| Institute of Botany, Chinese Academy of Sciences Beijing 100093The developments of molecular biology of cyanobacteria have made it on the frontier of biology. Recent years, the great progress in the genetic engineering of cyanobacteria has promised to use transgenic cyanobacteria to produce medicines or to solve environmental problems. However, the expression efficiencies of foreign genes in cyanobacteria have still been low. In order to make better social and economic benefits of transgenic cyanobacteria, we must improve the expression efficiency of foreign genes in cyanobacteria and elevate photosynthesis efficiency to stimulate their growth.Human Tumor Necrosis Factor a (abbreviated as hTNFa) was selected as target forign gene and it is a sort of multi-functional protein cytokine, which was generated by macrophage or monocyte under stimulus. TNFa has many physiological functions, plays a role of signal transductor, and most important is killing tumor tissue and tumor cells directly, specifically and wide spectrum. So, it was a quite hopeful nature factor to make into anti-tumor medicine. However, the recombinant product of Escherichia coli must be purified strictly, and usually for vein injection. Because of strong toxic side effects, the research had been stopped at clinical phase for more than ten years. We tried to express TNFa in cyanobacteria to make oral-taken medicine to reduce toxic effects. Now the transgenic Anabaena has been constructed, and the expression product has biological activities of inhibition of tumor. However, the expression efficiency was not desirable and its expression reduced the growth of the hosts.Since cyanobacteria are prokaryotic, their regulations of gene expression mainly focus on transcription level and translation level. So, looking for high effective promoters in cyanobacteria, changing the structure of SD sequence are two ways of improving expression efficiency in cyanobacteria. This work cloned TNFa cDNA containing different SD sequences into the downstream of promoter PpsbA of shuttle-expression vector pRL-489, constructed two vectors (pMD-489-TNF1, pMD-489-TNF2), and transferred into Anabaena sp. PCC 7120 by tri-parental conjugation. The quantitive assay of TNFa expression in Anabaena sp. PCC 7120 indicated that the expression efficiency of TNFa had improved effectively. The content of TNFa is 2.1 - 2.9% and 0.15% based on solubleproteins, respectively. The expression efficiencies are improved 21 - 29 times and 1.5 time respectively.While cultivated transgenic Anabaena, some changes have taken place on morphology and physiology, these indicated that the expression of TNFa affected photosynthesis of the hosts.By optical microscope and scanning electron microscope, we found that the number of heterocytes of transgenic Anabaena decreased about 30%, the vegetation cells transformed by pRL-489 turned to bigger, the heterocytes of Anabaena transformed by pMD-489-TNF bigger and vegetation cells smaller. After 15 days, the vegetation cells of Anabaena transformed by pMD-489-TNF increase their size obviously, their size as large as heterocytes. By emission electron microscope, we found that the structure of thylakoid membrane was clearer in transgenic Anabaena.The comparision of growth curves of transgenic Anabaena showed that there were no obvious effects on Anabaena transformed by pRL-489, while the growth decreased transformed by pMD-489-TNF. The cell numbers increased very slowly when the expression of TNFa was high. And in the later phase, the cell numbers somewhat decrease. It seems that expressing TNFa has some side effects on growth of hosts.Assaying photosynthetic light curve proved that transgenic Anabaena had lower light satuation points and stronger respiration. Perhaps, the expression of TNFa enhanced the light sensitivity of host, while aggraviate the metabolism burden. Over light satuation point, the true photosynthsis of them are almost same, so expression of TNFa didn't damage their photosynthetic reaction centers.From the ass... |
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