Preliminary Study On Epiphytic Bacteria Of Ulva Prolifera

Epiphytic bacteria play a key role in the formation of nutrient absorption and morphological structure in algae life history.Because only 1% of marine bacteria can be cultivated,and a large number of them are unknown bacteria,there are fewer research reports on endophytes compare to epiphytes.Some large seaweeds already have a well-established endophyte[1-2] and because the bacteria live inside the algae,so it doesn't change very much with the environment.Aires et al.analyzed the endophytes bacterial community structure of C.taxifolia through high-throughputs equencing in molecular biology techniques and found that endophytes bacterial community structure remained stable regardless of external environment[3-4].The results of these studies have shown that the algae's epiphytes will change with the area,time,and living conditions.The composition of endophytes is more stable and specific,and plays a key role in the growth of algae.This suggests that there is a need to distinguish between endophytes and epiphytes[4].Now we can use the methods of bacterial diversity analysis to clearly know the types and amounts of bacteria that are symbiotic inside and outside the algae.The composition analysis of the internal and external symbiotic bacteria of Ulva prolifera was carried out.The analysis of flora diversity revealed intuitively that there were some inherited equences in the symbionts of Brassica and existing databases.Relatively distant strains.The analysis showed that Alternaria spp.Occupied preponderance in both internal and external symbiotic bacteria,followed by chloroplast-norank and polaribacter4.It has been further shown that the intracellular and extracellular bacterial species of algae need to be studied differently,and the bacteria that are symbiotic around the Enteromorpha are the result of their mutual selection in the living environment.In this paper,the culturable CFB strains were isolated and identified,and an aseptic experimental system for Ulva prolifera was established by antibiotic method,which will be used to verify the MG function of the strain.The culturable extracellular symbiotic bacteria were isolated and purified,and 16 S r DNA gene sequence alignment methods was used to identify symbiotic bacteria belonging to genus Rhodobacter,Marinobacter,Marivita,and Rosevirus.They were all had reported in the literature.It may be the bacteria that have an important influence on the morphogenesis of Ulva.In order to detect the effect of extracellular symbiotic bacteria on the morphogenesis of Ulva prolifera,the explants were isolated and purified from the laboratory cultivated Ulva prolifera.Then used antibiotics to treat the algae aseptically.The morphogenetic process of Ulva prolifera.A temporary sterility was observed under the scanning electron microscope.Under aseptic conditions,the morphology of Ulva prolifera changed,becoming irregular and extremely slow,whereas with the growth of Ulva prolifera,the leaves experienced morphological abnormalities.To the more normal process,and the sterile dead algal body will not be degraded.Therefore,it is inferred that the morphological changes in the survival of Ulva prolifera in life history may be related to epiphytes.At the same time,it is known that a Flavobacterium symbiotic with green algae can secrete extracellular enzymes that can degrade polysaccharides in the cell walls of green algae to obtain functional oligosaccharides,and as a result,the bacteria are found to be new.The species[5]has not been reported before.Based on the various advantages of enzymatic preparation of oligosaccharides,we want to artificially synthesize the nucleic acid sequences of the enzyme proteins produced by extracellular commensal bacteria by means of genetic engineering and protein engineering,and express them heterologously through prokaryotic expression methods.And compare the prokaryotic expression vectors and transforming the recombinant receptors expression effect of ulvan lyase gene.The artificially synthesized ulvan lyase gene fragment was cut into prokaryotic expression vector p Cold I plasmid and p ET-28a(+)plasmid,respectively,to obtain recombinant plasmids p Cold I-859 and p ET-28a(+)-859(859 represents ulvan lyase).The ulvan lyase gene was identified by double digestion and sequencing.The plasmid p Cold I-859 was transformed into E.coli RP(DE3)competent cells,and the plasmid p ET-28a(+)-859 was transformed into E.coli BL21(DE3)Lys S.to expressed.The results showed that the target protein was not clearly expressed in the former but the target protein was expressed in the latter.The expression of the target protein was higher after isolation,purification,electrophoresis and western blot.In this experiment,preliminary studies on epiphytes of the Ulva prolifera showed that there are many kinds of substances and information exchanges between bacteria and algae.This subtle algal environment affects the life history of algae.At the same time,a comparative study has yielded a prokaryotic expression system that can fully express the ulvan lyase of Flavobacterium,which may replace the cumbersome process of separating and collecting extracellular enzymes secreted by Brassica symbiosis,and also replace chemical methods to some extent.The preparation of oligosaccharidesprovides a basis for the exploration of heterologous expression of marine algae tools.It also provides a reference for the preparation of low-cost,high-purity ulvan lyase protein in batches.