| Anabaena sp. PCC 7120 belongs to the eubacteria class which has the capacity of nitrogen fixation and photosynthetic oxygen production. It is a kind of autotrophic filose prokaryote which is gram-negative and multicellular. With so many advantages as simple structure, easy cultivation and the convenience for genetically engineering improvement,Anabaena sp. PCC 7120 has now become an important model organism and a stable gene expression host in the study of molecular biology and genetic engineering. In addition,Anabaena sp. PCC 7120 is an ideal protein resource that can be developed and utilized as animal feed, as it is rich in protein, nucleic acid, lipid and other nutrients. In this study,based on the achievements of previous genetic researches on Anabaena sp. PCC 7120 and the fact that the animal feed additive is an important terminal market of amino acid and using the Anabaena sp. PCC 7120 preferred codon, the researcher designs the target exogenous gene segments HEP234 that can specifically express the small peptide composed of various essential amino acids, genetically improves the target gene segment and brings it into the fusion gene sequence and thereby constructs the fusion gene sequence in vitro that contains the mentioned target gene segments and the shuttle plasmid p HEP130 of the strong promoter by means of genetic engineering, transforms the the Anabaena sp.PCC 7120 by means of triparental conjugation, develops positive transgenetic algal strains of plasmid stability through screening and purifying, conducts molecular detection, induced protein expression and Western-Blot to the transgenic Anabaena sp. PCC 7120, so as to confirm the existence of the target exogenous gene in the transgenetic algal strains. The researcher also optimizes the growth conditions of the transgenic Anabaena sp. PCC 7120 separately to find a significant improvement of the expression efficiency of the target exogenous genes of the transgenic algae under the optimized conditions. Major research results are as follows:(1) The target exogenous gene segment HEP234 is designed based on Anabaena sp.PCC 7120 preferred codon, which, at a length of 78 bp, can specifically express the small peptide composed of 13 types of essential amino acids. On this basis, HEP234 is genetically improved with TEV protease recognition sequence and GFP sequence introduced into N-terminal and His-tag into C-terminal. The fusion sequence containing the target exogenous gene segments has a total length of 819 bp. At the initial stage of the experiment, the fusion sequence that contains the mentioned target exogenous gene segments and the shuttle plasmid p HEP130 of the strong promoter Lac C are successfully constructed, Anabaena sp. PCC 7120 is transformed by means of triparental conjugation and the positive transgenic algal strains are developed after three purifying and screening.(2) Plasmid stability of the genetically engineered bacterium sets a theoretical basis for the efficient expression of the target exogenous gene products. In the experiment, the plate dilution method and the plate count method are adopted respectively to comprehensively compare the growth situation of the serially subcultured E.coli NEB 10?::p HEP130 and PCC 7120::p HEP130 under the cultivation condition under or not under selective pressure;and monoclonal plasmids of 5 generations are randomly selected for PCR identification.Results confirm that no plasmid deficiency occurs in E.coli NEB 10?::p HEP130 of the 147 generations and that the plasmid of PCC 7120::p HEP130 stays stable during 10 serial passages.(3) Under regular laboratory cultivation conditions(temperature 30 ?, continuous illumination 70 ?E m-2 s-1, oscillation 110 rpm), the growth curve of transgenic Anabaena sp. PCC 7120 under the non-nitrogen cultivation condition is figured out. In addition, the researcher finds that the transgenic algae growing in the nitrogen cultivation condition shows significantly higher growth rate that growing in the non-nitrogen cultivation condition within 5~17 days. Therefore, the transgenic Anabaena sp. PCC 7120 cultivated in the nitrogen condition for 15 days is selected for fusion protein extraction from the target gene.(4) By means of induced expression, smashing extraction, Ni column purification,affinity chromatography and other technological means, target gen fusion protein can be extracted from the efficiently expressed purified positive transgenosis algal strains. Theoptimum extraction conditions include 1m inducer for inducing and ultrasonication for smashing extraction. Finally, the target small peptide HEP234 Protein is obtained through identification by TEV protease digestion.(5) Single-factor control variable method is used to explore the effect of seed solution,temperature, p H, illumination, exogenous carbon source and nitrogen source on the biomass of transgenic Anabaena sp. PCC 7120 and on the expression efficiency of target gene fusion protein. The possible optimal cultivation conditions can be summarized as inoculating the seed solution at subcultivation logarithmic phase into fluid medium AA/8N,with inoculum size of 6%, temperature at 28?, continuous light intensity of 1500 Lux,vibration at constant velocity of 110 rpm and with exogenous carbon source(with carbon content of 50 mmol/L) like glucose and saccharose added. In the optimized cultivation condition rather than the conventional cultivation, the expression quantity of the target gene fusion protein can be improved significantly. The biomass of the final transgenosis algae cultivated in the optimized cultivation condition with glucose added as mentioned above is43.8% higher than that cultivated in conventional cultivation condition, which provides reference for mass production. |
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