密码子优化对豆血红蛋白基因在莱茵衣藻叶绿体中表达和产氢的影响
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摘要
莱茵衣藻(Chlamydomonas reinhardtii)结构简单,遗传学背景清晰,易于进行分子操作,培养容易且成本较低,尤其是氢化酶的活性高,能够利用太阳能和水产生氢气,被认为是目前最有开发潜力的生物制氢的模式物种。
莱茵衣藻的氢化酶活性高但是对氧气特别敏感,而氧气又是光合作用的产物,所以在产氢的过程中,氢化酶很容易受氧气抑制而失活,这是衣藻产氢的最大障碍。要想提高产氢效率达到工业化生产的目的,就要降低衣藻细胞内的氧气含量,保证氢化酶的活性。目前最常用的办法是去除培养基中的硫元素或加入某些抑制剂来抑制光系统Ⅱ的活性,以减少光合作用产生的氧气量,但是同时这样也会降低光解水产生的电子量,使光合产氢的电子来源减少,导致产氢量降低。如何降低细胞内的氧气含量又不影响电子的供应是提高莱茵衣藻产氢的关键问题。
豆血红蛋白(leghemoglaobin,Lb)与氧气具有很高的亲和力和快速的氧周转率,能帮助降低根瘤细胞内的氧气浓度和保证氧敏感的固氮酶的活性,没有豆血红蛋白的根瘤没有固氮作用。
本实验室前期工作已经尝试分别将大豆血红蛋白的球蛋白基因lba和血红素-球蛋白基因hemH-lba分别转化到莱茵衣藻的叶绿体中表达,以帮助降低细胞内氧气含量和提高产氢量。由于衣藻叶绿体中蛋白表达具有密码子AT偏向性,本实验尝试将hemH-lba基因进行密码子优化并转入莱茵衣藻的叶绿体中表达,以提高外源蛋白表达量和更好地发挥豆血红蛋白的作用,提高衣藻产氢量。
本论文的主要内容及结果如下:
1.将密码子优化后的豆血红蛋白基因送公司合成,并成功构建表达载体cg401-1-hemHc-lbac。该表达载体中的hemH基因和lbac基因设计为多顺反子表达形式。
2.利用基因枪法将载体cg401-1-hemHc-lbac转化到衣藻藻株cc849(以下简称对照藻849)的叶绿体中,用抗生素——壮观霉素筛选出转基因藻。
3.通过PCR和RT-PCR对转基因衣藻hemHc-lbac进行DNA和RNA水平的检测,PCR结果表明hemHc和lbac基因片段已异质化整合到转基因衣藻叶绿体基因组中,RT-PCR结果表明hemHc和lbac基因在在叶绿体中成功转录。
4.通过Western blotting的方法对转基因衣藻hemHc-lbac进行蛋白水平的检测,结果表明正常培养条件和产氢培养条件下hemHc基因和lbac基因的蛋白在衣藻叶绿体中都得到了表达,且都在第五天达到了最大值,表达量都是未优化密码子转基因藻hemH-lba的6.8倍。在非变性胶上的Western blotting杂交结果表明两个多顺反子表达的亚基电泳分离后在同一位置。
5.用定量Real-time PCR的方法对氢化酶的表达量进行检测表明,产氢条件下转基因藻hemH-lba和hemHc-lbac的氢化酶转录水平比对照藻849都有所提高。
6.对对照藻849和转基因藻hemH-lba、hemHc-lbac的生长情况进行检测表明:对照藻849饱和时的最大OD750值是3.2左右,转基因衣藻hemH-lba饱和时的最大OD750值是3.5,转基因衣藻hemHc-lbac饱和时的最大OD750值是3.3。达到饱和期时转基因衣藻hemHc-lbac、hemH-lba的最大叶绿素含量均为41mg/L左右,而对照藻849的最大叶绿素含量为35mg/L左右。说明转基因藻的生长并未受到抑制,其叶绿素含量反而有所增加。
7.用气相色谱对转基因藻和对照藻产氢量和耗氧量进行检测表明无论在黑暗耗氧法除氧还是冲氩法除氧的条件下转基因衣藻hemHc-lbac比hemH-lba和对照藻的产氢量都高。
8.用乙醚萃取血红素,利用紫外分光光度计扫描样品,在550nm处转基因衣藻hemH-lba和hemHc-lbac有小的血红素吸收峰,表明有微量血红素合成。
9.通过对对照藻849和转基因藻hemH-lba、hemHc-lbac的呼吸速率和光合放氧速率的检测表明,两种转基因藻的净光合放氧速率都比对照藻的偏低,而hemHc-lbac的净光合放氧速率比hemH-lba的更要低。两种转基因藻的呼吸速率比对照藻的都高,尤其是转密码子优化的藻hemHc-lbac的呼吸速率最高。
Chlamydomonas reinhardtii, with simple structure, clear molecular genetic background, easy operation and low cost on culturation, especially with high activity of hydrogenase which can produce hydrogen using solar energy and water, is considered as the most potential algal species for development of biological hydrogen production in the future.
Hydrogenase of C. reinhardtii has high catalytic activity, but particularly sensitive to oxygen which is the main byproduct of algal photosynthesis. Therefore, the process of hydrogen production and hydrogenase activity is very susceptible to oxygen inhibition and inactivation, which is the maximum obstacle for hydrogen production of C. reinhardtii. To improve the biohydrogen production of C. reinhardtii, the yield of oxygen must be reduced in C. reinhardtii cells to ensure the activity of hydrogenase. The most common way at present is to remove sulfur in the culture medium or by adding some of inhibitors to inhibit the activity of photosystem II. But at the same time it will also reduce the electronic source from the water photolysis process and result in low hydrogen production. Therefore, how to reduce the oxygen content in algal cells without affecting the electronic supply for hydrogenase is a key issue to increase hydrogen production of C. reinhardtii.
Leghemoglobin (Lb) has high oxygen affinity and rapid turnover rate. It helps to reduce the oxygen concentration inside the nodule cells and ensures the oxygen-sensitive nitrogenase activity. No Lbs, no nitrogen fixation activities in the soybean nodules.
We have tried to transfer soybean Lb protein genes lba and hemH-lba into the chloroplast of C. reinhardtii in our previous work. Since the expression of protein in the C. reinhardtii chloroplast has AT bias, in this experiment, the codon-optimized Lb genes lbac and hemHc-lbac were transfered into the chloroplast of C. reinhardtii in order to increase recombinant protein expression levels and hydrogen production. The contents and results of this thesis are as follows:
1. The codon-optimized soybean Lb genes lbac and hemHc were synthezed in the company and the chloroplast expression vector cg401-1-hemHc-lbac with the design of the polycistronic hemHc and lbac genes was successfully constructed.
2. The plasmid DNA of cg401-1-hemHc-lbac was transfered into C. reinhardtii chloroplasts with the particle bombartment method. Transformants were screened by the resistance of spectinomycin and the well-growth one was picked up and named as transgenic alga hemHc-lbac after screening.
3. PCR assay based on DNA templates of the transgenic alga hemHc-lbac showed that the hemHc-lbac gene fragments had been heteroplasmicly integrated into the chloroplast genome DNA of C. reinhardtii. RT-PCR assay based on RNA templates of the transgenic algae showed that the hemHc and lbac genes transcripted successfully in algal chloroplasts.
4. By the method of Western blotting, the recombinant hemHc and lbac proteins had been successfully expressed in the chloroplasts of transgenic algae hemHc-lbac under both normal culture condition and hydrogen production condition. On the fifth days, the expression of recombinant protein reached to the maximum level and the expression level of the codon-optimized recombinant proteins was 6.8 times of the non-optimzied one. The Western blotting result on the native gel electrophoresis showed that the two Lb subunits, hemH and Lba were in the same hybridyzation band with their own specific antibodies .
5. By the quantitative real-time PCR assay, the transcriptions of hydrogenase genes in both codon-optimized and non-optimized transgenic algae were increased than those in the control algal strain cc849.
6. The growth curves of the control algae 849 and transgenic algae hemH-lba, and hemHc-lbac showed that the growth of control algae 849 reached saturation with the maximum OD750 value of 3.2, while that of the transgenic algae hemH-lba was 3.5 and transgenic alga hemHc-lbac was 3.3. At saturation phase, the maximum chlorophyll contents of the transgenic alga hemHc-lbac and hemH-lba were all about 41mg / L, while it was about 35mg / L for the control algae 849. The results showed the growth of the transgenic algae was not inhibited. Moreover, their chlorophyll contents were increased slightly.
7. Hydrogen production and oxygen consumption of both transgenic algae and the control alga were detected. The results showed that the hydrogen production of transgenic algae hemHc-lbac and hemH-lba were higher than that of the control algae nomatter under full argon condition or under the dark anaerobic condition.
8. Heme was extracted with ether from both transgenic algae and scaned by using UV spectrophotometer. There was a small absorption peak at 550nm for both transgenic algae hemH-lba and hemHc-lbac, indicating that a trace heme might be synthesized.
9. Respiratory rate and photosynthetic rate of the control and transgenic algae were measured. The results showed that net photosynthetic oxygen evolution rate of transgenic algae hemHc-lbac was lower than transgenic algae hemH-lba and both were significantly lower than that of the control algae. On the other hand, the respiration rates of both transgenic algae were higher than that of the control algae, particularly for that of the codon-optimized algae hemHc-lbac.
莱茵衣藻的氢化酶活性高但是对氧气特别敏感,而氧气又是光合作用的产物,所以在产氢的过程中,氢化酶很容易受氧气抑制而失活,这是衣藻产氢的最大障碍。要想提高产氢效率达到工业化生产的目的,就要降低衣藻细胞内的氧气含量,保证氢化酶的活性。目前最常用的办法是去除培养基中的硫元素或加入某些抑制剂来抑制光系统Ⅱ的活性,以减少光合作用产生的氧气量,但是同时这样也会降低光解水产生的电子量,使光合产氢的电子来源减少,导致产氢量降低。如何降低细胞内的氧气含量又不影响电子的供应是提高莱茵衣藻产氢的关键问题。
豆血红蛋白(leghemoglaobin,Lb)与氧气具有很高的亲和力和快速的氧周转率,能帮助降低根瘤细胞内的氧气浓度和保证氧敏感的固氮酶的活性,没有豆血红蛋白的根瘤没有固氮作用。
本实验室前期工作已经尝试分别将大豆血红蛋白的球蛋白基因lba和血红素-球蛋白基因hemH-lba分别转化到莱茵衣藻的叶绿体中表达,以帮助降低细胞内氧气含量和提高产氢量。由于衣藻叶绿体中蛋白表达具有密码子AT偏向性,本实验尝试将hemH-lba基因进行密码子优化并转入莱茵衣藻的叶绿体中表达,以提高外源蛋白表达量和更好地发挥豆血红蛋白的作用,提高衣藻产氢量。
本论文的主要内容及结果如下:
1.将密码子优化后的豆血红蛋白基因送公司合成,并成功构建表达载体cg401-1-hemHc-lbac。该表达载体中的hemH基因和lbac基因设计为多顺反子表达形式。
2.利用基因枪法将载体cg401-1-hemHc-lbac转化到衣藻藻株cc849(以下简称对照藻849)的叶绿体中,用抗生素——壮观霉素筛选出转基因藻。
3.通过PCR和RT-PCR对转基因衣藻hemHc-lbac进行DNA和RNA水平的检测,PCR结果表明hemHc和lbac基因片段已异质化整合到转基因衣藻叶绿体基因组中,RT-PCR结果表明hemHc和lbac基因在在叶绿体中成功转录。
4.通过Western blotting的方法对转基因衣藻hemHc-lbac进行蛋白水平的检测,结果表明正常培养条件和产氢培养条件下hemHc基因和lbac基因的蛋白在衣藻叶绿体中都得到了表达,且都在第五天达到了最大值,表达量都是未优化密码子转基因藻hemH-lba的6.8倍。在非变性胶上的Western blotting杂交结果表明两个多顺反子表达的亚基电泳分离后在同一位置。
5.用定量Real-time PCR的方法对氢化酶的表达量进行检测表明,产氢条件下转基因藻hemH-lba和hemHc-lbac的氢化酶转录水平比对照藻849都有所提高。
6.对对照藻849和转基因藻hemH-lba、hemHc-lbac的生长情况进行检测表明:对照藻849饱和时的最大OD750值是3.2左右,转基因衣藻hemH-lba饱和时的最大OD750值是3.5,转基因衣藻hemHc-lbac饱和时的最大OD750值是3.3。达到饱和期时转基因衣藻hemHc-lbac、hemH-lba的最大叶绿素含量均为41mg/L左右,而对照藻849的最大叶绿素含量为35mg/L左右。说明转基因藻的生长并未受到抑制,其叶绿素含量反而有所增加。
7.用气相色谱对转基因藻和对照藻产氢量和耗氧量进行检测表明无论在黑暗耗氧法除氧还是冲氩法除氧的条件下转基因衣藻hemHc-lbac比hemH-lba和对照藻的产氢量都高。
8.用乙醚萃取血红素,利用紫外分光光度计扫描样品,在550nm处转基因衣藻hemH-lba和hemHc-lbac有小的血红素吸收峰,表明有微量血红素合成。
9.通过对对照藻849和转基因藻hemH-lba、hemHc-lbac的呼吸速率和光合放氧速率的检测表明,两种转基因藻的净光合放氧速率都比对照藻的偏低,而hemHc-lbac的净光合放氧速率比hemH-lba的更要低。两种转基因藻的呼吸速率比对照藻的都高,尤其是转密码子优化的藻hemHc-lbac的呼吸速率最高。
Chlamydomonas reinhardtii, with simple structure, clear molecular genetic background, easy operation and low cost on culturation, especially with high activity of hydrogenase which can produce hydrogen using solar energy and water, is considered as the most potential algal species for development of biological hydrogen production in the future.
Hydrogenase of C. reinhardtii has high catalytic activity, but particularly sensitive to oxygen which is the main byproduct of algal photosynthesis. Therefore, the process of hydrogen production and hydrogenase activity is very susceptible to oxygen inhibition and inactivation, which is the maximum obstacle for hydrogen production of C. reinhardtii. To improve the biohydrogen production of C. reinhardtii, the yield of oxygen must be reduced in C. reinhardtii cells to ensure the activity of hydrogenase. The most common way at present is to remove sulfur in the culture medium or by adding some of inhibitors to inhibit the activity of photosystem II. But at the same time it will also reduce the electronic source from the water photolysis process and result in low hydrogen production. Therefore, how to reduce the oxygen content in algal cells without affecting the electronic supply for hydrogenase is a key issue to increase hydrogen production of C. reinhardtii.
Leghemoglobin (Lb) has high oxygen affinity and rapid turnover rate. It helps to reduce the oxygen concentration inside the nodule cells and ensures the oxygen-sensitive nitrogenase activity. No Lbs, no nitrogen fixation activities in the soybean nodules.
We have tried to transfer soybean Lb protein genes lba and hemH-lba into the chloroplast of C. reinhardtii in our previous work. Since the expression of protein in the C. reinhardtii chloroplast has AT bias, in this experiment, the codon-optimized Lb genes lbac and hemHc-lbac were transfered into the chloroplast of C. reinhardtii in order to increase recombinant protein expression levels and hydrogen production. The contents and results of this thesis are as follows:
1. The codon-optimized soybean Lb genes lbac and hemHc were synthezed in the company and the chloroplast expression vector cg401-1-hemHc-lbac with the design of the polycistronic hemHc and lbac genes was successfully constructed.
2. The plasmid DNA of cg401-1-hemHc-lbac was transfered into C. reinhardtii chloroplasts with the particle bombartment method. Transformants were screened by the resistance of spectinomycin and the well-growth one was picked up and named as transgenic alga hemHc-lbac after screening.
3. PCR assay based on DNA templates of the transgenic alga hemHc-lbac showed that the hemHc-lbac gene fragments had been heteroplasmicly integrated into the chloroplast genome DNA of C. reinhardtii. RT-PCR assay based on RNA templates of the transgenic algae showed that the hemHc and lbac genes transcripted successfully in algal chloroplasts.
4. By the method of Western blotting, the recombinant hemHc and lbac proteins had been successfully expressed in the chloroplasts of transgenic algae hemHc-lbac under both normal culture condition and hydrogen production condition. On the fifth days, the expression of recombinant protein reached to the maximum level and the expression level of the codon-optimized recombinant proteins was 6.8 times of the non-optimzied one. The Western blotting result on the native gel electrophoresis showed that the two Lb subunits, hemH and Lba were in the same hybridyzation band with their own specific antibodies .
5. By the quantitative real-time PCR assay, the transcriptions of hydrogenase genes in both codon-optimized and non-optimized transgenic algae were increased than those in the control algal strain cc849.
6. The growth curves of the control algae 849 and transgenic algae hemH-lba, and hemHc-lbac showed that the growth of control algae 849 reached saturation with the maximum OD750 value of 3.2, while that of the transgenic algae hemH-lba was 3.5 and transgenic alga hemHc-lbac was 3.3. At saturation phase, the maximum chlorophyll contents of the transgenic alga hemHc-lbac and hemH-lba were all about 41mg / L, while it was about 35mg / L for the control algae 849. The results showed the growth of the transgenic algae was not inhibited. Moreover, their chlorophyll contents were increased slightly.
7. Hydrogen production and oxygen consumption of both transgenic algae and the control alga were detected. The results showed that the hydrogen production of transgenic algae hemHc-lbac and hemH-lba were higher than that of the control algae nomatter under full argon condition or under the dark anaerobic condition.
8. Heme was extracted with ether from both transgenic algae and scaned by using UV spectrophotometer. There was a small absorption peak at 550nm for both transgenic algae hemH-lba and hemHc-lbac, indicating that a trace heme might be synthesized.
9. Respiratory rate and photosynthetic rate of the control and transgenic algae were measured. The results showed that net photosynthetic oxygen evolution rate of transgenic algae hemHc-lbac was lower than transgenic algae hemH-lba and both were significantly lower than that of the control algae. On the other hand, the respiration rates of both transgenic algae were higher than that of the control algae, particularly for that of the codon-optimized algae hemHc-lbac.
引文
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