於 Pseudomonas syringae pv. tomato DC3000 中染色體定點插入應用於基因表現之研究

Abstract

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000) 為造成番茄細菌性斑點病的植物病原菌，由於其相關的研究頗多，加上其基因體序列已於2003年解序完成，故成為研究植物與病原菌交互作用的模式系統生物。一般來說，為了探討特定基因的功能性，常以刪除 (knock-out) 基因方式來觀察表型 (phenotype) 的變化，接者再以互補方式 (complementation) 確認所觀察到的表型變化確實為該基因所致。在細菌的研究上，常以質體攜帶被刪除基因的方式來建構互補菌株，但已有研究顯示，利用質體來表現互補基因時，其表現量有時並未與野生株相似，或是會有質體穩定性上的問題。為解決此問題，本篇研究以發展能將欲表現基因插入染色體定點位置的新方法為目的，來建構 Pst DC3000 的互補菌株。首先利用生物資訊學分析找出在 Pst DC3000 染色體上可能具有潛力之區域，將該區域以PCR增殖後，選殖入自殺載體中，建構出 pK18msLP，以利用 sacB 基因特性為原理的策略將基因插入預定位置。接著以在 Pst DC3000 上研究頗多的第三型及第六型分泌系統為對象，測試該策略之可行性及互補效果。結果顯示此法可成功建構互補菌株，且在生長競爭試驗中其互補效果較以質體互補佳。另一方面，前人研究提到在植物分析中質體可能有喪失的情形，在本研究中確實也觀察到此現象，攜帶 avrPtoB 基因的質體互補菌株在感染感病品種番茄及菸草後第五天該質體保留率皆下降 12~20% 左右。以目前的結果看來，利用 pK18msLP 確實能將基因成功插入 Pst DC3000 定點的位置上，較利用質體建構的互補菌株更能穩定地存在於細胞中，且互補基因的表現量與功能也較趨近於野生株。

Pseudomonas syringae pv. tomato DC3000 (Pst DC3000), the causing agent of tomato speck disease, is a model pathogen for the studies of the molecular mechanisms underlying gene-for-gene resistance and disease susceptibility. Its genome sequence has been completed in 2003; thus, it is important to know the function of each gene in the post genomic era. In general, to explore the function of genes, we often use loss- or gain-of-function strategies. For studying bacteria, complementation of a mutant strain was usually performed using a plasmid-based method. However, some properties of the plasmid, such as multicopy and instability, may hamper the utilization of plasmid to carry the gene for complementation. In order to solve this problem, we developed a new site-specific chromosomal integration strategy for genetic expression in Pst DC3000. First, potential regions suitable for integration of genes in the genome of Pst DC3000 was identified using bioinformatic analysis. Then a sacB-based strategy was applied using a suicide vector pK18msLP to integrate gene-of-interest into the designed region. The efficacy of this method was assessed by using mutants deficient in certain T3SS and T6SS effectors. Our data revealed that the chromosome-integrated method was better than the plasmid-borne method for complementation of hcp1 and hcp2, two T6SS effectors in a growth competition assay. In addition, a plasmid carrying avrPtoB was not stable in Pst DC3000 as we observed 12-20% decrease in the plasmid retention ratio when it was inoculated into the susceptible tomato lines and N. benthamiana. In conclusion, we have successfully established a stable chromosomal integration strategy for gene expression with similar or even better effects than the plasmid-encoded strategy.