Changes In Diversity Of Phyllosphere Microorganisms In Transgenic Bt Corn

Use of transgenic crop is increasing at a rapid rate in worldwide.Field and labaoatory studies of transgenic Bt crops have been carried out to assess their risks to environment. The insertion of exogenous gene (including the target gene, the marker gene and promoter) distrupted the existing network of plants, which may alter the physiological and biochemical or the structure. In this thesis, a field planted with transgenic Bt corn (MON810) and its parental variety (DK647) grow under normal agriculture for two yeas from2010to2011. The phyllosphere of7growth stages such as seeding stage, jointing stage, trumpet stage, tasseling stage, silking stage, milk stage and full ripe satge were examined with the leaves od different sampling times. The methods were PLFA and PCR-DGGE analysis.1、Phospholipid fatty acid analysis (PLFA) was used to study the diversity of Phyllosphere microorganisms community structure during2011maize growing season,23kinds PLFAs were detected, the dominated PFLAs were the saturated fatty acid and branched fatty acid; the contents of polyunsaturated fatty acid and cyclopropane fatty acid were relatively small in amount. The bacteria PLFAs in the leaves were much higher than those of fungi and actinomycete, accounting for70%of the total PLFA. Little difference showed the types of PLFA during periods, Phyllosphere microorganisms total PLFA, bacteria PLFA and fungi PLFA in the Bt corn were less than non-Bt corn. The effect of Bt corn to GN is apparent, reaching a significant level in tasseling stage. Principal component analysis (PCA) indicated that there were significant differences (P>0.05) between Bt corn and non-Bt corn in seedling, milk, full ripe stages, however, other stages were having high similarity. The Bt gene had no significant influence on the Phyllosphere microorganisms’community.2、The results showed that both four methods could got appropriate fragment for PCR,but the yield and purity of DNA were different and the best method was Kit method.Except Chemical method,other methods could get products of PCR. Primers F357/R518and ITS1-F/ITS2which also had the largest amplification were best for16S rRNA and ITS sequence amplified,which has provided a guarantee for the smooth progress of the follow-up DGGE. 3、Denaturing gradient gel electrophoresis (DGGE) analysis was used to analyze whether Bt corn can influence the diversity of soil microbial genetic communities. Clonging and sequencing were subsequently studied to further determine the microbial community of the major microorganism species and specific species of microorganism. The sequencing results of DGGE band patterns showed that most of the Phyllo-sphere microorganisms were uncultured, The main bacteria are devided into four major clusters, Proteobacteria has the largest proportion, then, Bacteroidetes, Firmicutes, Actinobacteria, The main fungi are devided into two major clusters, Ascomycota and Basidiomycota. Phyllosphere microorganisms of corn had similar tendency in two years from2010to2011. In2010years, the bacteria species corn leaves were richer than fungi, and, the differences were significant, which may because of the frequently rain causing fungi were unable to do long-term colonization on the corn leaves. Continuous two years samples show that more or less specific bands appeared in the DGGE band patterns of two genotypes maize at different stages. Specfic bands representatives of microbial species were different in different stages or the same stage of different years, did not had apparent regularity. In all, phyllosphere microorganisms were influenced heavily by the different growth stages and prevailing environmental conditions. Most of the growth stages, phyllosphere microorganisms were had high similarity in community genetic diversity between Bt corn and non-Bt corn.