Department of Plant Pathology, University of California, One Shields Ave., Davis 95616, USA.
Mol Plant Microbe Interact. 2010 Feb;23(2):176-86. doi: 10.1094/MPMI-23-2-0176.
A 4.5-kb genomic DNA containing a Monilinia fructicola cutinase gene, MfCUT1, and its flanking regions were isolated and characterized. Sequence analysis revealed that the genomic MfCUT1 carries a 63-bp intron and a promoter region with several transcription factor binding sites that may confer redox regulation of MfCUT1 expression. Redox regulation is indicated by the effect of antioxidants, shown previously to inhibit MfCUT1 gene expression in cutin-induced cultures, and in the present study, where H(2)O(2) enhanced MfCUT1 gene expression. A beta-glucuronidase (GUS) reporter gene (gusA) was fused to MfCUT1 under the control of the MfCUT1 promoter, and this construct was then used to generate an MfCUT1-GUS strain by Agrobacterium spp.-mediated transformation. The appearance of GUS activity in response to cutin and suppression of GUS activity by glucose in cutinase-inducing medium verified that the MfCUT1-GUS fusion protein was expressed correctly under the control of the MfCUT1 promoter. MfCUT1-GUS expression was detected following inoculation of peach and apple fruit, peach flower petals, and onion epidermis, and during brown rot symptom development on nectarine fruit at a relatively late stage of infection (24 h postinoculation). However, semiquantitative reverse-transcriptase polymerase chain reaction provided sensitive detection of MfCUT1 expression within 5 h of inoculation in both almond and peach petals. MfCUT1-GUS transformants expressed MfCUT1 transcripts at twice the level as the wild type and caused more severe symptoms on Prunus flower petals, consistent with MfCUT1 contributing to the virulence of M. fructicola.
一段含有梅渍病菌角质酶基因（MfCUT1）及其侧翼序列的 4.5kb 基因组 DNA 被分离并进行了特征分析。序列分析表明，基因组 MfCUT1 携带一个 63bp 的内含子和一个启动子区域，该区域有几个转录因子结合位点，可能赋予 MfCUT1 表达的氧化还原调控。氧化还原调控是通过抗氧化剂的作用来指示的，先前的研究表明抗氧化剂抑制了在角质诱导培养物中 MfCUT1 基因的表达，在本研究中，H 2 O 2 增强了 MfCUT1 基因的表达。β-葡萄糖醛酸酶（GUS）报告基因（gusA）被融合到 MfCUT1 的启动子控制下，然后通过根癌农杆菌介导的转化生成 MfCUT1-GUS 菌株。GUS 活性在角质响应中的出现和在角质酶诱导培养基中葡萄糖对 GUS 活性的抑制证实了 MfCUT1-GUS 融合蛋白在 MfCUT1 启动子的控制下正确表达。在桃和苹果果实、桃花瓣、洋葱表皮接种后，以及在油桃果实褐腐病症状发展的相对晚期（接种后 24 小时），检测到 MfCUT1-GUS 表达。然而，半定量逆转录聚合酶链反应在接种后 5 小时内对杏仁和桃花瓣中的 MfCUT1 表达进行了敏感检测。MfCUT1-GUS 转化体的 MfCUT1 转录物表达水平是野生型的两倍，在李属花瓣上引起更严重的症状，这与 MfCUT1 有助于梅渍病菌的毒力一致。
