Cloning Of Flagellin Genes Of Bacillus Cereus A-47 And Preliminary Analysis Of Corelation To Colonization

Endophytic bacteria are referred to the kind of microbe colonizing within plant tissues and establishing compatible relationship with their hosts. Many research results have demonstrated a part of endophytic bacteria are beneficial to their hosts. Therefore research on these bacteria has become a hotspot in China and in many other conturies.Bacillus cereus A-47 is a yield-increasing endophytic strain isolated from wheat roots and colonizes the rhizosphere and the interior of wheat roots at the same time, but the mechanism of endophytic colonization is still not clear. The objective of this experiment is to clone genes of flagellin and get a mutant with insertion. By comparing the population on or in the roots between the parental strain and the mutant, some indirect evidences about colonization have been acquired.The genome DNA of Bacillus cereus A-47 was extracted as the templet, and primers were designed according to the homologous genes of flagellins. Two genes of flagellins named fla/K and flaB were acquired by PCR. A segment of 780bp from flaA was connected to the suicide vector pHSG299, and the recombinant was transformed into Bacillus cereus A-47 via electroporation and a mutant was obtained which flagellin gene was inserted by the suicide plasmid. Wheats were inoculated with the mutant and wide-type strain A-47 respectively. Roots, 14 days later, were sampled from 6 replicates to evaluate the colonization of the surface of roots by the mutant or the wild-type strain. Results showed that population levels of them were 7.4 and 8.2 (Log10 of cfu/g root fresh weight) respectively. Roots were also sampled from 12 replicates to evaluate the endophytic colonization by the mutant and the wild-type strain respectively. Results indicated that population levels of them were 5.0 and 6.1 (LoglO of cfu/g root fresh weight) respectively. It seems that the endophytic population decrease with the decline on the surface of roots and the flagella have little fuction in the phase of endophyitc colonization, but we can't draw a certain conclusion until more work is completed.