Colonization Pattern Of Azospirillum Brasilense Yu62 On Maize Roots Isolation Of Nitrogen-fixing Bacilli And Study Of GlnB Gene

Part one of this work was about the colonization pattern of Azospirillum brasilense Yu62 on maize roots. Plasmid pVK1001 which carried the gfp gene of pGFPmut2 , a mutant of GFP , was introduced into A. brasilense Yu62 by electroporation. Maize seedlings were inoculated with the GFP-labelled bacteria and grown gnotobiotically in flask with semi- solid agar medium. Observations were performed with confocal laser scanning microscopy (CLSM) and electron microscopy , respectively , at 8 d and 12 d /10d after inoculation. Confocal laser scanning microscopy showed that A. brasilense Yu62 could penetrate into the cortex tissue , colonizing in the intercellular spaces of the parenchyma cells of the cortex tissue. Transmission(TEM) and scanning electron microscopy (SEM) showed that the majority of the bacteria colonized on the root surface and only a minority of them resided in the root interior.Part two of this work was isolation and identification of two strains of nitrogen-fixing Bacilli. Soil samples from rhizosphere of different kinds of crop and officinal were collected. Primary screening was done as sterilized the suspension of soil, diluted and plated on selected medium(nitrogen free), isolated and purified the colonies. After these, 15 strains were obtained. Then PCR amplification using two degenerate primers for nitrogenase Fe protein gene was performed with chromosomal DNA from these 15 strains. The ability of nitrogenase of these 15 isolates were analized using liquid medium. Also Southern blot was done to detect whether the genome of the 15 strains possess the nifH gene. Finally, the results implied that 2 of the 15 isolations were nitrogen-fixing Bacilli. 16S rDNA sequencing suggested that the 2 isolations were Bacillus megaterium C4 and Paenibacillus polymyxa N4. Constructed X phage genome library of C4.Part three of this work was using two degenerate primers amplified glnB gene fragment from C4 and G2( isolated by Y. Q. Ding). Translated nucleic acid sequences aligned with P_Ⅱ sequences from other bacteria suggested that the translation of C4 showed high similarity with the P_Ⅱ homologous of Rhodospirillum, Magnetospirillum and Azospirillum, while high similarity shared between the translation of G2 and the P_Ⅱ homologous of Pseudomonas, Xanthomonas, Burkholderia and Azoarcus. The identity of the translation of C4 and G2 with NrgB, the unique P_Ⅱ homologous obtained from Bacilli, was lower than 50%. Constructed several integration vectors carrying different selected marker of Gram positive bacteria to obtain single-crossover mutants. One of it was done with pE194 which possessed a thermosensitive replicon. A fusion between pE194 and pUC19 at their Pst\ sites was obtained. It could replicate in both E. coli and Bacillus. To raise the efficiency of electroporation several improvements were done, including changing the component of washing buffer, controlling the time of incubation, adding cell wall-weaken material, adding high- osmotic reagent and changing the voltage. With the optimum condition the efficiency of electroporation could obtain 10~3 -10~4 transforments /μgDNA in G2. While no transforments could obtain in C4. Failed to obtain mutant by transformed integration vectors and by incubated G2 carried pE194 derivant at non-permission temperature may rise the hypotheses that a homologous shorter than 300bp were not enough for homologous-dependent recombination.