【作者】 黄瑞；

【导师】 吴双秀； 王全喜；

【作者基本信息】 上海师范大学 ， 水生生物学， 2010， 硕士

【摘要】 在无氧条件下,莱茵衣藻（Chlamydomonas reinhardtii）中的氢化酶（H2ase）能将光合作用过程中产生的H+和e-或细胞内有机物分解产生的H+和e-合成H2,释放到细胞外。莱茵衣藻的氢化酶（H2ase）活性高,而且衣藻培养容易、遗传学背景清晰、生长速度快、实验操作容易,能利用太阳能和水制氢,被认为是目前非常有开发潜力的生物制氢的模式藻种。虽然衣藻的氢化酶（H2ase）活性高,但是衣藻的氢化酶（H2ase）活性很容易受氧气的抑制而失去活性,而氧气又是光合作用的主要产物,这就导致了莱茵衣藻产氢效率低,限制了工业化产氢的发展。目前降低衣藻细胞内氧气含量的方法主要是通过去除培养基中硫元素来抑制光合系统II（PSII）活性、进而降低光解水产生的氧气,但是该方法同时抑制了光解水所产生的电子产量,限制了产氢的电子来源,所以最终导致产氢效率低。因此,要提高衣藻的产氢效率就需要既降低细胞内的氧浓度又要保障电子的供应。大豆根瘤中的豆血红蛋白（leghemoglaobin,Lb）,它与氧气有很高的亲和能力,能帮助降低细胞内氧浓度和调节呼吸作用,因而能使大豆根瘤中同样对氧气敏感的固氮酶具有高效固氮效率,生物发酵工程研究中已经有应用血红蛋白体外表达而使产量提高的报导。豆血红蛋白由两部分组成,一部分是球蛋白（globin）,是由大豆（Glycine max）中的豆血红蛋白（Lbs）基因合成;另一部分是血红素,是由与大豆共生的慢生大豆根瘤菌（Bradyrhizobium japonicum）合成。在根瘤菌中,hemH基因编码合成的亚铁螯合酶（Ferrochelatase）它催化血红素合成的最后一步反应,而血红素的合成前体和代谢途径与高等植物和衣藻中叶绿素的合成相同,由5-aminolevulinic acid（ALA）经一系列反应合成而来,所以衣藻中含有充足的血红素合成的前体。本实验尝试将慢生大豆根瘤菌中的亚铁螯合酶hemH基因和大豆中的豆血红球蛋白lba基因同时转入衣藻藻种cc849的叶绿体中,通过hemH基因表达亚铁螯合酶（Ferrochelatase）催化合成血红素,与球蛋白lba一起帮助降低叶绿体内氧气含量,增加产氢酶H2ase活性;同时结合部分恢复PSⅡ活性以增加电子供应量,实现PSⅡ和氢酶同时最大限度行使其功能,为实现在常规条件下利用太阳能持续生产氢能的研究奠定实验基础。本论文的主要内容及结果如下:1.提取慢生大豆根瘤菌的总DNA,从总DNA中克隆出了表达亚铁螯合酶的基因hemH,与本实验室前期克隆的球蛋白基因lba一起成功地构建了衣藻叶绿体转化表达载体cg401-1-hemH-lba。2.通过基因枪法将载体cg401-1-hemH-lba转入到莱茵衣藻叶绿体中,通过壮观霉素筛选及液固反复继代培养,获得转基因藻株。3.通过PCR及RT-PCR扩增检测的方法对转基因衣藻hemH-lba进行DNA及RNA水平的检测,PCR结果证实hemH和lba编码区DNA已整合到部分衣藻叶绿体基因组中,RT-PCR的结果表明hemH和lba基因在RNA水平上得到转录。4.通过Western Blot方法对转基因衣藻hemH-lba进行蛋白水平的检测,正常培养条件下hemH基因和lba基因的蛋白在衣藻叶绿体中都得到了表达,第六天（饱和期）表达量最大,在产氢培养第五天时蛋白量达到最大。5.对野生藻849和转基因衣藻hemH-lba的生长情况进行检测表明:野生藻849饱和时的最大OD750值是3.5;转基因衣藻hemH-lba饱和时的最大OD750值是3,略低于野生藻849。饱和期时转基因衣藻hemH-lba的最大叶绿素含量为40mg/L左右,而野生藻849的最大叶绿素含量约为36mg/L左右,比转基因衣藻hemH-lba略低。结果表明,基因hemH-lba的转入影响了莱茵衣藻的生长速度,但是叶绿素含量没有受到影响。6.利用紫外分光光度计扫描野生藻和转基因衣藻hemH-lba在500nm到600nm的OD值,在550nm处转基因衣藻hemH-lba有血红素吸收峰,表明有微量血红素合成。7.比较了野生藻849和转基因衣藻hemH-lba在不同含硫浓度0、12.5、25、50μM下的氢气产量,氧气含量和产氢速率。结果表明,在各个含硫浓度下转基因衣藻hemH-lba的氧气量都是比野生藻849的氧气量下降的快,产氢量比野生藻849有所提高。8.通过对转基因衣藻hemH-lba及其野生藻849进行光照强度、叶绿素含量、含硫量三因素三水平的正交实验,确定了以TAP为培养基的转基因衣藻hemH-lba及野生藻849的最佳产氢条件。实验结果表明,在培养条件25℃下,转基因衣藻hemH-lba的最佳产氢条件为光照强度60μmol m^-2 s^-1,叶绿素含量0.5mg,含硫量12.5μmol/L;而野生藻849的最佳产氢条件为光照强度60μmol m^-2 s^-1,叶绿素含量0.5mg,含硫量0mol/L。9.根据密码子的简并性修改了亚铁螯合酶基因（hemH）的碱基,使得hemH碱基对中的AT含量达到了63%,利用已修改碱基的大豆球蛋白基因lba-M成功地构建了另一个衣藻叶绿体转化表达载体cg401-1-hemH-lba-M,现已获得转基因藻株。更多还原

【Abstract】 In anaerobic conditions, the hydrogenase （H2ase） in the Chlamydomonas reinhardtii produces hydrogen using electrons and protons which come from photosynthesis and organic decomposition and releases into the extracellular. The H2ase activity of C. reinhardtii is high and C. reinhardtii grows fast, is easy to culture, has clearly genetic background and easy for experiment. Therefore, C. reinhardti is the model algal species for the study of biohydrogen production by using solar energy and water and has great potential to be utilized in the industry in the future.But the H₂ ase of C. reinhardtii is extremely oxygen （O₂）-sensitive enzyme, while the oxygen is the main product of photosynthesis, which leading to the low hydrogen yield and limiting the development of its industrial hydrogen production. Currently the method to lower the oxygen content in C. reinhardtii cells is to remove sulfate in the culture medium to suppress the photosynthetic system II （PSII） activity, thereby reducing the oxygen produced by photolysis of water. But this method also inhibites electronic production by the photolysis of water, which limiting the electron source for the hydrogen production and finally leading to the low hydrogen production of C. reinhardtii. Therefore, to improve the efficiency of hydrogen production of C. reinhardtii requires not only reducing the oxygen concentration inside the cells but also maintaining the supply of electrons.Leghemoglobin （ Lb） in soybean root nodules has a high affinity with oxygen and capabilities. It can reduce intracellular oxygen concentration and regulate respiration, thus enable the oxygen-sensitive nitrogenase enzyme in the soybean root nodules has a highly efficient nitrogen-fixing efficiency. In several biofermentation engineering researches, the hemoglobin has been applied to express in vitro and increased production of the cultures. Lb consists of two parts. One part is globulin （globin）, encoded by soybean （Glycine max）. Another part is encoded by the symbiotic Bradyrhizobium japonicum. In the B.japonicum, hemH gene encodes the ferrochelatase which catalyzes the final step in heme synthesis and is necessary in the heme （heme） synthesis. The precursors of heme are similar to those of chlorophylls in C. reinhardtii and higher plants via the 5-aminolevulinic acid （ALA） pathway. Therefore, in C. reinhardtii cells there are adequate precursors for heme synthesis.This study attempts to transfer the soybean rhizobia hemH gene and soybean lba gene into the chloroplasts of C. reinhardtii strain cc 849 to synthesize the active Lb, which would help to reduce the oxygen content inside the chloroplast and increase activity of H2ase. With the helps of partial restoration of PSⅡactivity, the improvement of the electron supply from PSII maight increase the hydrogen yield of the transgenic algae. The work is a new attempt to improvement of hydrogen yield by using the characteristics of leghemoglobin.The main results of this thesis are as follows:1. Total DNA of B. japonicum has been extracted. The coding region of both the ferrochelatase gene, hemH, from B. japonicum, and the leghemoglobin gene, lba, from Glycine max, were transferred into chloroplast of C.reinhardtii. Chloroplast transformation vector cg401-1-hemH-lba was constructed.2. Transgenic C. reinhardtii was gotted by the gene particle method and screened on the solid spectinomycin mediu. Subcultured under liquid-solid medium with spectinomycin repeatedly to maintain the transgenic algae.3. Transgenic C. reinhardtii were identified by the PCR and RT-PCR amplification based on total DNA and RNA respectively. PCR results confirmed hemH and lba coding region DNAs have been integrated into part of algal chloroplast genomes. RT-PCR results showed that hemH and lba genes are transcribed at the RNA level.4. Western blotting results confirmed that ferrochelatase and lba protein expressed successfully in C. reinhardtii chloroplasts both in the normal culture condition and in the anaerobic condition. The maximum expression level appeared on the sixth day of normal culture condition and on the fifth day of the anaerobic condition, respectively.5. By comparison of the growth of wild and transgenic, the OD750 of transgenic algae is 3, about 14% slightly lower than that of strain cc849. But, the chlorophyll content of the transgenic algae was about 40 mg/L, higher than that of strain cc849. The results revealed that the transformation of hemH-lba gene affected the growth and chlorophyll content of transgenic algae.6. Using UV spectrophotometer to scan the heme extraction of strain cc849 and transgenic alga hemH-lba between 500nm and 600nm, there is a small absorption peak in 550nm. The result showed that transgenic alga hemH-lba synthesized small quantity of heme.7. To compare the amount of hydrogen yield, oxygen content and hydrogen production rate of strain cc849 and transgenic alga hemH-lba at different sulfur concentrations of 0, 12.5, 25, 50, the results show that in various sulfur concentration, the oxygen consumption rate of transgenic alga hemH-lba are larger than that of strain cc849 and hydrogen production increased compared to wild algae 849.8. Through the orthogonal experiment of light intensity, chlorophyll content, sulfur content （three factors and three levels）, the best conditions for hydrogen production of transgenic alga hemH-lba and wild algae 849 were identified respectively. In culture conditions at 25℃, 60μmol m^-2 s^-1 light intensity, 0.5mg bottle-1 of the chlorophyll content and , 12.5μmol L-1 of sulfur content favored transgenic alga hemH-lba‘s hydrogen production. While it was 60μmol m^-2 s^-1 light intensity, 0.5mg bottle-1 of the chlorophyll content, 0mol L-1 of the sulfur content best for strain cc849‘s hydrogen production.9. According to codon bias of the chloroplast DNA of C. reinhardtii, the nuclear acids of hemH gene and lba gene were modified to make the AT content reaching 63%. The modified hemH gene and lba gene were also successfully transformed into the chloroplast of C. reinhardtii.更多还原