Functional Characterization Of Cyanobacterium Hemoglobin Gene (CHB) In Bacterium, Yeast And Plants

Rapeseed is a worldwide main oil crop,its oil is for cook,and its cake is for feeding the animal.In recent years,the oil quality is continually refined and gradually applied in chemical industry,and bio-energy.Submergence stress as a result of flooding for days or longer often compromises plant growth and dramatically decreases the crop yields.Furthermore,a prolonged submergence treatment can lead to the death of plant.Therefore,the plant's tolerance to flooding/submergence stress is important trait for crop.Oxygen has become one of the major constraint factors to increase yield in the microbial industry.Using genetically engineered microorganisms is an effective strategy to solve these issues.Hemoglobin in animal cell can bound and transport oxygen in order to improve provision of oxygen,fluence of electrons in respiratory chain,vigor of ATPase;and hemoglobin in microbacteria can promote growth of microbacteria,synthesis of protein and production of metabolite.We have transformed CHB gene into Escherichia coli BL21,Saccharomyces cerevisia,Arabidopsis thaliana and Brassica napus and identified the functions.The major results are as follows:1.CHB gene was ligated into the prokaryotic expression vector pET30a and expressed in Escherichia coli BL21.CHB was successfully expressed in E.coli(BL21 strain) as a fusion protein.This protein was further confirmed by Western blot using anti-6 x His monoclonal antibody.The growth curve of Escherichia coli BL21 was measured in both hypoxia and enough oxygen conditions.The results showed that the CHB gene can not only enhance the growth speed of bacteria but also increase the yield of bacteria both in hypoxia and enough oxygen conditions.2.CHB gene was ligated into the yeast expression vector pYES2 and expressed in the protease-A-deficient(pep4) strain of Saccharomyces cerevisia.CHB expression was confirmed by reverse transcriptase-polymerase chain reaction(RT-PCR) using CHB-specific primers.The result of RT-PCR showed that the recombinant plasmid could be transcripted correctly in Saccharomyces cerevisia pep4.The growth curve of Saccharomyces cerevisia pep4 was measured.The result showed that CHB gene can enhance the growth speed of pep4.The expression of CHB decreased the yeast oil content,but did not change the yeast phospholipids.3.The vector pCAMBIA1300-CaMV35S-CHB was transformed into Arabidopsis thaliana mediated by A.tumefactiens strain GV3101.Transgenic plants were selected by antibiotic and identified with PCR.CHB can enhance growth and shorten life span of transgenic plants.4.The vector pCAMBIA1300-CaMV35S-CHB was transformed into Brassica napus mediated by A.tumefactiens strain LBA4404,and transgenic plants were identified with PCR analysis.Transcription of the gene was confirmed by reverse transcriptase-polymerase chain reaction(RT-PCR) using CHB primers.Results showed that the CHB gene was integrated into the genome of the host,transcribed and translated in B.napus.Heterologous expression of CHB reduced germination time, promoted plant growth and shorten life cycle.Furthermore,transgenic plants tolerated waterlogging.