玉米大斑病菌聚酮体合成酶基因 StPKS 功能分析

 【目的】利用RNA干扰技术初步探讨玉米大斑病菌聚酮体合成酶基因（StPKS）的功能。【方法】本试验将玉米大斑病菌StPKS基因片段连接到pSilent-1载体中，构建StPKS基因的RNA干扰载体pSilent-StPKS1-2。通过聚乙二醇介导的方法转入玉米大斑病菌野生型菌株01-23的原生质体，利用潮霉素筛选得到转化子，采用半定量RT-PCR方法分析转化子StPKS基因的表达情况，显微观察转化子与野生型菌丝形态的差异。【结果】本研究构建的StPKS基因RNA干扰载体对沉默该基因表达是有效的，经潮霉素抗性筛选，得到了30株阳性转化子，对其中5株黑色素积累下降、菌落颜色变浅的转化子进行了半定量PCR分析发现，5株转化子的StPKS基因表达量均有所下降；显微观察发现5株转化子的菌丝形态很不规则，发生了膨大、变形、分枝等现象。【结论】StPKS基因在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，试验同时发现该基因与菌丝形态密切相关。

Obtaining and analysis on silent transformants of Polyketide synthase gene(StPKS) in Setosphaeria turcica

【Objective】RNA interference technology was used to investigate the function of a polyketide synthase gene (StPKS) in Setosphaeria turcica.【Method】The silent vector, pSilent-StPKS1-2, was constructed by inserting StPKS fragments into plasmid pSilent-1 then trandformed into S. turcica 01-23 protoplasts through PEG. Transformants were selected on potato dextrose agar (PDA) containing 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.【Results】The RNAi vector was effective to reduce the gene expression. Thirty hygromycin-resistant transformants were isolated and five melanin-deficient transformants were analysised by Semiquantitative RT-PCR. The results showed that StPKS mRNA was weakly expressed in white-color phenotypes. The transformants hypae morphology was anomalistic, including intumescent, anamorphic and ramose.【Conclusion】StPKS gene involved in the DHN-melanin biosynthesis and related to the mycelial morphology. The DHN-melanin biosynthesis was readuced when StPKS gene weakly expression.