雨生红球藻和小球藻间的原生质体融合与糖异养融合子筛选
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摘要
为了增强雨生红球藻的异养生长能力,提高规模化培养效率,本研究基于PEG介导的原生质体融合技术,在雨生红球藻SCCAP K-0084和小球藻SAG211.11a间开展了细胞融合以及糖异养融合子筛选研究,筛选中通过限制性糖异养条件和添加潮霉素分别抑制野生型的雨生红球藻和小球藻的生长,提高筛选效率。结果表明,小球藻SAG211.11a具有极强的异养生长能力,能够利用葡萄糖、果糖、乙酸钠、甘油、苹果酸以及琥珀酸进行异养生长,而雨生红球藻则仅能够利用乙酸盐进行异养生长,此外雨生红球藻SCCAP K-0084还具有极强的潮霉素耐受能力,可以被应用于融合子筛选中。研究中,雨生红球藻和小球藻的原生质体制备效率分别达到72.11%±3.94%和42.07%±3.73%,满足细胞融合需要,融合过程中观察到雨生红球藻和小球藻间的典型融合现象,并且在限制性糖异养筛选下获得了雨生红球藻形态的融合子克隆,融合子的生长速率相比于野生型雨生红球藻有明显的增强,三个融合子在10 d培养后细胞密度分别达到6.7×10~4、8.7×10~4、6.5×10~4个/mL,明显高于野生型的2.45×10~4个/mL,但是相比于小球藻10 d后1.4×10~6个/mL的细胞密度而言,异养生长能力仍然有限,而且在连续传代过程中融合子经常会出现表型分化的现象。这一研究证明,通过细胞融合杂交的方式能够使雨生红球藻获得糖异养的生长能力,但是异源基因组间重组效率有待进一步提高。
To improve heterotrophic growth ability of Haematococcus pluvialis,improve the efficiency of large-scale cultivation,cell hybridization and fusant screening between H.pluvialis SCCAP K-0084 and heterotrophic Chlorella SAG211.11 a were carried out through PEG-mediated protoplast fusion. A screening condition only permiting heterotrophic growth based on glucose along with addition of hygromycin was utilized to improve the screening efficiency,which could inhibite growth of wild-typle H.pluvialis and C.kessleri,respectively. Results showed that,Chlorella kessleri SAG211.11 a had strong heterotrophic growth capacity,which could metabolite glucose,fructose,acetate,glycerol,malic acid and succinic acid in its heterotrophic growth,while three H.pluvialis strains could just utilize acetate for its growth. And H.pluvialis SCCAP K-0084 exhibited strong hygromycin-resistant ability. These characters could be applied in the process of fusant screening. Protoplast-releasing rates respectively reached 72.11%±3.94% and 42.07%±3.73% in H.pluvialis and C.kessleri. And fusants with Haematococcus form were obtained on the screening condition only permiting heterotrophic growth on glucose after effective cell fusion identified through microscopic check. These fusants had faster growth than wild type H.pluvialis in the condition with glucose as substrates. After 10 d cultivation,the cell densities respectively arrived at 6.7×10~4,8.7×10~4 and 6.5×10~4 mL~(-1) in three independent fusants,obviously higher than 2.45×10~4 in wild type H.pluvialis. However,they were obviousy lower than 1.4×10~6 mL~(-1) in C.kessleri,which meant a low-efficient heterotrophic growth on glucose. It was also found that,many fusants were easy to lose their capacity of glucose utilization with extended inoculation. That might be due to inefficient recombination between two genomes.
To improve heterotrophic growth ability of Haematococcus pluvialis,improve the efficiency of large-scale cultivation,cell hybridization and fusant screening between H.pluvialis SCCAP K-0084 and heterotrophic Chlorella SAG211.11 a were carried out through PEG-mediated protoplast fusion. A screening condition only permiting heterotrophic growth based on glucose along with addition of hygromycin was utilized to improve the screening efficiency,which could inhibite growth of wild-typle H.pluvialis and C.kessleri,respectively. Results showed that,Chlorella kessleri SAG211.11 a had strong heterotrophic growth capacity,which could metabolite glucose,fructose,acetate,glycerol,malic acid and succinic acid in its heterotrophic growth,while three H.pluvialis strains could just utilize acetate for its growth. And H.pluvialis SCCAP K-0084 exhibited strong hygromycin-resistant ability. These characters could be applied in the process of fusant screening. Protoplast-releasing rates respectively reached 72.11%±3.94% and 42.07%±3.73% in H.pluvialis and C.kessleri. And fusants with Haematococcus form were obtained on the screening condition only permiting heterotrophic growth on glucose after effective cell fusion identified through microscopic check. These fusants had faster growth than wild type H.pluvialis in the condition with glucose as substrates. After 10 d cultivation,the cell densities respectively arrived at 6.7×10~4,8.7×10~4 and 6.5×10~4 mL~(-1) in three independent fusants,obviously higher than 2.45×10~4 in wild type H.pluvialis. However,they were obviousy lower than 1.4×10~6 mL~(-1) in C.kessleri,which meant a low-efficient heterotrophic growth on glucose. It was also found that,many fusants were easy to lose their capacity of glucose utilization with extended inoculation. That might be due to inefficient recombination between two genomes.
引文
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[15]Fischer H,Robl I,Sumper M,et al.Targeting and covalent modification of cell wall and membrane proteins heterologously expressed in the diatom Cylindrotheca fusiformis(Bacillariophyceae)[J].Journal of Phycology,1999,35(1):113-120.
[16]Chen G,Chen F.Growing phototrophics cells without light[J].Biotechnology Letters,2006,28:607-616.
[17]Wood A,Aurikko J,Kelly D.A challenge for 21st century molecular biology and biochemistry:What are the causes of obligate autotrophy and methanoltrophy?[J].FEMS Microbiology Reviews,2004,28:335-352.
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[19]Sharon-Gojman R,Maimon E,Leu S,et al.Advanced methods for genetic engineering of Haematococcus pluvialis(Chlorophyceae,Volvocales)[J].Algal Research-Biomass Biofuels and Bioproducts,2015,10:8-15.
[20]Perez-Garcia O,Escalante F M E,de-Bashan L E,et al.Heterotrophic cultures of microalgae:Metabolism and potential products[J].Water Research,2011,45(1):11-36.
[21]Stainier R Y,Kunisawa R,Mandel M,et al.Purification and properties of unicellular blue-green algae(order Chroococcales)[J].Bacteriology Reviews,1971,35(2):171-205.
[22]Cheng T,Xu X,Zhang W,et al.Protoplast preparation from enriched flagellates and resting cells of Haematococcus pluvialis[J].Journal of Applied Microbiology,2018,124(2):469-479.
[23]Rosen B H,Berliner M D,Petro M J.Protoplast induction in Chlorella pyrenoidosa[J].Plant Science,1985,41(1):23-30.
[24]Tjahjono A E,Kakizono T,Hayama Y,et al.Formation and regeneration of protoplast from a unicellular green alga Haematococcus pluvialis[J].Journal of Fermentation and Bioengineering,1993,75(3):196-200.
[25]陈波.两种微型绿藻营养生理及原生质体制备与杂交育种的研究[D].广州:暨南大学,2000.
[26]Morales-Sánchez D,Martinez-Rodriguez O A,Kyndt J,et al.Heterotrophic growth of microalgae:metabolic aspects[J].World Journal of Microbiology and Biotechnology,2015,31:1-9.
[27]徐晓莹,程天佑,陈林,等.两种培养基间雨生红球藻细胞生长分化差异及磷的作用[J].过程工程学报,2016,16(5):840-848.
[28]Yamada T,Sakaguchi K.Protoplast induction in Chlorella species[J].Agricultural and Biological Chemistry,1981,45(8):1905-1909.
[29]Yamada T,Sakaguchi K.Comparative studies on Chlorella cell walls:Induction of protoplast formation[J].Archives of Microbiology,1982,132(1):10-13.
[30]沈继红,林学政,刘发义,等.细胞融合法构建EPA和DHA高产异养藻株的研究[J].中国水产科学,2001,8(6):63-66.
[31]江胜滔,施巧琴,黄建忠,等.酿酒酵母与雨生红球藻的细胞融合子的RAPD鉴定[J].福建师范大学学报:自然科学版,2004(4):76-79.
[2]高桂玲,成家杨,马炯.雨生红球藻和虾青素的研究[J].水产学报,2014,38(2):297-304.
[3]殷明焱,刘建国,张京浦,等.雨生红球藻和虾青素研究述评[J].海洋湖沼通报,1998(2):53-62.
[4]Han D X,Li Y T,Hu Q.Astaxanthin in microalgae:Pathways,functions and biotechnological implications[J].Algae,2013,28(2):131-147.
[5]蔡明刚,李峰.雨生红球藻规模化培养技术的研发进展[J].厦门大学学报:自然科学版,2016,55(5):733-741.
[6]Del Campo J A,Garcia-Gonzalez M,Guerrero M G.Outdoor cultivation of microalgae for carotenoid production:Current state and perspectives[J].Applied Microbiology and Biotechnology,2007,74(6):1163-1174.
[7]郭晓茜,任丹丹,张继红,等.不同微藻培养方式产生虾青素及其影响因素的研究现状[J].食品工业科技,2016,37(17):381-384.
[8]龙元薷,刘建国,张立涛.兼养对雨生红球藻细胞生长的促进作用及藻株差异性[J].海洋科学,2014,38(12):1-7.
[9]庄惠如,陈必链,王明兹,等.雨生红球藻混合营养与异养培养研究[J].微生物学通报,2000(3):198-201.
[10]Kobayashi M,Kakizono T,Yamaguchi K,et al.Growth and astaxanthin formation of Haematococcus pluvialis in heterotrophic and mixotrophic conditions[J].Journal of Fermentation and Bioengineering,1992,74(1):17-20.
[11]Hata N,Ogbonna J C,Hasegawa Y,et al.Production of astaxanthin by Haematococcus pluvialis in a sequential heterotrophic-photoautotrophic culture[J].Journal of Applied Phycology,2001,13(5):395-402.
[12]Wan M X,Zhang Z,Wang J,et al.Sequential heterotrophy-dilution-photoinduction cultivation of Haematococcus pluvialis for efficient production of astaxanthin[J].Bioresource Technology,2015,198:557-563.
[13]Zaslavskaia L A,Lippmeier J C,Shih C,et al.Trophic obligate conversion of an photoautotrophic organism through metabolic engineering[J].Science,2001,292(5524):2073-2075.
[14]Hallmann A,Sumper M.The Chlorella hexose H+ symporter is a useful selectable marker and biochemical reagent when expressed in Volvox[J].Proceedings of the National Academy of Sciences of the United States of America,1996,93(2):669-673.
[15]Fischer H,Robl I,Sumper M,et al.Targeting and covalent modification of cell wall and membrane proteins heterologously expressed in the diatom Cylindrotheca fusiformis(Bacillariophyceae)[J].Journal of Phycology,1999,35(1):113-120.
[16]Chen G,Chen F.Growing phototrophics cells without light[J].Biotechnology Letters,2006,28:607-616.
[17]Wood A,Aurikko J,Kelly D.A challenge for 21st century molecular biology and biochemistry:What are the causes of obligate autotrophy and methanoltrophy?[J].FEMS Microbiology Reviews,2004,28:335-352.
[18]Benedict C.Nature of obligate photoautotrophy[J].Annual Review of Plant Physiology,1978,29:67-93.
[19]Sharon-Gojman R,Maimon E,Leu S,et al.Advanced methods for genetic engineering of Haematococcus pluvialis(Chlorophyceae,Volvocales)[J].Algal Research-Biomass Biofuels and Bioproducts,2015,10:8-15.
[20]Perez-Garcia O,Escalante F M E,de-Bashan L E,et al.Heterotrophic cultures of microalgae:Metabolism and potential products[J].Water Research,2011,45(1):11-36.
[21]Stainier R Y,Kunisawa R,Mandel M,et al.Purification and properties of unicellular blue-green algae(order Chroococcales)[J].Bacteriology Reviews,1971,35(2):171-205.
[22]Cheng T,Xu X,Zhang W,et al.Protoplast preparation from enriched flagellates and resting cells of Haematococcus pluvialis[J].Journal of Applied Microbiology,2018,124(2):469-479.
[23]Rosen B H,Berliner M D,Petro M J.Protoplast induction in Chlorella pyrenoidosa[J].Plant Science,1985,41(1):23-30.
[24]Tjahjono A E,Kakizono T,Hayama Y,et al.Formation and regeneration of protoplast from a unicellular green alga Haematococcus pluvialis[J].Journal of Fermentation and Bioengineering,1993,75(3):196-200.
[25]陈波.两种微型绿藻营养生理及原生质体制备与杂交育种的研究[D].广州:暨南大学,2000.
[26]Morales-Sánchez D,Martinez-Rodriguez O A,Kyndt J,et al.Heterotrophic growth of microalgae:metabolic aspects[J].World Journal of Microbiology and Biotechnology,2015,31:1-9.
[27]徐晓莹,程天佑,陈林,等.两种培养基间雨生红球藻细胞生长分化差异及磷的作用[J].过程工程学报,2016,16(5):840-848.
[28]Yamada T,Sakaguchi K.Protoplast induction in Chlorella species[J].Agricultural and Biological Chemistry,1981,45(8):1905-1909.
[29]Yamada T,Sakaguchi K.Comparative studies on Chlorella cell walls:Induction of protoplast formation[J].Archives of Microbiology,1982,132(1):10-13.
[30]沈继红,林学政,刘发义,等.细胞融合法构建EPA和DHA高产异养藻株的研究[J].中国水产科学,2001,8(6):63-66.
[31]江胜滔,施巧琴,黄建忠,等.酿酒酵母与雨生红球藻的细胞融合子的RAPD鉴定[J].福建师范大学学报:自然科学版,2004(4):76-79.