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In this study carbon assimilation stage three successive catalytic enzymes: fructose 1,6 - bisphosphatase (Fructose-1, 6-bisphosphatase, referred FBPase), fructose 1,6 - bisphosphate aldolase enzyme (Fructose-1 , 6-bisphosphate Aldolase, ALD) and triose phosphate isomerase (Triosephosphate Isomerase, referred to as the TPI) as the target enzyme, the use of the cyanobacterial genetic engineering to study the regulation of the photosynthesis, the adjustment control for the in-depth understanding of photosynthesis rule and in accordance with these The law of transformation of the human needs of plants have a significant and far-reaching significance, the same time, it may also be an effective control or a way to reduce the concentration of CO2 in the atmosphere. This thesis, this goal, mainly in the transformation of one, two and three target gene into the blue-green algae, study how they expanded regulation cyanobacteria photosynthesis main line of work. First, by PCR cloned FBPase gene, and is connected with the expression vector pRL-439, and then cloned into the downstream of a strong promoter (PpsbA) shuttle vector pDC-08 Construction of the shuttle vector expressing the PDC-FBP, by three The conjugative transfer method to import Anabaena sp. Transgenic Anabaena 7120 FBPase expression efficiency improve by 16.8% as compared with wild Anabaena 7120 single activity of 1.34 times. Series ALD and TPI gene, and cloned into the shuttle expression vector pRL-489 downstream of the strong promoter (PpsbA) build shuttle expression vector pRL-AT1 and pRL-AT2, triparental conjugative transfer was introduced into Anabaena 7120. Compared with wild Anabaena 7120, transgenic Anabaena 7120 ALD and TPI expression efficiency were increased by 58.9% and 53.3%, dual enzyme activity increased 2.59 times. Moreover, when the exogenous gene of the operon and the vector plasmid is greater than a 1:1 ratio to build, it is possible to significantly enhance the expression of the exogenous genes and the corresponding enzymatic activity. Concatenating the three genes, and is connected to it with the expression vector pRL-439, and cloned into the shuttle vector pDC-08 downstream of the strong promoter (PpsbA) and then its constructed shuttle vector expressing PDC-ATF, by triparental The conjugative transfer method import Anabaena sp. Compared with wild Anabaena 7120 transgenic Anabaena 7120 ALD, TPI and FBPase expression efficiency increased by 22.8%, 20.9% and 6.2%, three-enzyme activity increased 1.96 times. CO2 concentration in the atmosphere for 360p.pm condition, improve the transgenic Anabaena 7120 photosynthetic oxygen evolution activity, CO2 assimilation rate and accelerate its growth. Compared with wild Anabaena 7120, the switch to a single gene of Anabaena 7120 net photosynthesis increased 17.2%, real photosynthesis increased 22.5%, corresponding CO2 assimilation rate increased by 18.1% and accelerate its growth; turn Anabaena Anabaena 7120 net photosynthesis dual enzyme dimer gene increased 45.5%, real photosynthesis increased by 41.6%, CO2 assimilation rate increased by 36.8%, and accelerate its growth; transfer three genes 7120 net photosynthesis increased 31.3%, the true photosynthesis increased by 38%, corresponding CO2 assimilation rate increased by 33.2% and accelerate its growth. lt; WP = 6 gt; from the transgenic Anabaena 7120 room temperature absorption spectrum of the data showed that the photosynthetic pigment composition compared with wild Anabaena 7120 transgenic Anabaena 7120 has not changed, photosynthetic structure was not affected. 77K fluorescence emission spectra data show that, compared with wild Anabaena 7120 transgenic Anabaena 7120 the PSII allocation of energy more and more likely to pass to the PSI; moreover, the transgenic Anabaena 7120PSI has higher activity, resulting More assimilation force, which is conducive to the conduct carbon assimilation stage. Kinetic fluorescence data indicate that, compared with wild Anabaena 7120, the the transgenic Anabaena 7120 has more the primordial solar energy conversion efficiency, that higher PSII activity. When 5mmol / L NaHCO3 added oxygen electrode reaction tank, along with the oxygen-evolving activity of transgenic Anabaena 7120 greatly increased, especially turn pRL-AT2 Anabaena 7120, compared with wild Anabaena 7120, its net photosynthesis, true photosynthesis were increased by 98%, 95.5%, and light saturation point, a 130.2 percent increase. This the transgenic Anabaena 7120 has a good prospect. Optical microscope observed in the same growth period, compared with wild Anabaena 7120, transgenic Anabaena 7120 individuals more, especially cell diameter wider; Moreover, transgenic Anabaena 7120 still has a late growth stage strong physiological activity, manifested in the form of still can not see the obvious cavity. So far, genetic engineering in cyanobacteria transformed the stage of the target gene of carbon assimilation in higher plant cells, improve transgenic cyanobacteria CO2 assimilation efficiency not seen any reports; to explore photosynthesis adjust cyanobacteria genetic engineering control law, and in accordance with these laws to transform human the needs of the plant, such as high CO2 assimilation transgenic cyanobacteria, are also not seen any reports.
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