| Endophyte is one of microorganism communities with abundant biodiversity. They have been living in healthy plant tissues without causing any disease symptoms and coevolving in their hosts. In recent years,with the study on the endophytic populations broadened and deepened,it has become one of the most popular research topics,because of its ecological and physiological functions and the great application potentiality in the fields of agriculture and medicine as biocontrol agents and foreign gene carrier. Therefore using endophytic bacteria as biological agents will become one of the future trend of development.In previous work , endophytic Bacillus subtilis strain BS2 and B. amyloliquefaciens strain TB2 have been proved to be promoting plant growth and inhibiting growth of plant pathogens,so that it has a promising prospect of application. To understand the infection mechanism of these potential biocontrol agents and their ecological characteristics in plant,this research was used gfp as a fluorescent biomarker to label endophytic bacteria BS2 and TB2. Based on the green fluorescence produced by gfp-tagged endophytic bacteria in the plant tissue,it could monitor the colonization pattern and investigate the biological characteristics,biocontrol function and colonization ability.Promoter 4412 of plasmid pGFP4412 was replaced by rpsD promoter of strain 168 (B. subtilis) to obtain a new vector pS4GFP in which the gfp gene could strongly express under the control of rpsD promoter. The plasmid pS4GFP was transformed into the endophytic bacteria strains BS2 and TB2. Two transformants,designated as BS2-gfp and TB2-gfp respectively,exhibited strong green fluorescence under fluorescence microscope,and were chosen for further study. The growth rates of two wild strains and two gfp-tagged strains were evaluated,and the gfp-tagged strains showed slightly slower growth rates than that of the wild strains. Results of bacteriostatic tests in vitro showed that the bacteriostatic ability had no significant change between wild strains and gfp-tagged strains. Moreover , for statistical evaluation,colonized population of gfp-tagged strains were not lower than that of the wild strains. The above test results showed that there were no large influence on the growth and antagonistic effect of endophytic bacteria,and it can meet the need for further experimental study.To clarify the mechanism of infection and colonization of endophytic bacteria, we clonedβ-1,4-endoglucanase genes and pectate lyase genes from strains BS2 and TB2,and discussed whether the two genes played a role in endophytic colonization. First,ORF ofβ-1,4-endoglucanase gene has been cloned from the two strains respectively,and we analyzed the gene and amino acid sequences. The result suggestes that theβ-1,4-endoglucanase gene contained 1500 bp nucleotides,which encodes 499 amino acids. The molecular weights of the two enzymes both are 55 kDa,their isoelectric points (pI) were calculated to be 7.49 and 7.83 respectively. BLAST search results showed that the nucleotide and amino acid sequences of theβ-1,4-endoglucanase gene coming from BS2 shared high homology with that of theβ-1,4-endoglucanase gene coming from TB2,and the both of similarities are 99%. Only one base was changed in the nucleotide sequences,the base A1015 in strain BS2 substitutes for G1015 in strain TB2,amino acid at the corresponding position changed from His to Arg. In addition,the nucleotide and amino acid sequences homology between the twoβ-1,4-endoglucanase genes and B. subtilis type strain 168 both were 93%. The twoβ-1 , 4-endoglucanases were successfully expressed in vector pET-29a(+) , consistent with the calculating result. SDS-PAGE electrophoresis analysis showed that the fusion protein molecular masses were about 59 kDa. Study on the biological characteristics of the two enzymes showed that the optimum pH for reaction of the enzyme were found at 6.4,showing that the two fusion enzymes belonged to neutral enzymes. The stable pH ranges were all 5.4~9.0. The optimum temperatures were all at 50℃,When the temperature was higher than 55℃,they were unstable. The enzyme activity at 65℃was only 31% of that at 50℃.In order to discuss the relationship betweenβ-1,4-endoglucanase gene from endophytic bacteria with endophytic colonization characteristic , two vectors containingβ-1,4-endoglucanase gene ORF or part ofβ-1,4-endoglucanase gene sequence were constructed and transformed to strain BS2 and TB2 respectively,resulting overproducing mutants and underproducing mutants. Furthermore ,overexpression vector was transformed into underproducing mutant , yielding complementation mutant. These different bacterial strains liquids were used to inoculate into Brassica chinensis roots respectively,then they were isolated at different time and in different tissue. According to the statistical data,it showed that the population of overproducing mutant had been maintained the largest quantity than underproducing mutant and gfp labeled strain. Whereas the number of underproducing mutants were the least. However , the bacterial numbers of complementation mutant were close to that of the overproducing mutant,and slightly higher than those labeled strains. All above results demonstratedβ-1,4-endoglucanase was one of the important factors that was involved in endophytic bacteria colonization process.Secondly,we cloned the pectate lyase genes from strain BS2 and TB2 respectivly. Analysed the nucleotide sequences and their deduced amino acid sequences of the two genes,we found that both of the two pectate lyase genes composed of promoter region and 1266 bp ORF region. Homologous analysis on two pectate lyase genes showed that the homologies of their nucleotide and amino acid sequences were 99%. Compared the nucleotide and amino acid sequences from two genes with B. subtilis type strain 168,the homology was 72%,but below 72% with other reported pectate lyases came from B. subtilis and B. licheniformis. The two pectate lyases were successfully expressed in vector pET-30a(+),SDS-PAGE electrophoresis analysis showed that the fusion protein molecular masses were about 50 kDa,which were agree with our prediction.In order to discuss the relationship between pectate lyase gene from endophytic bacteria with endophytic colonization characteristic,overexpression vector was constructed which obtained through connecting the shuttle vector and the whole length pectate lyase gene. After transforming overexpression vector to wild type strain,we screened the overproducing mutants,named BS2-pex and TB2-pex respectively. Using the bacterial liquid from mutants and labeled strains to inoculate into B. chinensis root respectivly,the number of endophytic bacteria from different tissue in B. chinensis during different time was observed. The result showed that in the initial period of invading,the number of endophytic bacteria from overproducing mutants were obviously more than that of labeled strains,and the difference became not obvious during the passage of time. Further research was necessary in determined the exact function of pectate lyase.In conclusion,β-1,4-endoglucanase is conserved in endophytic bacteria,and is the one of the important factors that are involved in endophytic bacteria colonization process. At the same time,the analysis shows that the pectate lyase produced by endophytic bacteria plays a key role in entering into plant at the initial stage. |
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