小麦PDS基因VIGS载体构建体系的优化及验证

Optimization and Validation of VIGS System with Wheat PDS Gene

为在小麦上进行VIGS体系的优化,根据GenBank中公布的小麦PDS基因序列(FJ517553.1)设计特异引物,利用RT-PCR技术克隆PDS基因片段,以BSMV:γ-2a为原载体,通过酶切将原载体中的2a基因片段替换为PDS基因片段,构建小麦PDS基因的VIGS重组载体BSMV:γ-PDS。经测序确认,其与GeneBank中登录的小麦PDS基因序列同源性为97%。重组载体经本实验室简化改良的方法进行转录模板的处理。与试剂盒所提供的方法相比,本研究将RNA酶孵育处理步骤整合入质粒提取的步骤中,省略了SDS-蛋白酶K孵育处理等步骤,降低了引入新杂质的风险,同时降低了实验成本,节省了时间和精力。体外转录的电泳结果表明,体外转录反应的产物浓度大,条带单一,可用于接种实验。抽穗期小麦穗部接种实验及实时定量PCR的结果均表明,小麦内源PDS基因被有效沉默,证实了优化的重组载体BSMV:γ-PDS体系的有效性,奠定了利用VIGS载体侵染抽穗期小麦体系优化的基础。

In order to obtain an optimized technical system for using virus induced gene silencing in wheat, special primers based on the sequence of wheat PDS gene（GenBank accession. FJ517553. 1） were designed and the partial sequence of wheat PDS gene was amplified through RT-PCR. Sequence analysis displayed that the similarity between the partial sequence and PDS in GenBank was 97~. BSMV.7-2a vector was used as the original vector. The 2a fragment of BSMV. γ-2a vector was re- placed by partial fragment of wheat PDS gene and the recombinant vector BSMV. 7-PDS was ob- tained. An modified purification procedure was used for the purification of the vector, and compared with the procedure provided by the transcription kit in order to obtain a less cost and quicker proce- dure. This procedure integrated the incubating process of RNase treatment in plasmid isolation proce- dure, and the linearization of the plasmid with restriction enzymes was conducted after plasmid isola- tion, so the process of treating with proteinase K and SDS was omitted. The electrophoresis analysis showed that the transcribed products were pure and had high concentration. Then the transcribed vi- rus RNA was used to inoculate wheat spikes at the heading stage. The results of inoculation and qRT- PCR showed that the optimized BSMV.γ-PDS system was effective as the transcript level of PDS gene were decreased in spikes. This work laid a solid foundation for using the VIGS system in wheat.