PSORA:一种基于高通量测序的T-DNA插入位点分析方法  

PSORA:A Strategy Based on High-Throughput Sequence for Analysis of T-DNA Insertion Sites

作　　者：马雪萌 余成敏 赛小玲 刘贞[1] 桑海洋 崔百明[1] MA XueMeng;YU ChengMin;SAI XiaoLing;LIU Zhen;SANG HaiYang;CUI BaiMing(College of Life Sciences,Shihezi University,Shihezi 832003,Xinjiang)

机构地区：[1]石河子大学生命科学学院,新疆石河子832003

出　　处：《中国农业科学》2022年第15期2875-2882,共8页Scientia Agricultura Sinica

基　　金：国家自然科学基金(31460466)。

摘　　要：【目的】建立一种批量分析T-DNA插入位点的简单、有效的方法。【方法】提供一种基于高通量测序技术的T-DNA插入位点的分析方法,将其命名为PSORA:Parallel sequencing of one round amplicons。首先对一轮交错式热不对称PCR(TAIL-PCR)的扩增产物进行高通量测序,随后通过生物信息学分析T-DNA插入位点,该方法降低了TAIL-PCR过程中对特异性扩增的要求。PSORA中使用的引物一侧为简并引物,另一侧为T-DNA特异性引物。在特异性引物的5’端设计6 nt的样品标签(Barcode),用于标记不同的转化事件。所使用的5个转基因烟草株系(L1、L6、L9、L15和L19)由农杆菌介导质粒pBI121转化获得。此外,通过标准PCR对PSORA的结果进行验证。【结果】利用PSORA对5个转基因株系的T-DNA插入位点进行分析,结果显示,L6含2个插入位点(NW_015801367的36316 bp处和NW_015950898的42202 bp处),L9、L15和L19各含1个插入位点(L9的插入位点为NW_015943682的235969 bp处;L15的插入位点为NW_015802951的60529 bp处;L19的插入位点为NW_015863435的12188 bp处),L1的插入位点未能成功获取。对生物信息学分析结果进行PCR验证,含不同插入位点的转基因株系之间可互为阴性对照,野生型(WT)作为空白对照,结果表明,在各转基因株系中均得到与预期一致的特异性扩增,该结果验证了PSORA的有效性。【结论】PSORA是一种分析T-DNA插入位点的有效方法,可以同时分析多个插入事件,相对于传统的染色体步移方法更简便、快速。【Objective】The purpose of this study was to establish a simple and efficient approach for identifying all T-DNA insertion sites.【Method】A T-DNA insertion sites analysis approach based on high-throughput sequence technologies was developed,called PSORA:Parallel sequencing of one round amplicons.The process involves high-throughput amplicon sequencing of a round of thermal asymmetric PCR(TAIL-PCR)and bioinformatics analysis of T-DNA insertion sites,which reduces concerns about the specificity of TAIL-PCR.In PSORA,only two primers are required,a degenerate primer and a T-DNA specific primer.A 6-nt Barcode was designed at the 5’end of the specific primers for labeling different transgenic events.All five transgenic events(L1,L6,L9,L15 and L19)of tobacco used in this study were produced via Agrobacterium mediated transformation with plasmids pBI121.In addition,the results of PSORA are confirmed by standard PCR.【Result】The T-DNA insertion sites of five transgenic events were analyzed by PSORA.The results showed that L6 contained two insertion sites(36316 bp on NW_015801367 and 42202 bp on NW_015950898),the lines of L9,L15 and L19 each contained one insertion site(The insertion site of L9 was located at 235969 bp on NW_015943682.The insertion site of L15 was located at 60529 bp on NW_015802951 and the insertion site of L19 was located at 12188 bp on NW_015863435),but the insertion site of L1 could not be detected.PCR was performed to validate the results from bioinformatics analysis,transgenic events with different insertion sites were used as negative controls for each other,and the wild type(WT)was used as a blank control.The results showed that specific amplification consistent with expectations was obtained in each transgenic event.The effectiveness of PSORA was successfully confirmed.【Conclusion】PSORA is an effective strategy to analyze T-DNA insertion sites.PSORA can parse the comprehensive molecular characteristics of all T-DNA insertion events simultaneously,making it simpler and faster than th

关 键 词：染色体步移 T-DNA插入位点 转基因植物 高通量测序 交错式热不对称PCR 

分 类 号：Q943.2[生物学—植物学]