Scientia Agricultura Sinica

Previous Articles    

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

MA XueMeng, YU ChengMin, SAI XiaoLing, LIU Zhen, SANG HaiYang, CUI BaiMing   

  1. College of Life Science, Shihezi University, Shihezi 832003, Xinjiang
  • Published:2022-06-21

Abstract: 【ObjectiveThe purpose of this study was to establish a simple and efficient approach for identifying all T-DNA insertion sites. MethodA 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. ResultThe T-DNA insertion sites of five transgenic events were analyzed by PSORA. The results showed that L6 contained two insertion sites (36 316 bp on NW_015801367 and 42 202 bp on NW_015950898), the lines of L9, L15 and L19 each contained one insertion site (The insertion site of L9 was located at 235 969 bp on NW_015943682. The insertion site of L15 was located at 60 529 bp on NW_015802951 and the insertion site of L19 was located at 12 188 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. ConclusionPSORA 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 the traditional methods of genome walking.


Key words: genome walking, T-DNA insertion sites, transgenic plant, high-throughput sequencing, TAIL-PCR

[1] MA XueMeng,YU ChengMin,SAI XiaoLing,LIU Zhen,SANG HaiYang,CUI BaiMing. PSORA: A Strategy Based on High-Throughput Sequence for Analysis of T-DNA Insertion Sites [J]. Scientia Agricultura Sinica, 2022, 55(15): 2875-2882.
[2] DU Yu,ZHU ZhiWei,WANG Jie,WANG XiuNa,JIANG HaiBin,FAN YuanChan,FAN XiaoXue,CHEN HuaZhi,LONG Qi,CAI ZongBing,XIONG CuiLing,ZHENG YanZhen,FU ZhongMin,CHEN DaFu,GUO Rui. Construction and Annotation of Ascosphaera apis Full-Length Transcriptome Utilizing Nanopore Third-Generation Long-Read Sequencing Technology [J]. Scientia Agricultura Sinica, 2021, 54(4): 864-876.
[3] SHAO MeiQi,ZHAO WeiSong,SU ZhenHe,DONG LiHong,GUO QingGang,MA Ping. Effect of Bacillus subtilis NCD-2 on the Growth of Tomato and the Microbial Community Structure of Rhizosphere Soil Under Salt Stress [J]. Scientia Agricultura Sinica, 2021, 54(21): 4573-4584.
[4] ZHAO WeiSong,GUO QingGang,SU ZhenHe,WANG PeiPei,DONG LiHong,HU Qing,LU XiuYun,ZHANG XiaoYun,LI SheZeng,MA Ping. Characterization of Fungal Community Structure in the Rhizosphere Soil of Healthy and Diseased-Verticillium Wilt Potato Plants and Carbon Source Utilization [J]. Scientia Agricultura Sinica, 2021, 54(2): 296-309.
[5] HUANG ZiYue,LIU WenJun,QIN RenLiu,PANG ShiChan,XIAO Jian,YANG ShangDong. Endophytic Bacterial Community Composition and PICRUSt Gene Functions in Different Pumpkin Varieties [J]. Scientia Agricultura Sinica, 2021, 54(18): 4018-4032.
[6] CHEN LuLu,WANG Hui,WANG JiKun,WANG JiaBo,CHAI ZhiXin,CHEN ZhiHua,ZHONG JinCheng. Comparative Analysis of miRNA Expression Profiles in the Hearts of Tibetan Cattle and Xuanhan Cattle [J]. Scientia Agricultura Sinica, 2020, 53(8): 1677-1687.
[7] ZHAO WeiSong,GUO QingGang,LI SheZeng,WANG PeiPei,LU XiuYun,SU ZhenHe,ZHANG XiaoYun,MA Ping. Effect of Wilt-Resistant and Wilt-Susceptible Cotton on Soil Bacterial Community Structure at Flowering and Boll Stage [J]. Scientia Agricultura Sinica, 2020, 53(5): 942-954.
[8] ZHAO YuanYuan,LI PengFei,XU QinZhi,AN QingMing,MENG JinZhu. Screening and Analysis of Follicular Development Related Genes in Goat [J]. Scientia Agricultura Sinica, 2020, 53(17): 3597-3605.
[9] LIU HaiYang, WANG Wei, ZHANG RenFu, RAXIDA ·ABDURAHMAN, YAO Ju. Fungal Community Structure of Cotton-Field Soil Under Different Incidences of Cotton Verticillium Wilt [J]. Scientia Agricultura Sinica, 2019, 52(3): 455-465.
[10] WeiSong ZHAO,QingGang GUO,SheZeng LI,YaJiao WANG,XiuYun LU,PeiPei WANG,ZhenHe SU,XiaoYun ZHANG,Ping MA. Control Efficacy of Broccoli Residues on Cotton Verticillium Wilt and Its Effect on Soil Bacterial Community at Different Growth Stages [J]. Scientia Agricultura Sinica, 2019, 52(24): 4505-4517.
[11] ZHANG XiaoLi,ZHANG HongYuan,LU Chuang,PANG HuanCheng,JIN CunWang,GAO Xi,CHENG AiPing,LI YuYi. Effects of the Different Autumn Irrigation Years on Soil Bacterial Community in Hetao Irrigation District [J]. Scientia Agricultura Sinica, 2019, 52(19): 3380-3392.
[12] LI WenGuang,YANG XiaoXiao,HUANG ChunGuo,XUE NaiWen,XIA Qing,LIU XiaoLi,ZHANG XiaoQi,YANG Si,YANG ZhenPing,GAO ZhiQiang. Effects of Rapeseed Green Manure on Soil Fertility and Bacterial Community in Dryland Wheat Field [J]. Scientia Agricultura Sinica, 2019, 52(15): 2664-2677.
[13] DAI HongCui,ZHANG Hui,XUE YanFang,GAO YingBo,QIAN Xin,ZHAO HaiJun,CHENG Hao,LI ZongXin,LIU KaiChang. Response of Fungal Community and Function to Different Tillage and Straw Returning Methods [J]. Scientia Agricultura Sinica, 2019, 52(13): 2280-2294.
[14] HOU JianWei,XING CunFang,LU ZhiHong,CHEN Fen,YU Gao. Effects of the Different Crop Straw Biochars on Soil Bacterial Community of Yellow Soil in Guizhou [J]. Scientia Agricultura Sinica, 2018, 51(23): 4485-4495.
[15] XU Xi, REN MingJian, LI LuHua, YANG XiCui, XU RuHong. Differential Expression of Grain Pigment Related Genes of Guizimai No.1 [J]. Scientia Agricultura Sinica, 2018, 51(2): 203-216.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed   
No Suggested Reading articles found!