杜氏盐藻异养转化藻株的鉴定及初步功能分析

Preliminary Functional Analysis and Characterization of Heterotrophic Transformation Algal Strains of Dunaliella Salina

作者: 专业:细胞生物学 导师:薛乐勋 年度:2010 学位:硕士  院校: 郑州大学

Keywords

Dunaliella salina, electroporation transformation, constitutive heterotrophic expression vector, inducible heterotrophic expression vector, copy number, glucose transport gene Glut1

        杜氏盐藻(Dunaliella salina, D. salina)是一种单细胞的真核绿藻,无细胞壁,属于光合自养型生物。杜氏盐藻本身有很多优点:可在0.05-5 M NaCl的极端环境中生存因而赋予其较强的抗污染能力;遗传操作简便;具有翻译后加工修饰;培养条件简单、成本低、生长周期短;自身营养价值很高;无毒无害等优点,使得盐藻成为一种表达外源基因的良好宿主。因此,开发盐藻作为生物反应器生产多种具有生物活性的外源蛋白如药用蛋白、血管抑素、抗体和疫苗等具有巨大的应用前景。盐藻的培养有两种方式:一种是开放池式的大规模培养,但是易受微生物污染、气候、光照、营养条件以及昼夜温差等因素的影响;另外一种是封闭式的光照生物反应器培养,但是利用光照生物反应器培养存在接种量大和光抑制效应等缺点。因此使得盐藻的生长速率和生物量难以大幅度提高,从而盐藻的利用受到了很大的限制。通过基因工程手段改变盐藻的营养模式(从光合自养型向异养型过渡)从某种程度上可以最大限度提高盐藻的生物量,为从根本上解决上述可能的难题提供理论依据。莱茵衣藻、小球藻、硅藻等藻类的异养研究发现,转入单个外源基因如小球藻的HUP1基因(H+/hexose cotransporter 1)、人红细胞的Glutl基因(Glucose transporterl)以及酿酒酵母的Hxtl、Hxt2和Hxt4基因等转变藻类的营养模式后,这些藻类可以利用外源的糖类进行异养生长,其生长速率和细胞密度均显著提高。葡萄糖转运蛋白(Glut1)是葡萄糖转运蛋白家族中的一种通道转运蛋白,位于细胞膜表面,是葡萄糖转运的主要载体。因此在本研究中我们尝试将Glut1基因转入盐藻细胞中以改变盐藻的营养模式,从而为提高盐藻的生长速度提供理论基础,为进一步建立杜氏盐藻生物反应器提供可行性思路。本课题组前期已经从人胎盘组织中克隆了人Glut1基因,并通过测序证实所克隆的Glut1基因序列的正确性,进一步利用基因重组技术构建了盐藻组成型和诱导型异养表达载体。本课题是在此基础上通过优化的电击转化方法将两种异养表达载体转入杜氏盐藻细胞中,利用草丁膦(Phosphinothricin, PPT)通过液体和固体筛选出杜氏盐藻异养转化藻株。结果表明,在PPT浓度为6μg/mL时对照野生藻全部死亡,而转化藻株生长良好,在此基础上共筛选出了3株组成型异养藻株和2株诱导型异养藻株,分别命名为:C1、C2、C3、I1和I2。进一步对筛选出的5株异养转化藻株进行RT-PCR鉴定,结果显示,5株转化藻株在相应大小的位置都出现了约为250bp的较为特异的DNA条带,提示外源基因Glutl已成功整合到杜氏盐藻异养转化藻株的基因组中。在转基因生物体中,转基因拷贝数对目标基因的表达水平和遗传稳定性有很大地影响,这使得转基因拷贝数的估算成为转基因生物研究中的重要内容和课题之一。实时荧光定量PCR (Real-time Quantitative PCR)和Southern botting技术是目前研究转基因拷贝数较为理想的方法,因此我们利用这两种技术分析5株异养转化藻株的拷贝数,结果发现3株盐藻组成型异养转化藻株C1、C2和C3的Glut1基因拷贝数分别为:2、1和3;2株杜氏盐藻诱导型异养转化藻株I1和I2的Glut1基因拷贝数分别为:4和4。Southern blotting检测C1、C2和C3的拷贝数与Real-time PCR结果一致,但在I1和I2中其拷贝数分别为3和2,稍低于Real-time PCR的结果。最后对异养转化藻株的葡萄糖转运进行了分析,结果表明在光照条件下各种不同的葡萄糖浓度对自养株、组成型异养株(C2)和诱导型异养株(I2)的生长均有明显的影响,而且各株不同的杜氏盐藻在5 mm的葡萄糖浓度作用下其生长明显强于其它葡萄糖浓度(P<0.05);同时发现在光照和葡萄糖(5 mm)共同作用能明显提高各株不同杜氏盐藻藻株的生长速度(P<0.05),在黑暗条件和葡萄糖的共同作用下,组成型异养株的生长速率明显高于自养株和诱导型异养株(P<0.05),自养株和诱导型异养株的生长几乎停滞,其原因可能是诱导型异养株的Glutl基因由于拷贝数多随机插入到杜氏盐藻染色体DNA上的位点不确定干扰了杜氏盐藻细胞基因组的自稳系统而导致生长速度降低或停滞,或出现转基因沉默效应。而组成型的异养株在光照和葡萄糖的共同作用下其生长速率也明显高于黑暗和葡萄糖共同作用下的生长速率,提示该异养藻株可能处于兼性异养,如何使其处于完全异养状态,为进一步大规模的发酵培养奠定基础,尚需要进一步的研究。总之,本研究初步建立了异养转化藻株,为杜氏盐藻生物反应器的建立提供理论基础。
    Dunaliella salina (D. salina) is a kind of photoautotrophic unicellular green algae lacking rigid cell wall. D.salina itself has many advantages as following: growing in extreme environment with salinities ranging from 0.05 to 5 M sodium chloride, and thus strong anti-pollution ability; easy genetic manipulation; post-translational processing; simple culture conditions, inexpensive cultivation cost and relative short growth cycle; high nutritional value; non-toxic harmless, et al. Therefore, it is very important prospect to develop the bioreactor of D.salina to product many biological active compounds, such as pharmaceutical protein, angiostatin, antibodies and vaccine, and so on.At present, there are two patterns in D.salina cultivation, one is in open pond culture systems which often overrun by other algae or bacteria, in which the growth of D.salina are often limited by light, climate, nutrient and temperature; the other is in closed photobioreactor systems which was restrained by large starter inoculums grown and light limitation. So it is difficult to increase the biomass of D.salina in large-scale, which also limited the utilization of D.salina. Trophic conversion of D.salina from an obligate photoautotrophic to heterotrophic promote the biomass of D.salina to some extent through genetic engineering, which will provide the theoretical basis for solving the problem above.The growth rate and cell density of algae were markedly enhanced after single gene (including HUP1 from Chlorella, Glutl from human erythrocyte and Hxtl, Hxt2 and Hxt4 from Saccharomyces cerevisiae, respectively) was introduced into algae including Chlamydomonas, Chlorella, Diatoms and other algae Glutl, a member of glucose transporter protein family, located in cell membrane surface, is the main carrier of glucose transporter. Thus, in this study, we attempted to change the trophic pattern by introducing Glutl into D.salina, which will provide will provide the theoretical basis for improving the growth rate of D.salina, and the feasibility ideas for further development of D. Salina bioreactor. Our research group has cloned Glutl gene from human placenta tissue, and has confirmed its correction by sequencing, and then constructed inducible heterotrophic expression vector pMDDGN-Bar and constitutive heterotrophic expression vector G5Glutl-Bar of D.salina through gene recombination technology. Based on the study above, in this study, the two heterotrophic vectors were transformed into D.salina through optimized electroporation methods, heterotrophic algal strains of D.salina were selected used the PPT by liquid and solid screening procedures. We found that the wild algae all died on the concentration of 6μg/mL of PPT, while transformed algal strains grew well. Finally,3 constitutive heterotrophic algal strains and 2 inducible heterotrophic algal strains was screened out, and named as Cl, C2, C3, I1 and I2, respectively. Further, the results of RT-PCR demonstrated that 5 strains of transformants all appeared about 250 bp of specific DNA bands, suggesting that foreign Glutl gene had been successfully integrated into the genome of heterotrophic strains of D. salina.In transgenic organisms, the transgene copy number can greatly influence the expression level and genetic stability of the target gene, and thus the estimation of transgene copy number is one of the important contents in the study of genetically modified organisms(GMOs). Currently, Real-time PCR and Southern blotting techniques are ideal methods for estimating transgene copy number. Thus, we analyzed the transgene copy number of the 5 strain transformants using the two technologies, respectively. The results revealed that the copy numbers of C1, C2, C3 I1 and I2 were 2,1,3,4 and 4, respectively. Finally, the glucose transport of transformants was investigated. The results demonstrated that different concentrations of glucose have significant impact on photoautotrophic strains, constitutive heterotrophic strain (C2) and inducible heterotrophic strain (I2) in light conditions, and growth of the three different strains of D.salina was stronger on the concentration of 5 mm glucose than other glucose concentrations(P<0.05). The results also showed that combined light and glucose (5 mm) obviously increased growth rates of different algal strains; while under the condition of dark combined with glucose (5 mm), the growth ratios of constitutive heterotrophic strain (C2) were significantly higher than that of inducible heterotrophic strain (I2) and photoautotrophic strain (P<0.05), whereas, the growth ratios of inducible heterotrophic strain and photoautotrophic strain remained almost stagnant, which may be evoked by many copy number of Glutl gene randomly inserted into the genome DNA of D. salina, further interferes with D.salina genome stability systems and results in transgene silencing effect. In addition, growth ratios of constitutive heterotrophic strain under the condition of light combined with glucose were significantly higher than that under the condition of dark combined with glucose, suggesting that constitutive heterotrophic strain may be in the facultative heterophic, however, how to make it completely heterophy remains further studied, which will lay a foundation for large-scale fermentation culture of D.salina. In conclusion, D.salina heterotrophic strain was preliminarily established, which will provide theoretical basis for construction of D.salina bioreactor.
        

杜氏盐藻异养转化藻株的鉴定及初步功能分析

摘要4-6
Abstract6-8
1 引言11-13
2 杜氏盐藻异养转化藻株的筛选及鉴定13-24
    2.1 材料13-17
        2.1.1 主要仪器设备13-14
        2.1.2 主要试剂14-15
        2.1.3 杜氏盐藻藻株、培养基和培养条件15
        2.1.4 载体15-17
        2.1.5 Glut1引物设计17
    2.2 方法17-20
        2.2.1 电击法转化盐藻细胞17
        2.2.2 盐藻异养转化藻株的液体和固体筛选17-18
        2.2.3 盐藻异养转化藻株和野生藻株总RNA的提取18-19
        2.2.4 杜氏盐藻异养转化藻株PCR鉴定19-20
    2.3 结果20-22
        2.3.1 盐藻异养转化藻株的筛选20-21
        2.3.2 异养转化藻株RNA质量鉴定21
        2.3.3 异养转化藻株的PCR鉴定21-22
    2.4 讨论22-23
    2.5 小结23-24
3 杜氏盐藻异养转化藻株的初步功能分析24-46
    3.1 材料25-26
        3.1.1 主要仪器设备25
        3.1.2 主要试剂25
        3.1.3 盐藻藻株、培养基和培养条件25
        3.1.4 主要溶液配制25-26
        3.1.5 引物设计26
    3.2 方法26-33
        3.2.1 实时荧光定量PCR检测转化藻株Glut1拷贝数27-29
        3.2.2 Southern blotting检测转化藻株Glut1拷贝数29-33
        3.2.3 异养转化藻株葡萄糖转运分析33
    3.3 结果33-42
        3.3.1 实时荧光定量PCR检测转化藻株Glut1拷贝数33-37
        3.3.2 Southern blotting法分析转化藻株Glut1拷贝数37-38
        3.3.3 实时荧光定量PCR与Southern blotting分析转化藻株Glut1拷贝数的比较38
        3.3.4 异养转化藻株葡萄糖转运分析38-42
    3.4 讨论42-45
    3.5 小结45-46
参考文献46-49
综述:微藻生物反应器的研究进展及其应用前景49-69
    参考文献65-69
致谢69-70
个人简历、在学期间发表的学术论文与研究成果70
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