高通量测序检测金针菇RNAi转化子插入位点及拷贝数

Detection of the integration sites and copy numbers in an RNAi transformant of Flammulina velutipes using high‐throughput sequencing

本研究对金针菇Flammulina velutipes的一个RNAi转化子菌株1382R3进行了高通量测序,以本实验室先前获得的野生型W23基因组数据为参考,分析了该转化子的基因插入位点以及拷贝数。转化子菌株1382R3是通过农杆菌介导将fv-hmg1-RNAi载体转化至金针菇菌株并通过PCR检测筛选标记而得到。通过BLAST将转化子测序的reads对外源载体和基因组定位,找到具有基因组序列(GS)和外源载体序列(ES)两种序列的临界reads,并据此使用PERL语言程序成功在转化子1382R3菌株中找到两个插入位点。对两个插入位置的序列分析表明:在插入位点1,T-DNA片段部分插入;在插入位点2,T-DNA全部插入到基因组。两个插入位点都对基因组内源基因的表达造成了一定的干扰。此方法拓宽了高通量测序技术的应用范围,将其运用到遗传转化插入位置和拷贝数的研究中,有利于食用菌的功能基因组及基因工程研究。

The integration behavior of an RNAi vector was analyzed based on re-sequencing of a mutant and comparison with a previously obtained Flammulina velutipes reference genome. Mutant strain F. velutipes 1382R3 was obtained through Agrobacterium tumefaciens-mediated transformation （ATMT） with RNAi vector fvhmg1-RNAi, and was re-sequenced after PeR-based confirmation of presence of the selection marker. Mapping sequences based on BLAST and new PERL scripts revealed two integration sites where sequence reads deviated from the reference genome sequence and overlapped with the RNAi vector sequence. Integration site 1 contained only a part of the expected transfer DNA （T-DNA） while integration site 2 contained the full T-DNA fragment. Therefore, two integrations could be interfering with endogenous gene expression. This new application of high throughput sequencing for rapid identification of locations and numbers of integration sites in ATMT mutants is an important extension of the available tools for functional genomics and genetic engineering.