Cloning, Analysis And Expression Of Phosphomannose Isomerase Gene (PMI) From Kelp

The development and utilization of marine biological resources has become thefocus of competition in the high-tech world in21st century, and it is particularlyimportant for the research and application of marine biological genetic resources.Saccharina is one of the most important large economic marine brown algae, whichbelong to Ochrophyta, Phaeophyceae, Laminariales, Laminariaceae. The economiccomponents such as mannitol, iodine and so on which are rich in kelp have beenwidely used in medicine, seaweed chemical and agricultural fertilizer industry. Thealgin and fucoidan are the fillers of algal cell wall with a variety of biological activtieswhich are higher contents in brown algae. However, the research of alginatesbiosynthesis pathway in brown algae is rarely been reported. Therefore, it is importantand great significant to study the key genes in alginates and fucoidan biosyntheticpathway, and make the results applied to the MAS breeding areas and to reveal thealgae evolutionary process.Phosphomannose isomerase is a metalloenzyme participated in the first stepreaction of alginates biosynthetic pathway, which catalyzes the reversibleisomerization of fructose6-phosphate and mannose-6-phosphate in vivo and it playsan important role in the biosynthesis of GDP-mannose, which is the precussor of alginand fucoidan. It has been found that there are three different PMI genes （type1） inEctocarpus siliculosus, which are named PMI1, PMI2and PMI4. In this study, usinghomologous amplification method, the cDNA sequences of phosphomannoseisomerase1and4genes （PMI1and PMI4） of "Rongfu" were obtained for the firsttime which used the cDNA obtained from kelp total RNA as a template, and the fulllength of PMI1and PMI4is1020bp and1881bp, which encoded626and339aminoacids, respectively. The DNA sequences of PMI2and PMI4were obtained for the firsttime using kelp total DNA as the template, and were assisted by the kelp genomicdatabase data. The length of PMI2is8010bp, which consist of11exons and10introns; the length of PMI4is5347bp, which consist of3exons and2introns. Inaddition to,32PMI CDS sequences from16brown algae were obtained by analysisof the transcriptome database from the1KP plan. These brown algae PMI genes of brown algae belongs to the type1of phosphomannose isomerase, and they are threedifferent copy types. Analysis of the sequence conservation between these brownalgae PMIs and reported PMIs in other organisms shows that PMIs of brown algae arerelatively conservative. There are three conserved regions, and the sites related withmetal-binding residues are same in the all brown algae PMIs and three of fourresidues are same which are related with proton transfer. Newly discovered brownalgae PMI genes have high level of amino acid sequence identity with Ectocarpussiliculosus PMI genes. The similarity between the different PMI copy types of thesame species are lower than the same PMI copy types of the different species, itimplies that the phenomenon of PMI gene duplication may occur in the era of thecommon ancestor of the brown algae.This study obtained kelp PMI4gene, and constructed the prokaryotic expressionplastid pET32a-PMI4and the secretory expression plastid pPICZaA-PMI4successfully, and then transformed them into E. coli strain BL21and Pichia pastorisX-33strain respectively. The fusion protein from Prokaryotic expression is58kDa,which is consistent with the expected size, and a large number of the target protein ispresent in the form of inclusion bodies, only a minority is presence in the supernatant.The fusion protein expressed from Pichia pastoris is37kDa. However, it is present inthe cell but not secrete to fermentation broth. Fusion proteins were purified by Niaffinity chromatography. The activity of PMI from Pichia pastoris was169.7U/mgand found that the optimum temperature and pH were15℃and8.5, respectivelywhich show that the enzyme is a loe-temperature enzyme and alkaline protein.Besides, Zn²⁺、Cu²⁺and Mn²⁺inhibited the activities of PMI, Ca²⁺and Mg²⁺had noinfluence on the enzyme activity, and Co²⁺promoted he activities of PMI.