利用RNAi技术鉴定哈茨木霉聚酮合酶基因pkst-1的功能

哈茨木霉(Trichoderma harzianum)是优良的生物防治真菌,能产生抗生性聚酮化合物(polyketides).目前没有关于催化木霉聚酮化合物合成的聚酮合成酶(polyketide synthase,PKS)的相关编码基因的报道,这限制了对聚酮化合物代谢合成途径的研究.该研究通过融合PCR技术获得哈茨木霉的聚酮合成酶pkst-1基因的编码区序列,成功构建了RNAi介导的pkst-1基因沉默表达载体,使pkst-1基因的表达量在mRNA水平上显著降低.pkst-1基因失活使转化子发酵液产生了类似基因敲除的抑菌效果,降低了哈茨木霉抑菌能力,研究表明pkst-1基因编码催化抗生性聚酮化合物合成的关键酶.此外,RNA干扰技术在研究中的成功应用为哈茨木霉或其它丝状真菌的次级代谢合成途径相关基因的功能鉴定提供新的研究思路.

A study on the function of polyketide synthase gene pkst-1 from T. harzianum using RNAi

Trichoderma harzianum is an excellent biocontrol agent with a well-known ability to produce a wide variety of antibiotic polyketides,but there is no relevant report on genes encoding polyketide synthase (PKS) from Trichoderma harzianum.In this study, a full-length coding sequence of PKS (pkst-1) gene of T.harzianum 88 was obtained from clones performed by In-Fusion technique,a plasmid expression vector coding for RNAi targeting mRNA of pkst-1 was constructed successfully,and the expression of pkst-1 gene was inhibited successfully by RNAi.The inactivation of pkst-1 gene made the fermentation of the transformant have a similar effect of inhibiting bacteria as gene-knockout,which led to a reduced inhibitory ability in Trichoderma harzianum,indicating that pkst-1 was the key enzyme of polyketide synthesis pathway.Additionally,the successful application of RNAi in this study provide new perspectives on the function identification of secondary metabolic genes from T.harzianum or other filamentous fungus.