪志平，陈声明，等．环境因子和γ射线对钝顶螺旋藻形态和生长的影响．浙江农业大学学报，1997，23（1）：36~40
[11]
龚小敏，胡鸿钧．60Co-γ射线诱变钝顶螺旋藻的研究．武汉植物学研究，1996，14（1）：58~66
[12]
秦松，童顺，等．钝顶螺旋藻质粒的电镜观察及杂交研究．海洋与湖泊，1994，25（5）：560~562
[13]
Ogawa T, Terui G.. Studies on the growth of Spirulina platensis, Ⅰ.On the pure culture of .J. Spirulina platensis Ferment. Technol., 1970,48(6):361~367
[14]
Brown A. D., Borowitakz L. J., Biochemistry and physiology of Protozoa, 1979, 2:139
[16]
华汝成．单细胞藻类的培养与利用．中国：农业出版社，1986，22~100
[15]
古玉环，陈军．盐生杜氏藻的生物学特性与培养研究．1995，31（4）：52~55
[17]
Oren A., Shilo M.．Population dynacics of Dunallella parva in the Dead sea．limnology and Oceanography 1982, 27: 201~211.
[18]
Zenvirth D., Kaplan A.．Uptake and efflux of inorganic carbon in Dunaliella salina．Palanta, 1981 152: 8~12
[19]
杨雪梅，吴超元．盐藻的研究与开发．生命科学，1994，6：（5）：8~10
[20]
Fisher M., Gokhman I., pick U., Zamir A.． A salt-resistant plasma membrane carbonic anhydrace is induced by salt in Dunaliella saline．J Biol chem., 1996, 271: 17718~17723
[21]
Mil’ko E. S.．Study of the requirements of two Dunaliella spp. in mineral and organic components of the medium．Moscow University Vestnik, Biologya, 1962, 6: 21~23
[22]
Grant B. R.．The effect of carbon dioxide concentration and buffer system on nitrate and nitrate and assimilation by Dunaliella tertiolecta．Jounal of General Microbiology, 1968, 54: 327~336
[23]
Grant B.R.．The effect of carbon dioxide concentration and buffer systerm on nitrate and nitrate and assimilation by Dunaliella tertiolecta．Jounal of General Microbiology, 1968, 54: 327~336
[24]
Grant B. R.．Action of light on nitrate and assimilation by the marine Chlorophyte Dunaliella tertiolecta (butcher)．Journal of General Microbiology, 1967, 48: 379~389
[25]
陈峰，姜悦．微藻生物技术．中国：轻工业出版社，1999，144~173
[26]
姜建国，姚汝华．氮磷比对盐藻生长及甘油和色素的积累的影响．热带海洋，1999，18（1）：68~72
[27]
郝建欣，孙钰，等．盐藻生长过程中氮磷利用与色素积累，海洋科学，2003，27（3）：41~44
[28]
耿德贵，王义琴，等．杜氏盐藻基因工程选择性标记的研究．生物技术，2001，11（5）：1~2
[29]
李杰，刘颖，等．G418和Cm作为转基因盐藻的抗生素筛选标记．生物技术，2003，13（3）：22~23
[30]
郑合明，薛乐勋．抗生素及PPT对盐藻生长的影响．郑州大学学报(医学版)，2002年，37（6）：790~793
[31]
Ben-Amotz A., Avron M.．Plant Physiol, 1973, 51:37
[32]
Belmans D., Van Laere A.．Plant Cell Environ, 1987, 10:135
[33]
Avron K., Schreiber U., Avron M.．salt-induceal. Metabolic change．sin, 1980
[34]
Borowitzka M.A., Borowitzka L.J.．Micro-alga Biotechnology. Cambridge: Cambridge- University press, 1988, 27~55
[35]
焦新之， 刘虹．杜氏盐藻质膜ATPase渗透调节的可能作用．植物生理学报，1991，17（4）333~341
[36]
李红萍，焦新之．杜氏盐藻渗透调节过程中的甘油代谢途径．植物生理学报，1994，20（1）：91~99
[37]
黄益群，焦新之．二羟丙酮还原酶在杜氏盐藻渗透调节过程中的特性．植物生理学报， 1993，19（30）：250~256
[38]
Gee R, Goyal A., Byerrum R.U.．Plant Physiolo, 1993, 103: 243
[39]
Sadaka A., Lers A., Zamir A., Arron M.．A critical examination of the role of the novel protein synthesis in the osmotic adaptation of the halotolerant alga Dunaliella. FEBS Letters, 1988, 244: 93~98
[40]
Sadaka A., Himmelhochs, Zamir A.．A 150-kilodalton cell surface protein is induced by salt in the halotolerant green alaga Dunliella salira, Plant Physiol, 1991, 95:822~831
[41]
Fisher M., Pick U., Zamir A., A salt induced 60-kilodalton plasma membrane protein plays a potential role in the extreme halotolerance of the alga Dunaliella, Plant Physiol, 1994,106: 1359~1365
[42]
Fisher M., Grokhma I., Pick U., Zamir A.．A structurally novel transferrin—like protein accumulates in the plasma membrane of the unicellular green alga Dunaliella salina grown in high salinities, J Boil Chem., 1997, 272: 1565~1570
[43]
徐坤，李琳，等．杜氏盐藻盐适应相关蛋白，植物生理学报，1998，24（4）：327-332
[44]
EonSeon Jin, Juergen E. W.．Polle, Anastasios Melis Involvement of zeaxanthin and of the Cbr protein in the repair of photosystem Ⅱfrom photoinhibition in the green alga Dunaliella salina．Biochimica et Biophysica Acta, 2001, 1506: 244~259
[45]
Luca Dondini, Stefania Bonazzi, Donaktella Serafini-Fracassini．RecovEm of growth capacity and of chloroplast transglutaminase avtivity induced by polyamines in a polyamine-deficient variant strain of Dunaliella salina．[J] Plant Physiol, 2000, 157: 473~480
[46]
Shaish A., Ben-Amotz A., Avron M.．Production and seletion of high ( β-carotene mutants of Dunaliella bardawil (Chlorophyta)．J. Ferment Bioeng., 1994, 77 (4) : 352~357
[47]
Hard B. C., Gilmour D.J.．A mutant of Dunaliella parva CCAP19/9 leaking large amounts of glycerol into the medium．[J] Appl. Phycol., 1991, 3 (4) : 376~372
[48]
刘广发，楼士林，等．杜氏藻与大肠杆菌融合初探．厦门大学学报， 1992，31（5）：572~574
[49]
刘广发，楼士林，等．杜氏藻与大肠杆菌跨界融合初探．海洋科学，1993，4：35~40
[50]
何永雷．建立杜氏盐藻的遗传转化系统和cDNA基因文库：［硕士学位论文］．上海：上海交通大学，1998，
[51]
Lee Y. K., Tan H. M.．Interphylum protoplast fusion and genetic recombination of the Algae Porphyridium cruentum and Dunalilla spp．J General Microbiology, 1988, 134(3):635
[52]
周冀明，詹 萍，等．盐藻3-磷酸甘油脱氢酶基因克隆．四川大学学报，1997， 34（1）：105~110
[53]
周冀明，白林含，等．盐藻磷酸甘油脱氢酶基因CDNA文库的构建．四川大学学报，1997，34（6）：839~840
[54]
杨志勇，张庆琪，等．盐藻基因组文库的构建．植物生理学报，2000，26（1）：75~78
[55]
张晓宁，万由衷，等．盐胁迫下盐藻特异表达基因片段的克隆．复旦大学学报，2001，40（5）：500~503
[56]
方孝东，林栖风，等．盐藻线粒体GIY-YIG族归巢内切酶基因受到盐胁迫时增强转录．中国生物化学与分子生物学报，2003，19（5）：625~629
[57]
Long Z. F., Wang S.Y, Nelson N.．cloning and nucleotide sequence analysis of gene cooling for the major chlorophyll-binding protein of the moss Phyacomitrella patens and halotolerant alga Dunaliella salina．1989,76:299~312
[58]
谈诤，林志新，等．盐生盐藻肌动蛋白基因及其5′上游片段的克隆．上海交通大学学报， 2001，25（7）：1058~1061
[59]
Ko J. H., Kim J. Y., Lee S H.．Molecular cloning and characterization of a cDNA encoding caltractin from Dunaliella salina．Plant Cell Physiol, 1999, 40(5): 457~461
[60]
姜国忠，袁保梅，等．简并引物在克隆盐藻热休克蛋白70基因家族中的应用．郑州大学学报，2003，38（6）：875~877
[61]
张晓宁，王昊，等．盐藻启动子活性片段的克隆及序列分析．复旦学报(自然科学版)，2002，41（22）：227~229
[62]
王天云，张贵星，薛乐勋．一种简便高效的改良降落PCR．中国生物工程杂志，2003，23（11）：80~82
[63]
耿德贵，王义琴，等．Gus基因在杜氏盐藻细胞中的瞬间表达．高技术通讯，2002，2：35~39
[64]
李杰，任宏伟，等．外源报告基因EGFP在盐藻中实现瞬时表达．中国生物化学与分子生物学报，2003，19（3）：338~342
[65]
周德庆．微生物学教程．北京：高等教育出版社，1993，99~106
[66]
李季伦，张伟心，等．微生物生理学．北京：北京农业大学出版社，1993，74~79
[67]
记明侯．海藻化学．北京：科学出版社，1997，466~467
[68]
张宪政，陈凤玉，王荣富．植物生理学实验技术．辽宁：科学技术出版社，1994，66-67
[69]
Вендт，В，П.，Дрокова Т.Г.У.К.Р.ботанлчнийЖ；1962，19，No.6，60～63
[70]
Tuchman N.C.．The role of heterotrophy in alga. In: R. J. Stevenson, M. L. Bothwell and R.L.Loweeds．Alga ecology, San Diego: Academic Press, 1996, 299~319
[71]
蔡传杰，陈善娜．植物激素的研究进展．云南大学学报（自然科学版），2001，23：99~101
[72]
雅可夫.莱什姆．植物生长调节的分子及激素基础．中国：科学出版社，1980，95~107
[73]
Evans L., Trewavas A.．Is algal development controlled by plant growth substance?． J. Phycol, 1991, 27: 322~326
[74]
Stirk W. A., et al.．J Appl. Phycol., 1998, 9: 327~330
[75]
潘瑞炽，董愚得．植物生理学（第三版）．北京：高等教育出版社，1993，109~203
[76]
欧阳英石．磷酸肌醇家族与细胞功能．生物物理学报，2000，6，16（2）：195~203
[77]
韩丽君．海藻中植物生长素组成的初步研究．海洋科学，2003，27（3）：70~72
[78]
韩丽君，范晓．海藻植物生长素的提取和分离纯化方法．海洋与湖沼2003，20（2）：70~76
[79]
张成武，唐乐同．硫酸铵对钝顶螺旋藻生长的影响．海洋湖沼通报，1992，（2）：35~39
[80]
Christine Oesteerhelt, Wolfgang Gross．Different Sugar Kinases are involved in the sugar sensing of Galdieria sulphuraria．Plant Physiol, 2002, 128 (1): 291~299
[81]
Ray P.M., Curry G. M.．Intermediates and competing reactions in the photodestruction of indoleacetic acid．Nature, 1958, 181: 895~896
[82]
Broun J., Tevini M.．Regulation of UV-protective pigment synthesis in the epidermal layer of rye seedling (Sescle cereale L.)．Photochem. Photobiol..1993, 57 ( 2 ): 38~323
[83]
Quaite F.E., Sutherland B.M., Sutherland J.C.．Action spectrum for DNA damage in alfalfa lowers predicted impact of ozone depletion．Nature, 1992, 358: 576~578
[84]
周俊初. 微生物遗传学（第一版）．中国：农业出版社，1996，31~34
[85]
王起华，张恩栋，等．盐生杜氏藻和青岛大扁藻的超低温保存．植物学报，2000，42（4）：399~402
Ettl H．Taxono mische Bemerkungen zuden phytononadina．Nova Hedvigia, 1983, 35: 731-736
[3]
Chardard R．Cryptogamie Algologie．1987, 8 (3 ) : 173~189
[3]
Pringsheim E. G.．Pure Culture of Algae．1949
[4]
汪志平，贾小明，等．不同形态钝顶螺旋藻藻丝体分离和纯化方法 浙江农业学报 1998，10（5）：275~277
[5]
唐欣昀，甘旭华，等．螺旋藻的无菌纯化．中国微生物学会五十周年会议摘要，2002
[6]
林伟．几种海洋微藻的无菌化培养．海洋科学，2000，24（10）：4~6
[7]
王高歌，张宝红，等．无菌钝顶螺旋藻单细胞的制备与再生．高技术通讯，2001，（4）
[8]
徐增富，刘晓勤，等．无菌短藻丝体钝顶螺旋藻藻株的获得．中山大学学报（自然科学版），1997，36（5）：123~126
[9]
Les protistes mesotrophes, C. R. Acad.．Sci. Paris, 1929, 185, 114
[10]
汪志平，陈声明，等．环境因子和γ射线对钝顶螺旋藻形态和生长的影响．浙江农业大学学报，1997，23（1）：36~40
[11]
龚小敏，胡鸿钧．60Co-γ射线诱变钝顶螺旋藻的研究．武汉植物学研究，1996，14（1）：58~66
[12]
秦松，童顺，等．钝顶螺旋藻质粒的电镜观察及杂交研究．海洋与湖泊，1994，25（5）：560~562
[13]
Ogawa T, Terui G.. Studies on the growth of Spirulina platensis, Ⅰ.On the pure culture of .J. Spirulina platensis Ferment. Technol., 1970,48(6):361~367
[14]
Brown A. D., Borowitakz L. J., Biochemistry and physiology of Protozoa, 1979, 2:139
[16]
华汝成．单细胞藻类的培养与利用．中国：农业出版社，1986，22~100
[15]
古玉环，陈军．盐生杜氏藻的生物学特性与培养研究．1995，31（4）：52~55
[17]
Oren A., Shilo M.．Population dynacics of Dunallella parva in the Dead sea．limnology and Oceanography 1982, 27: 201~211.
[18]
Zenvirth D., Kaplan A.．Uptake and efflux of inorganic carbon in Dunaliella salina．Palanta, 1981 152: 8~12
[19]
杨雪梅，吴超元．盐藻的研究与开发．生命科学，1994，6：（5）：8~10
[20]
Fisher M., Gokhman I., pick U., Zamir A.． A salt-resistant plasma membrane carbonic anhydrace is induced by salt in Dunaliella saline．J Biol chem., 1996, 271: 17718~17723
[21]
Mil’ko E. S.．Study of the requirements of two Dunaliella spp. in mineral and organic components of the medium．Moscow University Vestnik, Biologya, 1962, 6: 21~23
[22]
Grant B. R.．The effect of carbon dioxide concentration and buffer system on nitrate and nitrate and assimilation by Dunaliella tertiolecta．Jounal of General Microbiology, 1968, 54: 327~336
[23]
Grant B.R.．The effect of carbon dioxide concentration and buffer systerm on nitrate and nitrate and assimilation by Dunaliella tertiolecta．Jounal of General Microbiology, 1968, 54: 327~336
[24]
Grant B. R.．Action of light on nitrate and assimilation by the marine Chlorophyte Dunaliella tertiolecta (butcher)．Journal of General Microbiology, 1967, 48: 379~389
[25]
陈峰，姜悦．微藻生物技术．中国：轻工业出版社，1999，144~173
[26]
姜建国，姚汝华．氮磷比对盐藻生长及甘油和色素的积累的影响．热带海洋，1999，18（1）：68~72
[27]
郝建欣，孙钰，等．盐藻生长过程中氮磷利用与色素积累，海洋科学，2003，27（3）：41~44
[28]
耿德贵，王义琴，等．杜氏盐藻基因工程选择性标记的研究．生物技术，2001，11（5）：1~2
[29]
李杰，刘颖，等．G418和Cm作为转基因盐藻的抗生素筛选标记．生物技术，2003，13（3）：22~23
[30]
郑合明，薛乐勋．抗生素及PPT对盐藻生长的影响．郑州大学学报(医学版)，2002年，37（6）：790~793
[31]
Ben-Amotz A., Avron M.．Plant Physiol, 1973, 51:37
[32]
Belmans D., Van Laere A.．Plant Cell Environ, 1987, 10:135
[33]
Avron K., Schreiber U., Avron M.．salt-induceal. Metabolic change．sin, 1980
[34]
Borowitzka M.A., Borowitzka L.J.．Micro-alga Biotechnology. Cambridge: Cambridge- University press, 1988, 27~55
[35]
焦新之， 刘虹．杜氏盐藻质膜ATPase渗透调节的可能作用．植物生理学报，1991，17（4）333~341
[36]
李红萍，焦新之．杜氏盐藻渗透调节过程中的甘油代谢途径．植物生理学报，1994，20（1）：91~99
[37]
黄益群，焦新之．二羟丙酮还原酶在杜氏盐藻渗透调节过程中的特性．植物生理学报， 1993，19（30）：250~256
[38]
Gee R, Goyal A., Byerrum R.U.．Plant Physiolo, 1993, 103: 243
[39]
Sadaka A., Lers A., Zamir A., Arron M.．A critical examination of the role of the novel protein synthesis in the osmotic adaptation of the halotolerant alga Dunaliella. FEBS Letters, 1988, 244: 93~98
[40]
Sadaka A., Himmelhochs, Zamir A.．A 150-kilodalton cell surface protein is induced by salt in the halotolerant green alaga Dunliella salira, Plant Physiol, 1991, 95:822~831
[41]
Fisher M., Pick U., Zamir A., A salt induced 60-kilodalton plasma membrane protein plays a potential role in the extreme halotolerance of the alga Dunaliella, Plant Physiol, 1994,106: 1359~1365
[42]
Fisher M., Grokhma I., Pick U., Zamir A.．A structurally novel transferrin—like protein accumulates in the plasma membrane of the unicellular green alga Dunaliella salina grown in high salinities, J Boil Chem., 1997, 272: 1565~1570
[43]
徐坤，李琳，等．杜氏盐藻盐适应相关蛋白，植物生理学报，1998，24（4）：327-332
[44]
EonSeon Jin, Juergen E. W.．Polle, Anastasios Melis Involvement of zeaxanthin and of the Cbr protein in the repair of photosystem Ⅱfrom photoinhibition in the green alga Dunaliella salina．Biochimica et Biophysica Acta, 2001, 1506: 244~259
[45]
Luca Dondini, Stefania Bonazzi, Donaktella Serafini-Fracassini．RecovEm of growth capacity and of chloroplast transglutaminase avtivity induced by polyamines in a polyamine-deficient variant strain of Dunaliella salina．[J] Plant Physiol, 2000, 157: 473~480
[46]
Shaish A., Ben-Amotz A., Avron M.．Production and seletion of high ( β-carotene mutants of Dunaliella bardawil (Chlorophyta)．J. Ferment Bioeng., 1994, 77 (4) : 352~357
[47]
Hard B. C., Gilmour D.J.．A mutant of Dunaliella parva CCAP19/9 leaking large amounts of glycerol into the medium．[J] Appl. Phycol., 1991, 3 (4) : 376~372
[48]
刘广发，楼士林，等．杜氏藻与大肠杆菌融合初探．厦门大学学报， 1992，31（5）：572~574
[49]
刘广发，楼士林，等．杜氏藻与大肠杆菌跨界融合初探．海洋科学，1993，4：35~40
[50]
何永雷．建立杜氏盐藻的遗传转化系统和cDNA基因文库：［硕士学位论文］．上海：上海交通大学，1998，
[51]
Lee Y. K., Tan H. M.．Interphylum protoplast fusion and genetic recombination of the Algae Porphyridium cruentum and Dunalilla spp．J General Microbiology, 1988, 134(3):635
[52]
周冀明，詹 萍，等．盐藻3-磷酸甘油脱氢酶基因克隆．四川大学学报，1997， 34（1）：105~110
[53]
周冀明，白林含，等．盐藻磷酸甘油脱氢酶基因CDNA文库的构建．四川大学学报，1997，34（6）：839~840
[54]
杨志勇，张庆琪，等．盐藻基因组文库的构建．植物生理学报，2000，26（1）：75~78
[55]
张晓宁，万由衷，等．盐胁迫下盐藻特异表达基因片段的克隆．复旦大学学报，2001，40（5）：500~503
[56]
方孝东，林栖风，等．盐藻线粒体GIY-YIG族归巢内切酶基因受到盐胁迫时增强转录．中国生物化学与分子生物学报，2003，19（5）：625~629
[57]
Long Z. F., Wang S.Y, Nelson N.．cloning and nucleotide sequence analysis of gene cooling for the major chlorophyll-binding protein of the moss Phyacomitrella patens and halotolerant alga Dunaliella salina．1989,76:299~312
[58]
谈诤，林志新，等．盐生盐藻肌动蛋白基因及其5′上游片段的克隆．上海交通大学学报， 2001，25（7）：1058~1061
[59]
Ko J. H., Kim J. Y., Lee S H.．Molecular cloning and characterization of a cDNA encoding caltractin from Dunaliella salina．Plant Cell Physiol, 1999, 40(5): 457~461
[60]
姜国忠，袁保梅，等．简并引物在克隆盐藻热休克蛋白70基因家族中的应用．郑州大学学报，2003，38（6）：875~877
[61]
张晓宁，王昊，等．盐藻启动子活性片段的克隆及序列分析．复旦学报(自然科学版)，2002，41（22）：227~229
[62]
王天云，张贵星，薛乐勋．一种简便高效的改良降落PCR．中国生物工程杂志，2003，23（11）：80~82
[63]
耿德贵，王义琴，等．Gus基因在杜氏盐藻细胞中的瞬间表达．高技术通讯，2002，2：35~39
[64]
李杰，任宏伟，等．外源报告基因EGFP在盐藻中实现瞬时表达．中国生物化学与分子生物学报，2003，19（3）：338~342
[65]
周德庆．微生物学教程．北京：高等教育出版社，1993，99~106
[66]
李季伦，张伟心，等．微生物生理学．北京：北京农业大学出版社，1993，74~79
[67]
记明侯．海藻化学．北京：科学出版社，1997，466~467
[68]
张宪政，陈凤玉，王荣富．植物生理学实验技术．辽宁：科学技术出版社，1994，66-67
[69]
Вендт，В，П.，Дрокова Т.Г.У.К.Р.ботанлчнийЖ；1962，19，No.6，60～63
[70]
Tuchman N.C.．The role of heterotrophy in alga. In: R. J. Stevenson, M. L. Bothwell and R.L.Loweeds．Alga ecology, San Diego: Academic Press, 1996, 299~319
[71]
蔡传杰，陈善娜．植物激素的研究进展．云南大学学报（自然科学版），2001，23：99~101
[72]
雅可夫.莱什姆．植物生长调节的分子及激素基础．中国：科学出版社，1980，95~107
[73]
Evans L., Trewavas A.．Is algal development controlled by plant growth substance?． J. Phycol, 1991, 27: 322~326
[74]
Stirk W. A., et al.．J Appl. Phycol., 1998, 9: 327~330
[75]
潘瑞炽，董愚得．植物生理学（第三版）．北京：高等教育出版社，1993，109~203
[76]
欧阳英石．磷酸肌醇家族与细胞功能．生物物理学报，2000，6，16（2）：195~203
[77]
韩丽君．海藻中植物生长素组成的初步研究．海洋科学，2003，27（3）：70~72
[78]
韩丽君，范晓．海藻植物生长素的提取和分离纯化方法．海洋与湖沼2003，20（2）：70~76
[79]
张成武，唐乐同．硫酸铵对钝顶螺旋藻生长的影响．海洋湖沼通报，1992，（2）：35~39
[80]
Christine Oesteerhelt, Wolfgang Gross．Different Sugar Kinases are involved in the sugar sensing of Galdieria sulphuraria．Plant Physiol, 2002, 128 (1): 291~299
[81]
Ray P.M., Curry G. M.．Intermediates and competing reactions in the photodestruction of indoleacetic acid．Nature, 1958, 181: 895~896
[82]
Broun J., Tevini M.．Regulation of UV-protective pigment synthesis in the epidermal layer of rye seedling (Sescle cereale L.)．Photochem. Photobiol..1993, 57 ( 2 ): 38~323
[83]
Quaite F.E., Sutherland B.M., Sutherland J.C.．Action spectrum for DNA damage in alfalfa lowers predicted impact of ozone depletion．Nature, 1992, 358: 576~578
[84]
周俊初. 微生物遗传学（第一版）．中国：农业出版社，1996，31~34
[85]
王起华，张恩栋，等．盐生杜氏藻和青岛大扁藻的超低温保存．植物学报，2000，42（4）：399~402