柑橘潰瘍病病原菌表現載體的構築

Abstract

本實驗室已利用線狀噬菌體Cflt能插入柑橘潰瘍病病原菌（Xnthomonas campestris pv. citri）染色體的Pst I至EcoR I片段，構築成一插入性載體（integration vector）pKSKP-4。由於目前尚未發展出適合柑橘潰瘍病病原菌及Cflt的基因表現系統，以便研究基因的功能，因此構想利用pKSKP-4開發?一表現載體（expression vector），期望能接入欲表現的外來基因，而又不致對菌體本身造成影響。 首先以南方氏轉漬法（Southern blot）證明pKSKP-4能插入柑橘潰瘍病病原菌的染色體中。再經測試，發現此載體的cat基因及ampr基因的?動子都能使接入的cat基因在菌體內表現，此即表現載體pKSKP-4/Cat的構築；隨後以CAT-ELISA測定?動子的表現能力：細菌抽出物中的CAT-enzyme佔總蛋白質濃度的0.0042％ 由生長曲線得知，柑橘潰瘍病病原菌野生菌株的生長速率較轉形菌株要快；進而再由質體穩定性測試得知，pKSKP-4能穩定存於柑橘潰瘍病病原菌染色體中28代，隨著染色體一起複製而不致遺失。最後再經電孔法（electroporation）將此載體送入六種植物致病菌中，測其寄主範圍（host range），初步認?這些菌種的轉形效率較X. c. pv. citri低。

An expression vector, pKSKP-4, was designed for the purpose of constructing a suitable gene expression system of Xanthomansa campestris pv. citri. pKSKP-4 contains the Pst I/ EcoR I fragment of fialmentous phage Cf1t, which makes it an integartion vector, too. The desire here was to take the advantage of pKSKP-4 for foreign gene experssion inside the X. c. pv. citri, and hopefully it wouldn't damage the growth condition of the bacteria. First, the result of the Southern blot showed that pKSKP-4 has the ability to integrate the chromosome of X. c. pv. citri. Then the inserted cat gene was found to express by the promoter of kanr and ampr. This is the construction of the expression vector pKSKP-4d/Cat. From the observation of the growth curves, we found that the wildtype X. c. pv. citri has faster growth rate than the transformant. Then the result of plamid stability test showed that the integrated pKSKP-4, followed the replication of chromosome, is still stable in the chromosome of X. c. pv. citri after culturing for twenty-eight generations. Finally we test the host range of pKSKP-4d/Cat by electroporating the vector into six plant pathogens. The results suggest that the transformation efficiency of X. c. pv. citri is higher than all the other bacteria.