Screening Of PKS Gene From Soil Metagenomics Library And Identification Of The Active Substance Against Nematodes

Screening of PKS gene from Soil Metagenomics Library and Identification of the Active Substance against NematodesThe soil is the most main habitats for microorganisms with estimated numbers of 4-5×1030 in per gram soil. However, it is difficult to obtain the whole information of unculturable microorganisms, due to over 99% of microorganisms could not be cultured by the traditional culturable methods. Metagenome break through the limitation of culturabe method, and bring a new technology for utilization for resources of non-culturable microorginasm in soil. In this papper, genes with nematocidal activity were screened from soil metagnomic library derived from Tibet and greenhouse in Huabei.1. Comparison of methods for DNA extraction from soilBecause of amount of organic matter and humic acid in soil, it is extremely difficult to extract and purify microbial DNA from soil. Comparison of various methods for extraction and purification of DNA showed that DNA of direct extraction were short fragments and large amounts, contained high impurities and fitted for analysis of microbial diversity. However, DNA of indirect extraction that combined with differential centrifugation and Nycodenz density gradient centrifugation were long fragments, high purity and fitted for construction of large insert fragment library after purified with gel electrophoresis. In addition, pH, available P, available K and organic matter in soil had influences to DNA quantities.2. Screening and functional analysis of PKS and NRPS from Tibet alpine meadow soil and HuabeiThe diversity of family of PKS gene and NRPS gene were revealed by PCR method with degenerate primer from DNA diectly extacted from two soil samples. The result showed that 37 PKS-KS genes (28 from Tibet,9 from north China) and 40 NRPS-A genes (24 from Tibet,16 from north China) were obtained and these genes have lower similarity with knowed genes by BLASTX (similarity to PKS from 57% to 82%, similarity to NRPS from 47% to 67%), which indicated that they were probably new genes.3. Construction of metagenomic fosmid library from Tibet and HuabeiTwo metagenomic fosmid libraries were constructed using microbial DNA indirect extracted from 2 soil samples. Fosmid library from Tibet contained 30 624 clones and Fosmid library the soil example from Huabei contained 31 008 clones. The average length of insert fragments were 30 kb, and the stability of that was good without losing or resetting. The results of end sequencing of clones indicated that the percent of no significant homo logy sequences in library of the soil example from Huabei were 92.75%, yet that in library of Tibet were 94.3%. This showed that the two libraries contained many unknowed microbial speics and would lay the foundation for exploiting novel functional gene from soil.4. Screening for clones contained PKS gene and evaluation of its nematictdal activityA total of 7 clones contained PKS gene were obtained from two metagenomic libraries by PCR sequence selection according to degenerate primer designed from KS domain of PKS gene (4 from Huabei library,3 from Tibet library), of which K99 clone contained 2 domains. The nematocidal activity for root knot nematode and pine wood namtode was evaluated by steeping method with cultural liquid of clones for 4 days. The result showed that the nematocidal activity of clone K99 was high than others, and the rate of knockdown to two speices nematode treated with cultured liquid of clone K99 for 12 hours was 100%. The result of pot culture experiment in greenhouse also revealed that control efficiency of clone K99 against root knot nematode was 89%.5. Identification of nematocidal active substance produced by clone K99The lethallty rate of pine wood nematode treated with crude extact from culture liquid by normal butanol and bacterial cell of clone K99 by methanol for 12 hours were 100% and 96%, respectively. The activity of crude extract treated with hot at 121℃for 20 minuties was unchanging, which implied that the active substance was thermal stability. The result of tracking method by thin-layer chromatography showed that the channels with medium polar including channels 1, channels 2 and channels 3 possessed nematocidal activity, but the channel 5 with small polar and channel 4 with more polar did not kill nematode. From the results above, it was draw some conclusions that the active substance produced by clone K99 probably was polyketides with medium polar and thermal stability.6. Evaluation of nematictdal activity of one strain actinomyces contained PKS genePKS gene and NRPS gene were amplified from one strain actinomyces F001 with nematocidal activity and the ferment condition for F001 was optimized. The result indicated that PKS gene and NRPS gene from F001 had high similarity to know gene from others actinomyces. The optimal ferment condition for F001 was culture under 28℃for 9 days, pH of medium was 7.0, rotational speed was 180 r/min and the liquid volume was 200 mL in 250 mL.In a conclusion, some clones with nematocidal activity were obtained from two metagenomic fosmid libraries, especially clone K99 with high nematocidal activity in vivo and in pot culture experiment in greenhouse. The active substance produced by clone K99 probably was polyketides with medium polar and thermal stability. It was worth to further study.