Studies On The Optimization Of A Rice Epiphytic Bacillus Brevis Expression System

The green fluorescent protein (GFP) of the jellyfish Aequorea Victoria is a usefulreporter molecule for monitoring gene expression in vivo in eukaryotic andprokaryotic cells. A GFP vector was constructed for in situ detection of the riceepiphyte Bacillus brevis strain DX01. The promoterless gfp-S65T gene wastranscriptionally fused to a strong B. brevis promoter pCP01 functional F1 fragment,which was subcloned from the genome of strain DX01 by PCR technique, and thenrespectively inserted into plasmid pUC118 and an Escherichia coli-Bacillus shuttlevector pHY300PLK leading to result in expression vectors pUC118-F1gfp andpHY300-F1gfp. The DX01 cells harboring the plasmid pHY300-F1gfp could producebright green fluorescence when they were examined by using fluorescent microscopy.The results were confirmed by western blot analysis. The transcription from F1remained constitutive although it was 280 bp shorter than in promoter pCP01.GFP-expressing cells were also sorted by fluorescence-activated cell sorting (FACS)analysis. The fluorescence intensity of cell populations of DX01::pHY300-F1gfp wastwo-fold higher than that of DH5a::pUC118-F1gfp, which implied that the GFPexpression vector pHY300-F1gfp was highly efficient in B. brevis DX01. Though F1showed no host cell specificity, it displayed higher transcriptional activity in B. brevisDX01 than in E. coli DH5 a.To improve the activity of F1, insertion mutagenesis of F1 based on in vitrotransposition reaction was performed. 227 insertion clones were screened from aninsertion library, 13 mutants were identified and sequenced. Their MIC of Cam andCAT synthesis were assayed. Seven mutants with enhanced transcription activity in E.coli DH5α were obtained. The MICs of Cam of Fm3 and Fm10 were 900 μg/mL,which is 2.6 fold higher than that of the control. Four mutants showed lower MIC ofCam, and two kept unaltered. In general, the MIC of Cam was positively related toCAT activity in E. coli DH5α cells. The insertions in DNA sequence of promoter F1resulted in different effects. Remarkably, the insertions in the neighbor sites led todiametrically opposing effects. The results of FACS analysis confirmed that theenhanced promoters showed similar high activities in B. brevis strain DX01.The anti-fungal protein 1 (Rs-AFP1) cDNA was derived from radish cultivar"Chuanxinhong" by reverse transcription PCR, and then the promoter F1 and the VIIImiddle-wall-protein signal peptide of B. brevis strain 47 were fused to its 5'end in acorrect orientation respectively. Subsequently, the terminator codon of the Rs-afp1gene was mutated and a T7 terminator was fused to its 3' resulting in the two endexpression vectors pUC118-F1SP' Agfp and pHY300-F1SP' Agfp. The fused proteinwas confirmed expressed in E. coli DH5α by Western blot analysis. RAPD analyses were conducted on genetic polymorphisms of twelve fir genotypesto identify their relationships. These genotypes included eight supposed hybridTaxodium mucronatum Tenore forest samples and the hybrid female-parent?T.mucronatum Tenore and the same class of hybrid male-parent? Cryptomeria fortuneiHooibrenk and sugi C. Japonica(L.f.) D.Don. 17 of 31 primers were selected to usefor separate amplification reactions with DNA from 12 genotypes. 164 RAPDfragments were produced and 98.8 % of them showed polymorphism. The resultsrevealed that the genetic relationship of sample No.11 in three Cryptomeria genotypesis the closest to the original male-parent; the samples No.1,4 and 9 are most possiblythe true hybrid T. mucronatum Tenore populations; the sample No.5 may be the falsehybrid. Conclusions could not be drawn from the data in hand for the rest samples.