高效靶向降解烟草花叶病毒核酸的dsRNA筛选与大量制备

徐翔,解屹,宋丽云,申莉莉,李莹,王勇,刘明宏,刘东阳,王小彦,赵存孝,王凤龙,杨金广

Screening and Large-Scale Preparation of dsRNA for Highly Targeted Degradation of Tobacco Mosaic Virus (TMV) Nucleic Acids

Xiang XU,Yi XIE,LiYun SONG,LiLi SHEN,Ying LI,Yong WANG,MingHong LIU,DongYang LIU,XiaoYan WANG,CunXiao ZHAO,FengLong WANG,JinGuang YANG

表1 本研究所用引物

Table 1 Primers used in this study

引物 Primer 引物序列 Primer sequence (5′-3′) 产物长度 Expected size (bp) 引物用途 Use of primer TMVCP-F 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGATGTCTTACAGTATCACTACTCC-3′ 548 克隆带启动子的CP基因 CP gene with promoter fragment cloning TMVCP-R 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGAGTTGCAGGACCAGAGG-3′ TMVMP-F 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGGGAAAGAGCCGACGAG-3′ 401 克隆带启动子的MP基因 MP gene with promoter fragment cloning TMVMP-R 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGGCAAGCCTGATTGACATA-3′ TMVP126-F 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGTCTTACCGTCGATGTTT-3′ 709 克隆带启动子的P126基因 P126 gene with promoter fragment cloning TMVP126-R 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGGTTCTTGTTCGGCACT-3′ TMVRdRP851-1238F 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGCTTACTTCCCGGCCTCTA-3′ 456 克隆带启动子的RdRP851-1238基因 RdRP851-1238 gene with promoter fragment cloning TMVRdRP851-1238R 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGGCTTTCGCCTGGTATGTT-3′ TMVRdRP1461-1774F 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGATTTCGCTGGCGTTTG-3′ 381 克隆带启动子的RdRP1461-1774基因 RdRP1461-1774 gene with promoter fragment cloning TMVRdRP1461-1774R 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGCTGCCGTCATTGGGTC-3′ TMVRdRP1573-2330F 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGTGACCTTCCACGACAGA-3′ 825 克隆带启动子的RdRP1573-2330基因 RdRP1573-2330 gene with promoter fragment cloning TMVRdRP1573-2330R 5′-ATTCTCTAGAAGCTTAATACGACTCACTATAGGGAGCGCCACATGATACTT-3′ L4440-dsRdRP1461-1774F 5′-GGAGACCGGCAGATCTATTTCGCTGGCGTTTGGG-3′ _ 重组表达载体的构建 Construction of recombinant expression vector L4440-dsRdRP1461-1774R 5′-TATAGGGCGAATTGGGTACCCTGCCGTCATTGGGTCAAC-3′ L4440-F 5′-CTTTATAGTCCTGTCGGGTTTCGC-3′ _ 重组表达载体测序 Sequencing of recombinant expression vector L4440-R 5′-GGCGAGAAAGGAAGGGAAGAAAG-3′ Actin-F 5′-CAAGGAAATCACCGCTTTGG-3′ _ Actin基因转录水平的检测 Detection of Actin gene transcript level Actin-R 5′-AAGGGATGCGAGGATGGA-3′ TMV-F 5′-GAGTAGACGACGCAACGG-3′ _ TMV CP基因转录水平检测 Detection of TMV CP transcript level TMV-R 5′-CCAGAGGTCCAAACCAAAC-3′