Dynamics In Bacterial Population And Shift Of NptⅡ Gene In The Phyllosphere Of Insect-resistant Transgenic Cotton

Insect-resistant transgenic cotton 33B, SGK321, GK12 and corresponding receptor cotton 33, Shiyuan 321, No.3 Simian cotton were sampled at different growing stages. Dynamics and diversity among bacterial populations in the phyllosphere were analyzed with traditional microbial methods and molecular biological techniques. In addition, a drift of the kanamycin resistant maker genes nptâ…¡ into the phyllosphere bacteria was monitored.Dynamics and diversity of bacterial population in the phyllospere of the six cotton varieties were studied by the means of conventional culture methods. In different sample time, significant differences were found among the total number and diversity index of phyllospere bacterial population, but little differences among six varieties at the same time. According to phenotype characters, the strains isolated from cotton phyllospere were classified into 104 strains. The ITS squence of 17 main strains were cloned, sequenced and analyzed by using BLAST. They were submitted to GenBank (Accession number DQ447751-DQ447767). They were divided into 10 genus including 9 bacteria families, 4 yeast families. The predominance familis in phyllospere of all six cotton varieties were Bacillus, Pseudomonas .Ribosomal Intergenic Spacer Analysis (RISA) was used to compare the diversity differences in phyllospere bacterial population between six cotton varieties. A clear fingerprint map was obtained. The phyllospere bacterial population structure presented complexity and rich diversity. Cluster analysis showed that the composition of phyllospere bacterial population displayed a great uneveness and random, suggesting that environment and climate factors affected the phyllosphere bacteria, but Insect-resistant transgenic cottons did not present visible impact.We studied the Dynamics and diversity of kanamycin-resistant bacteria population in the phyllosphere of the 6 cottons varieties, the result indicated that at different sample time, significant differences were found among the total number and diversity index of phyllospere bacterial population, but slight differences among six varieties when the sampling time was same. At the same time, we discovered the positive segment of bacteria in the phyllosphere of insect-resistant transgenic cotton SKG321, GK12 and receptor cotton No.3 Simian cotton by germ PCR to monitor the nptâ…¡ gene drift. The unique primer was designed from the squence of nptâ…¡ gene, the CK is pBI121 plasmids. We suggested that nptâ…¡ gene in insect-resistant transgenic cottons could drift into the phyllosphere bacteria.