玉米大斑病菌聚酮体合成酶基因StPKS功能分析  

Functional Analysis of Polyketide Synthase Gene StPKS in Setosphaeria turcica

作　　者：张鑫[1] 曹志艳[1] 刘士伟[1] 郭丽媛[1] 董金皋[1] 

机构地区：[1]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

出　　处：《中国农业科学》2011年第8期1603-1609,共7页Scientia Agricultura Sinica

基　　金：国家自然科学基金项目(30771390)

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。【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 transformants were melanin-deficient.Result of semiquantitative RT-PCR showed that StPKS mRNA was weakly expressed in white-color phenotypes.Melanin production was significantly declined.The transformants hypae morphology was anomalistic,including intumescent,anamorphic and ramose.【Conclusion】StPKS involved in the DHN-melanin biosynthesis and melanin production was readuced when StPKS weakly expressed.

关 键 词：玉米大斑病菌 聚酮体合成酶基因 DHN黑色素 RNA干扰 

分 类 号：S435.131[农业科学—农业昆虫与害虫防治]