| Ralstonia solanacearum is the causal agent of bacterial wilt on solanaceous plants,including 450 plants in 54 families.The model strain GMI1000 does not cause disease in tobacco plants,which is jointly determined by the type Ⅲ effectors RipP1 and RipAA.The effector coding genes are usually co-expressed with the type Ⅲ secretion system,and regulated by type Ⅲ regulatory genes.In R.solanacearum GMI1000,the transcription of RipP1 showe don difference in hrpB mutant,the type Ⅲ system regulator.This thesis studied the transcription mechanism of ripP1 to elucidate its specific expression profile.On the one hand,the results will provide a clue to understand the particular transcription events for other effectors.The main research progress of this paper is as follows:（1）Based on bioinformatics analysis,it was found that ripP1 gene contains two putative promoter sequencess.The promoter P1 located close to the translation initiation codon was located at-135 to-216 bp and harbored the PIP-box(TTCG-N16-TTCG)essential for Hrp B regulation.The P2 promoter far from the translation initiation codon was located at-593 to-644 bp position without Hrp B-regulated elements.（2）Diverse promoter deletion mutant was created to evaluate their contributions to ripP1 expression.In GMI1000 strain,P1,P2 and full-sequence（P1P2）promoter deletion mutants were constructed,the transcription level of ripP1 gene was subsequently analyzed,and the effects of each promoter on transcription expression were compared.After the three promoter fragments wer saparatly fused with Lac Z gene,the promoter activity was tested in wild-type and hrpB mutants.It was found that the P1 promoter was regulated by hrpB,while the activities of P2 and full-length P1P2 promoter were not affected by hrpB.（3）Cloning the gene candidate regulating the P2 promoter in R.solanacearum by means of promoter trapping method.The P2 promoter Lac Z fusion was introduced into wild-type GMI1000.The Tn5transposition system was used to screen mutants with significantly reducedβ-galactosidase activity.Finally,the RS01937 gene was verified to exhibit regulatory role in P2 promoter activity.Based on bioinformatics analysis,the transcriptional expression mechanism of ripP1 gene of R.solanacearum was studied in this paper.It was found that Hrp B and RS01937 co-regulates ripP1 gene,which theoretically clarified that why the ripP1 gene is not co-expressed with type Ⅲ secretion system.Because the ripP1 gene is an important effector determining host range,this research will provide a scientific clue to understand bacterial adaptation in different hosts. |
