Metagenomic Technology Platform And It's Application For The Research And Exploitation Of Microbial Resources In Mangrove Soil

Microbes inhabiting in special intertidal mangrove soil niches possess the enormous genetic diversity and immense potential to produce new secondary metabolites. Paying attention to the research mangrove soil microbes would accelerate the rate of discovering novel bioactive molecules. Culture-independent metagenomic technology provides a powerful tool to explore microbial resources with high throughput in diverse niches, however, some hurdles have not yet overcomed when it applied to mangrove soil niches. ( i) Mangrove soil is high in clay and rich in organic matter, the abundant high molecular weight inhibitors such as humic acid and humic compounds in soil samples are difficult to remove, and are always co-extracted with soil DNA, subsequent molecular diversity analysis would be hindered because of the poor quality of the isolated DNA. (ii) In the lysis step, DNA liberated from soil microbes, especially the large fragment DNA, was likely to adsorb to the soil clay. Therefore, the total extracts with low percentage of large fragment DNA failed to construct large insert environmental libraries for biotechnology use. Towards overcoming these hurdles, the method of extraction of mangrove soil DNA for microbial diversity research and the method of construction of large fragment metagenome library of mangrove soil were studied in this paper. The results were as follows:1. NaCl posed significance effect on yield of coextracted humic compounds (YCHC). YCHC decreased when the concentration of NaCl in the extraction buffer increased. Whereas, the effect of Na₃PO₄ on YCHC was just on the opposite; when polyvinylpyrrolidone (PVP) was used pre-lysis or in lysis, YCHC increased, whereas, when PVP was used after lysis, the quality of crude extracts was improved; when polyvinylpolypyrrolidone( PVPP) was used pre- lysis or in lysis, YCHC decreased, whereas, when PVPP wae used after lysis, it has no positive effect on the quality of crude extracts; Whenever CTAB was used pre lysis, in lysis or after lysis,, the quality of crude extracts was improved; When the concentration of SDS in the extraction buffer increased or the lysis time lasted more longer, the YCHC increased; When the concentration of CTAB in the extraction buffer increased, the YCHC decreased; The relationship between YCHC and lysis time with CTAB was as follows, the YCHC increased in the first two hours, then decreased gradually; The addition of CTAB in the extraction buffer not only resulted in lysing microbial cells but also removing the humic compounds, the latter one was the main function; Whenever PVP, PVPP, CTAB were used pre lysis, in lysis or after lysis, it does not resulted in the loss of microbial diversity; The quality of mangrove soil DNA without further purification was adequate for PCR when NaCl[1.5mol.L^-1], PVPP[2%] and CTAB[2%] introduced in the extraction buffer.2. Applying our optimized direct extraction method, the percentage of large fragment DNA in the total extracted mangrove soil DNA was significantly increased, and the large fragment metagenome library derived from natural mangrove soil over four seasons was successfully constructed by the optimized DNA extraction and electro elution purification method. This library produced 9570 cosmid clone, the average insert size for this library was larger than 35kbp. At least 335Mbp valuable genetic information of mangrove soil microbes was encompassed in this culturing-independent library. A clone containing anti-tetracylin activity, a clone producing brown pigment and a clone containing amylase activity were detected by function- driven screening method.3. Experiments were designed to investigate the capability of capturing the indigenous microbial genome of SDS, lysomyze, shaking with bead beating, sonication, microwave and froze-thawn cycles in this study. Results showed, (i) Each methods could lyse the indigenous bacteria and archaea of mangrove soil, but could not lyse the indigenous actinobacteria and fungi. ( ii ) Indigenous fungi lysed when applying the the combination of SDS, lysomyze and shaking with bead beating. (iii) Indigenous actinobacteria lysed when applying the the combination of SDS, lysomyze, shaking with bead beating and microwave.4. DGGE was used to compare the mangrove soil community produced by six methods respectively. Results showed, none method could produce whole bacterial community, the DGGE profile produced by soniction, microwave and lysomyze were similar, the DGGE profile produced by SDS, shaking with bead beating, froze-thawn cycles and froze-thawn cycles contained special band respectively.5. The mangrove soil microbial extracellular DNA diversity was evaluated by PCR and the seasonal variation of bacterial diversity in the Avicennia marina rhizosphere was analyzed by DGGE. Results showed, DNA derived from bacteria and archaea were involved in extracellular DNA, but DNA derived from actinobacteria and fungi were excluded. The bacterial communities in the Avicennia marina rhizosphere of different depth in different seasons were similar.In summary, Establishment of metagenomic technology platform allowed mining of valuable intertidal microbial resource to become a reality. It is a recommended method for those researchers who have still not circumvented the large insert environmental libraries or for those beginning research in this field, so as to avoid them attempting repetitive, fussy work.