3种小球藻DNA提取方法的比较

单细胞真核藻类小球藻已成为藻类分子生物学研究的热点,但其特殊的细胞壁组分给DNA的提取带来了一定的困难。文章比较了3种从小球藻中提取DNA的方法,即纤维素酶法、作者改进的CATB法和目前单细胞藻类常用的裂解法。通过所提取的DNA进行浓度、纯度分析,认为CTAB法是一种简单、快速和高效的适合于小球藻的DNA提取方法。     

一种纯化高等植物叶绿体DNA的有效方法

１９８４年,Ｂｏｏｋｊａｎｓ ｅｔ ａｌ．建立了高盐介质纯叶绿体ＤＮＡ的方法。在我们进行的高粱叶绿体光系统基因研究中发现,用Ｂｏｏｋｊａｎｓ的方法提取的ｃｔＤＮＡ酶切效果欠佳,产率太低。应用我们反进的高盐低ＰＨ方法,纯化了高粱、小麦、水稻和速效豌豆等植物的ｃｔＤＮＡ。结果表明,这种改进的方法具有有得率高,酶切效果好和操作简便等优点。     

水稻叶绿体DNA的提取和纯化

以籼稻品种珍讪97B为材料,采用溶液捣碎和不连续蔗糖梯度离心的方法提取了籼稻的叶绿体DNA,DNA经限制性内切酶酶解和琼脂糖胶电泳可以得到清晰的条带,来自蚕豆的核酮糖—1,5—二磷酸羧化氧合酶大亚基基因探针和23SrRNA基因探针可以与酶切条带杂交,由此确定了含这二种基因的BamHI酶切片段。     

叶绿体DNA的发现历程

叶绿体是半自主性的细胞器,含有自己的DNA和遗传信息表达系统,可自身转录和翻译部分必需的蛋白.20世纪60年代叶绿体DNA的发现为内共生假说提供了有力的证据.回顾了叶绿体DNA的发现历史,介绍了科学家们运用遗传杂交、组织化学染色、电子显微镜观察以及生物化学等各种生物学实验手段证明叶绿体DNA存在的过程.     

中国野生葡萄叶绿体分离及叶绿体DNA提取的研究

以中国野生刺葡萄、山葡萄、桑叶葡萄和东南葡萄的成熟叶片为材料,比较柱式植物叶绿体DNAout试剂盒和改良的高盐-低pH法分离叶绿体及提取cpDNA效果。结果显示:(1)2种方法均分离得到了中国野生葡萄的叶绿体,但与柱式植物叶绿体DNAout试剂盒相比,改良的高盐-低pH法得到的叶绿体浓度高、杂质少,更适合中国野生葡萄叶绿体分离。(2)柱式植物叶绿体DNAout试剂盒提取的cpDNA,其OD260/OD280在1.28~1.36之间,质量浓度为4.2~7.8ng·μL~(-1),质量低,不能满足后续测序要求;而改良的高盐-低pH法提取的cpDNA,其OD260/OD280值在1.84~1.90之间,质量浓度为2 514.4~4 133.7ng·μL~(-1),质量高,纯度好,能满足后续测序要求。研究表明,改良的高盐-低pH法能简便、快速获得中国野生葡萄完整叶绿体及高质量cpDNA,并且提取的cpDNA可满足后续测序要求,为葡萄属植物叶绿体基因组的深入研究奠定了基础。     

栽培草莓叶绿体分离与cpDNA的提取方法

以栽培草莓‘红颊’(Fragaria×ananassa Duch.‘Benihoppe’)新鲜幼嫩叶片为材料,利用高盐-低pH的缓冲液结合Percoll密度梯度离心分离纯化叶绿体,再用SDS-蛋白酶K法提取叶绿体DNA(cpDNA)。经荧光显微镜、琼脂糖凝胶电泳和PCR扩增检测,结果表明:该方法分离的草莓叶绿体完整性高,叶绿体DNA质量好,纯度高,能够满足后续基因组测序等实验的基本要求,为草莓属及其他草本植物cpDNA的提取提供参考。     

利用DNA芯片技术高效合成OstreococcusTauri叶绿体基因组

目的：21世纪以来,随着合成生物学的高速发展及其所遇到的问题,开发下一代DNA合成技术已经成为了必然趋势。基因芯片技术和DNA大片段组装技术是建立下一代DNA合成平台的关键技术力量。方法：为了开发具有工业化标准的DNA芯片一基因组合成平台,我们首次利用电化学DNA芯片和DNA大片段组装技术合成了72kb的Ostreococcusmud的全叶绿体基因组。结果：首先,我们使用电化学DNA芯片合成仪合成了564条150bp的OligoMix,并成功扩增分离了其中96％的Oligo序列,剩下的基因组序列是通过传统的固相亚磷酰胺三脂合成法合成。在此基础上,我们利用DNA重组技术将564条150bpOligo片段分三步克隆到了一个pGSYN系统。通过高通量测序,我们证实叶绿体基因组被成功地人工合成。整个合成成本大约是目前传统基因合成成本的10％．20％。结论：研究证实基因芯片技术和DNA大片段组装技术的应用是能够明显的降低现阶段基因组合成工艺的成本。新技术的成熟推广和成本的有效控制也会进一步加速科学家对基因组功能的深入研究以及合成生物学的质的飞跃。     

猕猴桃干叶片DNA的提取及叶绿体基因PCR-RFLP反应

采用CTAB法猕猴桃干叶片提取总DNA,运用PCR技术扩增出rbcL和psbA两个叶绿体基因片段,分别用两种限制性内切酶对它们进行酶切分析,实验结果表明,当CTAB提取液体积数为750ul,猕猴桃叶片质量为10mg时,所得到的猕猴桃DNA的质量较为理想：DNA模板量为250ng时,扩增到的特异性条带明亮,无拖尾,2个基因的酶切结果共得到25个限制性位点,其中24个具有多态性,为从cpDNA水平探讨猕猴桃属植物的系统发育打下了坚实的基础。     

高等植物的一种叶绿体转录系统

引言 近几年来,在DNA序列水平上对质体基因组的结构做了广泛的研究。与之相比,对于叶绿体基因表达机理,为叶绿体RNA聚合酶所使用的促动子的组成分,以及对转录活性的时间和发育控制的调节区域却知道得很少。随着具有转录活性的叶绿体DNA蛋白质复合体--有转录活性的染色体(TAC)的分离,更详细地研究叶绿体核蛋白 。     

热带普通小球藻培养模式的筛选及其培养基的优化

【背景】从海南热带海区中分离得到一株微藻,其生长速度快、适应力强,经鉴定该微藻为普通小球藻。【目的】提高热带普通小球藻的生长速率。【方法】以"宁波大学3#微藻培养液配方"为基础培养液,分别添加有机碳(C6H12O6和CH3COONa)对热带普通小球藻进行自养、兼养及异养培养,获得促进热带普通小球藻快速生长的培养方式。在"宁波大学3#微藻培养液配方"的基础上对热带普通小球藻的兼养培养基配方进行优化,并用优化兼养培养基与"宁波大学3#微藻培养基"对比培养热带普通小球藻。【结果】添加6 g/L CH3COONa的兼养模式促进热带普通小球藻生长效果最好;优化的兼养培养基配方为:6 g/L CH3COONa,20 mg/L(NH4)2SO4-N,5 mg/L Na H2PO4-P,3 mg/L Fe SO4-Fe,1 mg/L Vitamin B1和0.000 5 mg/L Vitamin B12。对比培养实验结果显示,培养第6天,兼养培养液收获的生物量(细胞密度)达4.20×107 cells/m L,是"宁波大学3#配方微藻培养液"的2.30倍。【结论】兼养培养模式为热带普通小球藻的最佳培养模式,优化的兼养培养基极显著地提高了热带普通小球藻的生物量(P0.01)。     

Electrotransformation of Chlorella vulgaris

Using hygromycin B resistance as a marker for selection, we have established the conditions required for the transformation of Chlorella vulgaris. The exponentially grown C. vulgaris cells were transformed by electroporation with plasmid pIG121-Hm, and transformants were selected with hygromycin B at a concentration of 50 μg/ml. Cell extracts prepared from the late-log cultures of the transformants exhibited glucuronidase activities as conferred by the gus gene on pIG121-Hm. The maintenance of plasmid in the algal cells seemed to be transient as many cultures derived from the hygromycin B-resistant colonies gradually lost the hygromycin resistance upon prolonged growth. The result of Southern blotting of the genomic DNAs prepared from transformant cultures exhibiting persistent hygromycin resistance showed that integration of part of the plasmid DNA into the host chromosome had taken place. Received: 19 December 1997 / Revision received: 5 October 1998 / Accepted: 27 October 1998     

响应面法优化普通小球藻混合营养培养基组成生产生物质

利用响应面法优化了混合营养培养普通小球藻生产生物质的培养基组成.首先采用Plackett-Burman设计对11个相关营养因素的效应进行了评价,并筛选出影响小球藻细胞生长的3个主要因素为KNO3、葡萄糖和NaC1;然后结合Box-Behnken设计建立了以小球藻浓度为响应值的二次回归方程模型,获得优化的培养基组成为KNO31.64g/L、葡萄糖45g/L、NaC1 1.57g/L;模型预测的最大浓度为5.28g/L,验证值为5.68g/L;验证结果表明,所建立模型预测精度较好,可用于优化小球藻的混养培养基组成.优化条件下混养小球藻细胞的蛋白质和色素含量较优化前降低,而可溶性糖和油脂含量提高,脂肪酸以棕榈酸和油酸为主;细胞组分分析结果显示,混养培养所得小球藻生物质具有作为生产微藻生物能源原料的潜力.     

磁处理对小球藻超微结构的影响

用透射电镜研究在不同强度磁场处理下,小球藻超微结构的变化。结果显示:磁处理强度不同,小球藻细胞亚显微结构变化程度不同。细胞壁、叶绿体、线粒体和液泡等部位是受磁处理影响的主要部位。研究发现在较高强度磁处理下,出现质壁分离,类囊体结构轻微破坏,液泡和线粒体增加,能量物质积累等现象,影响小球藻的正常代谢。     

小球藻对镉离子的吸附速率方程

研究了小球藻对Cd^2 的吸附动力学。研究表明,小球藻对Cd^2 有一定的吸附作用,在吸附的起始阶段,吸附速率较快,且随温度的升高吸附速率增大,吸附过程在20min内达到平衡。结果还表明,该吸附是简单的一级反应,并由此建立了吸附反应的速率方程,推算出吸附反应的活化能Ea=31．42kl／mol。     

小球藻细胞活性物质的提取及对啤酒酵母的生理效应

小妹藻（Chlorella vulgaris）为绿藻门普生性单细胞藻类。因其具有极为丰富均衡的营养成分,常作为医疗保健品,或用于食品、轻工业以及饲料添加剂,并可用于重金属的生物吸附、污水处理及环境评价方面．小球藻生长因子（Chlorena Growth Factor,CGF）又称小球藻精,为其细胞活性物质,含有氨基酸、核酸、多糖、多肽、蛋白质、酶、维生素、矿物质等成分,被誉为“类荷尔蒙”。     

Investigation of mixotrophic,heterotrophic, and autotrophic growth of Chlorella vulgaris under agricultural waste medium

Growth of Chlorella vulgaris and its lipid production were investigated under autotrophic, heterotrophic, and mixotrophic conditions. Cheap agricultural waste molasses and corn steep liquor from industries were used as carbon and nitrogen sources, respectively. Chlorella vulgaris grew remarkably under this agricultural waste medium, which resulted in a reduction in the final cost of the biodiesel production. Maximum dry weight of 2.62 g L^?1 was obtained in mixotrophic growth with the highest lipid concentration of 0.86 g L^?1. These biomass and lipid concentrations were, respectively, 140% and 170% higher than autotrophic growth and 300% and 1200% higher than heterotrophic growth. In mixotrophic growth, independent or simultaneous occurrence of autotrophic and heterotrophic metabolisms was investigated. The growth of the microalgae was observed to take place first heterotrophically to a minimum substrate concentration with a little fraction in growth under autotrophic metabolism, and then the cells grew more autotrophically. It was found that mixotrophic growth was not a simple combination of heterotrophic and autotrophic growth.     

Chloroplast DNA diversity in Calluna vulgaris (heather) populations in Europe

Variation in the chloroplast genome of Calluna vulgaris (heather), the dominant species of northwest European heath communities, was analysed using polymerase chain reaction-restriction fragment length polymorphisms (PCR-RFLPs) and microsatellites. No length polymorphisms were detected in the 100-200 base pair (bp) fragments amplified by the conserved microsatellite primers, and sequencing revealed that the repeat regions were interrupted relative to the corresponding sequence in Nicotiana tabacum. In contrast, PCR-RFLP analysis revealed high levels of haplotype diversity within populations (hS = 0.443, hT = 0.842), as well as substantial differentiation between populations (GST = 0.473). Diversity and differentiation were higher in southern Europe than in northern Europe. Interpreted in the light of data from allozyme studies and pollen core records, the results suggest that the main glacial refugia for C. vulgaris were located in southwest Europe, including northern Spain, the Pyrenees and the Massif Central region of France. There is also evidence for diffuse survival of the species at more northerly latitudes.     

利用市政污水培养Chlorella vulgaris生产生物柴油

为了考察利用南昌市政污水规模化培养富油微藻生产生物柴油,同时达到净化污水的目的,取南昌市青山湖污水处理厂未经任何处理的市政污水作为普通小球藻(Chlorella vulgaris)生长的培养液。监测了C.vulgaris在市政污水中连续培养10 d的特定生长率、生物质产量以及与之相关的市政污水中氨氮(NH4+-N)、总磷(TP)、化学需氧量(COD)、总悬浮固体(TSS)和挥发性悬浮固体(VSS)的清除情况。实验表明:营养物质的水平显著地影响了C.vulgaris的生长。C.vulgaris的生长率在培养8 d后达到最大,OD680为2.856,总的生物质产量日均最大积累速率为0.01 g/L,油脂含量为干质量的18%,油脂的平均日产量为0.001 g/L。培养10 d内NH4+-N、TP和COD的去除率分别为50.0%、32.1%和26.0%,TSS和VSS的日平均去除速率分别为0.01 g/L和0.006 1 g/L。     

Use of Chlorella vulgaris for CO(2) mitigation in a photobioreactor

Carbon dioxide (CO₂) is a colorless gas that exists at a concentration of approximately 330 ppm in the atmosphere and is released in great quantities when fossil fuels are burned. The current flux of carbon out of fossil fuels is about 600 times greater than that into fossil fuels. With increased concerns about global warming and greenhouse gas emissions, there have been several approaches proposed for managing the levels of CO₂ emitted into the atmosphere. One of the most understudied methods for CO₂ mitigation is the use of biological processes in engineered systems such as photobioreactors. This research project describes the effectiveness of Chlorella vulgaris, used in a photobioreactor with a very short gas residence time, in sequestering CO₂ from an elevated CO₂ airstream. We evaluated a flow-through photobioreactor's operational parameters, as well as the growth characteristics of the C. vulgaris inoculum when exposed to an airstream with over 1850 ppm CO₂. When using dry weight, chlorophyll, and direct microscopic measurements, it was apparent that the photobioreactor's algal inoculum responded well to the elevated CO₂ levels and there was no build-up of CO₂ or carbonic acid in the photobioreactor. The photobioreactor, with a gas residence time of approximately 2 s, was able to remove up to 74% of the CO₂ in the airstream to ambient levels. This corresponded to a 63.9-g/m³/h bulk removal for the experimental photobioreactor. Consequently, this photobioreactor shows that biological processes may have some promise for treating point source emissions of CO₂ and deserve further study. Received 25 April 2002/ Accepted in revised form 27 July 2002