西瓜噬酸菌铜代谢相关基因copZ功能分析

 瓜类细菌性果斑病(Bacterial fruit blotch,BFB)病原菌为西瓜噬酸菌(Acidovorax citrulli,Ac),主要为害西瓜、甜瓜等葫芦科作物,是瓜类生产上主要细菌性病害。目前生产上对该病害的防治手段主要以化学防治为主,其中铜制剂的防治效果较为显著。本研究以西瓜噬酸菌野生型菌株Aac-5中铜代谢相关基因copZ为对象,探究copZ基因缺失对西瓜噬酸菌抗铜性、生长能力等表型的影响;采用RT-qPCR技术测定在不添加和添加外源铜离子条件下,基因copZ缺失后对其他铜代谢相关基因表达量的影响。成功构建了西瓜噬酸菌copZ基因缺失突变菌株ΔcopZ和互补菌株ΔcopZ-comp。结果表明,野生型菌株Aac-5与ΔcopZ-comp菌株在添加1.25 mmol·L^-1 CuSO₄的生长能力与不添加CuSO₄相似,而ΔcopZ菌株的生长能力减弱;当添加CuSO₄至2.50 mmol·L^-1时,野生型菌株Aac-5、ΔcopZ菌株和ΔcopZ-comp菌株的生长能力均减弱。基因copZ缺失在不含铜和含铜条件下均能使西瓜噬酸菌生物膜形成能力减弱;在不含铜条件下基因copZ不影响西瓜噬酸菌的运动能力,在含有1.25 mmol·L^-1 CuSO₄的运动性培养基中各菌株运动能力丧失。ΔcopZ菌株在西瓜子叶内的生长能力相较于野生型菌株Aac-5显著增强,但在西瓜幼苗上的致病力差异不显著,仍具有引起烟草过敏性反应的能力。在不同铜浓度处理后,铜代谢相关基因cueR、copA表达量在野生型菌株中的变化趋势与ΔcopZ菌株存在差异;基因cusA、hylD在野生型菌株中表达量变化趋势与ΔcopZ菌株一致。这表明基因copZ在西瓜噬酸菌铜代谢过程中具有重要作用。

Functional analysis of copZ involved in copper metabolism of Acidovorax citrulli

Bacterial fruit blotch (BFB) of watermelon and melon, caused by Acidovorax citrulli (Ac), damages watermelon, melon and other cucurbit crops being a threat to cucurbits industry. At present, the control methods of BFB are mainly based on chemicals, among which the copper-based bactericides have the most significant control effect. The copZ gene plays critical roles in the copper-metabolism of A. citrulli. In this study, to explore the functions of copZ in A. citrulli, we used A. citrulli Aac-5 as the wild-type strain to construct mutant strain ΔcopZ, and its complementary strain ΔcopZ-comp. The copper resistance, pathogenicity, mobility and hypersensitive response of the subsequent strains were determined using tobacco plants.. In addition to the phenotypes, we also analyzed the expression levels of the selected genes in the mutant strain by RT-qPCR, including those related to copper-metabolism genes. The results showed that the copZ deletion mutant strain ΔcopZ and complementary strain ΔcopZ-comp of A. citrulli strain Aac-5 were successfully constructed. The growth ability of wild-type strain Aac-5 and complementary strain ΔcopZ-comp incubated in KB liquid medium with 1.25 mmol·L^-1 CuSO₄ was similar to that of the two strains incubated without CuSO₄, while the growth ability of mutant strain ΔcopZ was significantly decreased when 1.25 mmol·L^-1 CuSO₄ was added into KB liquid medium. When incubated in KB liquid medium with 2.50 mmol·L^-1 CuSO₄, the growth ability of Aac-5, ΔcopZ and ΔcopZ-comp was significantly decreased. The biofilm formation of A. citrulli strains was decreased when incubated in KB liquid medium with 0 mmol·L^-1, 1.25 mmol·L^-1 and 2.50 mmol·L^-1 CuSO₄. The elimination of copZ did not affect the swimming ability of A. citrulli, which were lost when copper sulfate was added into medium. In our pathogenicity tests, the growth ability of mutant strain ΔcopZ in watermelon cotyledons was significantly higher than that of wild-type strain Aac-5, but the difference in pathogenicity on watermelon seedlings was not significant, and it retained the ability to induce hypersensitive response when infiltrated into tobacco leaves. When incubated in liquid KB medium containing different concentrations of copper sulfate, the expression of copper-metabolism-related genes cueR and copA in wild-type strain Aac-5 were different from those in mutant strain ΔcopZ. The patterns of the expression level of genes cusA and hylD in wild-type strain were consistent with those of mutant strain ΔcopZ. The above results indicate that gene copZ plays an important role in the copper-metabolism of A. citrulli.