玉米大斑病菌聚酮体合成酶基因StPKS功能分析.pdfVIP

中国农业科学 2011,44(8):1603-1609 Scientia Agricultura Sinica doi: 10.3864/j.issn.0578-1752.2011.08.008 玉米大斑病菌聚酮体合成酶基因 StPKS 功能分析 张 鑫，曹志艳，刘士伟，郭丽媛，董金皋 （河北农业大学生命科学学院植物分子病理学实验室河北保定 071001 ） 摘要：【目的】利用RNA干扰技术探讨玉米大斑病菌聚酮体合成酶基因StPKS 的功能。【方法】将玉米大斑病 菌 StPKS 基因片段连接到pSilent-1载体中，构建 StPKS 的RNA干扰载体pSilent-StPKS1-2。利用聚乙二醇（PEG） 介导的方法将之转入玉米大斑病菌野生型菌株 01-23 的原生质体中，通过潮霉素筛选得到转化子，采用半定量 RT-PCR 方法分析转化子中 StPKS 的表达情况，显微观察转化子与野生型菌丝形态的差异。【结果】本研究构建了 StPKS RNA 干扰载体并进行了成功转化，经潮霉素抗性筛选和 PCR 验证最终获得了 9 个阳性转化子，其中 5 个转 化子菌落颜色变浅。对转化子进行半定量 PCR 分析发现，5 株转化子的 StPKS 表达量均有所下降；黑色素产量显 著降低；菌丝呈不规则状，发生了膨大、变形、分枝等现象。【结论】 StPKS 在DHN黑色素合成途径中起作用，其 表达量下降会减少黑色素的产生。 关键词：玉米大斑病菌；聚酮体合成酶基因；DHN黑色素；RNA干扰 Functional Analysis of Polyketide Synthase Gene StPKS in Setosphaeria turcica ZHANG Xin, CAO Zhi-yan, LIU Shi-wei, GUO Li-yuan, DONG Jin-gao (Molecular Plant Pathology Laboratory, College of Life Science, Agricultural University of Hebei, Baoding 071001, Hebei) Abstract: 【Objective 】The objective of this study is to discuss the function of polyketide synthase gene StPKS in Setosphaeria turcica by RNA interference technology. 【Method 】 The silent vector, pSilent-StPKS1-2, was constructed by inserting StPKS fragments into plasmid pSilent-1 and trandformed into S. turcica 01-23 protoplasts by PEG. Transformants were selected using hygromycin and the expression levels of StPKS was analysised by semiquantitative RT-PCR. The hypae morphology of transformants and wide type was observed by microscope. 【Result 】The RNAi vector was constructed and effectively transformed. Nine positive transformants were screened by hygromycin and PCR. Five of nine transfo