Construction of a model system for expression of lytic genes using plant-inducible promoters

• Main Authors: Yi-Ning Cheng, 鄭宜寧
• Other Authors: Shu-Fen Weng
• Format: Others
• Language: zh-TW
• Published: 2012
• Online Access: http://ndltd.ncl.edu.tw/handle/58974005210544366842

碩士 === 國立中興大學 === 分子生物學研究所 === 100 === Gram-negative plant pathogenic bacteria have hypersensitive response and pathogenicity (hrp) genes. Products of these genes lead to formation of symptoms in a susceptible host plant and induce hypersensitive reaction (HR) in resistant host or nonhost plants. There are six hrp gene clusters (hrpA to hrpF) in Xanthomonads. Plant-inducible promoter (PIP) box (TTCGC-N15-TTCGC) is present in the promoter region of these hrp gene clusters, subject to regulation by regulatory proteins HrpG and HrpX. PIP usually can be activated in plant or poor media (such as XVM2), but has a weak promoter activity in rich media. Lytic phage phiL7 infects and lyses X. campestris pv. campestris (Xcc) specifically, involving the function of at least four lytic genes, p27 (possibly encodes holin), p28 (encodes lysozyme), p29 and p29.1 (similar to the Lamda phage Rz and RZ1 genes, respectively). Previous studies in our laboratory showed that transformation of the recombinant plasmid containing p27-p28-p29-p29.1 into E. coli and Xcc leads to cell lysis. Replicative form (RF) DNA of phiLf, a filamentous phage infecting Xcc, is small and easy to manipulate. In this thesis, several phiLf RF DNA-based recombinant phages were constructed, using plant-inducible promoters of hrpB, hrpE and hrpF to transcribe phiL7 lysis gene cluster or p27 gene only. Promoter activity assay showed that all 9 tested promoters (including those with a PIP promoter, hpa2, hrpB, hrpC, hrpD, hrpE, hrpF and XcvhrpF, and those without a PIP promoter, hpa1 and hpaR) can be induced by growing in a poor medium. During construction of p27-carrying recombinant phages, it was found that only the clone with p27 oriented directly, phiLfDIG-N-p27(R), was obtained but not the one oriented inversely,phiLfDIG-N-p27(F). This suggests that orientation of the inserted p27 may affect viability of the host. Titers of the recombinant phages with a genome size less than 7.5 kb were about 6.5 × 109, similar to that of the parental phiLfDI. However, the titers reduced by 90% and 45% as genome sizes of the recombinant phages were greater than 7.5 kb. In addition, the latter recombinant phages, phiLfDIG-hrpB-p27(R), phiLfDIG-hrpE-p27(R) and phiLfDIG-hrpF-p27(R), formed smaller plaques. However, no lytic effect was observed after infection of P20H cultured in XVM2 with these recombinant phages. It was also found that activities of the Xc17 hrpB, hrpE and hrpF promoters could not be induced in Xc11 and P20H grown in a poor medium. RT-PCR analysis showed that expression of hrpX was very low in Xc11 and Xc11A. Recombinant plasmid expressing hrpX constitutively has been transformed into Xc11A to evaluate the expression and function of hrpX.