Methodological Innovation For Analysis Of The Bacterial Community Structure And The Study Of CFB Group Associated With Ulva Prolifera

There is only one dominant ecotype of Ulva prolifera for the consecutive blooming green tides in the Yellow Sea, which indicates that the biological traits of the floating U. prolifera are in important position of all blooming reasons. Bacterial communities were revealed to play key roles in terms of nutrition absorption and morphorgenesis. The study of U. prolifera associated bacteria provides new angle for tracing the source and the blooming mechanism of the green tides. Compared to epiphytic bacteria, more highly specific association between endophytic bacteria and macroalgae exists. Consequently, it’s necessary to study epiphytic and endophytic bacteria separately. However, an effective method meeting the needs for the study is still absent.In this paper, we compared the bacterial community structures of floating and attached U. prolifera by universal primer of 16 S rRNA. We found some rules and problems: 1) There was obvious difference between floating and attached U. prolifera at the level of family and phyla, and similarity exists between the two floating strains. 2) We also found some functional group such as CFB and Planctomycetes. Axenic system should be created to to verify the function of CFB and obvious difference existed of the percentage of Planctomycetes compared with the published paper, which prompt that we should analyse the epi- and endophytic bacterial community structures separately. 3) Specific primers should be designed to avoid the disturbance of choloroplast 16 S.At first, we obtained no epiphytes U. prolifera by newly mechanical method, i.e. co-vortex of Ulva thalli with certain particle size of silica sand and opitimize the parameter. According to the DGGE analysis, this method cause minimal disturbance to endophytes and could be used to analyze the epi- and endo- bacterial communities separately.In order to avoid the interaction of chloroplast 16 S to the endophytic community structure analysis, two ways for the optimization of primers were established. For the first, we optimized the primer 799 F from 3 aspects including primer design, annealing temperature and hot start. As a result, the percentage of chloroplast 16 S dropped dramatically after optimization, which revealed the mechanism of PCR specificity. Secondly, On the basis of the 2 nucleotide mismatch in the conserved region founded by comparing 31 phyla of bacteria and the sequence of chloroplast 16 S, we designed a new primer 1525 R. This primer could be used to analyze the endophytic community structure without chloroplast amplification according to the sequencing results.One dramatically character distinguishes the floating U. prolifera from the attached one is the highly branches morphology which contributes to its floating life. CFB bacteria were an important group taking part in the morphology of Ulva. In this paper, we compared CFB community structure of 8 strains of floating and attached U. prolifera by specific CFB primers.In addition, we separated CFB strains and established the sterile system of U. prolifera using antibiotics, which could be used to verify the function of CFB strains.In conclusion, we analyzed the bacterial community structure of floating and attached U. prolifera primarily and we developed a series of methods which laying the foundation of analyzing the epi- and endo- bacterial communities structures separately. In addition, we analysed the CFB community structure and established the sterile system of U. prolifera which could be used to verify the function of CFB strains.