Analysis of Total RNA in CMV Infected Tobacco by Ion Pair Reverse Phase High Performance Liquid Chromatography

Abstract

Abstract: To study the impact of plant virus on host plant at the RNA level gene expression, in this study differential expressing of total RNA induced by cucumber mosaic virus（CMV）infection in tobacco through ion pair reversed phase liquid chromatography（RP-HPLC） was analyzed. Ion pair RP-HPLC has been applied for RNA isolation, purification and analysis in recent years, and has the advantages of direct injection, good reproducibility, and rapid analysis. Suitable ion pair reagents as the hexylamine/1, 1, 1, 3, 3, 3-Hexafluoro-2-propanol（HA/ HFIP）system was optimized to achieve a acceptable total RNA separation, the separation peak differences between healthy and diseased plant were observed, but needed further experiments to verify. Therefore, this paper provided an alternative method for study the viral effects on gene expression of host plants at the RNA level and plant virus pathogenic mechanisms.

Key words: Ion pair RP-HPLC, Cucumber mosaic virus（CMV）, Total RNA, Differential gene expression

Qiao Wenjie, Lei Rong, Jiang Hongshan, Hu Fan, Li Zhihong, Zhu Shuifang. Analysis of Total RNA in CMV Infected Tobacco by Ion Pair Reverse Phase High Performance Liquid Chromatography[J]. Biotechnology Bulletin, 2013, 0(4): 90-95.

References

［1］薛春生, 肖淑芹, 王国英, 等. 利用cDNA-AFLP 技术分析玉米自交系黄早四甘蔗花叶病毒侵染后基因差异表达[J]. 植物病理学报, 2005, 35（3）：229-234.
［2］周以飞, 戴艳, 潘大仁, 等. 花叶病毒侵染的果蔗（Badila）的基因表达[J]. 福建农林大学学报：自然科学版, 2006, 35（3）: 288-291.
［3］ Ding SW. RNA-based antiviral immunity [J]. Nature Review Immunology, 2010, 10（9）：632-644.
［4］张新华, 李富军. 病毒诱导的基因沉默技术及其在植物中的研究进展[J]. 西北植物学报, 2012, 32（2）：419-424.
［5］黄冰艳, 吉万全, 郭蔼光, 等. 转录后基因沉默（PTGS）及其在作物遗传改良中的应用[J]. 中国生物工程杂志, 2005, 25 （5）：1-5.
［6］ Mlotshwa S, Voinnet O, Mette MF, et al. RNA silencing and the mobile silencing signal [J]. Plant Cell, 2002, 14（Suppl. 1）: S289-S301.
［7］ Meister G, Tuschl T. Mechanisms of gene silencing by doublestranded RNA [J]. Nature, 2004, 431（7006）：343-349.
［8］ Liang P, Pardee AB. Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction [J]. Science, 1992, 257（5072）：967-971.
［9］ Ivanova NB, Belyavsky AV. Identification of differentialy expressed genes by restriction endonuclease-based gene expression fingerprinting [J]. Nucleic Acids Research, 1995, 23（15）：2954-2958.
［10］ Oefner PJ, Huber CG. A decade of high-resolution liquid chromatography of nucleic acids on styrene-divinylbenzene copolymers [J].
Journal of Chromatography B, 2002, 782（1）：27-55.
［11］ Azarani A, Hecker KH. RNA analysis by ion-pair reversed-phase high performance liquid chromatography [J]. Nucleic Acids Research, 2001, 29（2）：e7.
［12］ Dickman MJ, Hornby DP. Enrichment and analysis of RNA centered on ion pair reverse phase methodology [J]. RNA-a Publication of the RNA Society, 2006, 12（4）：691-696.
［13］ Dickman MJ. Ion pair reverse-phase chromatography ：A versatile platform for the analysis of RNA [J]. Chromatography Today, 2011 ：22-26.
［14］ Noll B, Seiffert S, Vornlocher HP, et al. Characterization of small interfering RNA by non-denaturing ion-pair reversed-phase liquid chromatography [J]. Journal of Chromatography A, 2011, 1218 （33）：5609-5617.
［15］ Seiffert S, Debelak H, Hadwiger P, et al. Characterization of side reactions during the annealing of small interfering RNAs [J].
Analytical Biochemistry, 2011, 414（1）：47-57.
［16］ Waghmare SP, Pousinis P, Hornby DP, et al. Studying the mechanism of RNA separations using RNA chromatography and its application in the analysis of ribosomal RNA and RNA ：RNA interactions [J]. Journal of Chromatography A, 2009, 1216（9）: 1377-1382.
［17］ Roossinck MJ, Zhang L, Hellwald KH. Rearrangements in the 5'nontranslated region and phylogenetic analyses of cucumber mosaic virus RNA 3 indicate radial evolution of three subgroups [J].
Journal of Virology, 1999, 73（8）：6752-6758.
［18］ McCarthy SM, Gilar M, Gebler J. Reversed-phase ion-pair liquid chromatography analysis and purification of small interfering RNA [J]. Analytical Biochemistry, 2009, 390（2）：181-188.
［19］ Transgenomic Optimized Purification of siRNA Oligonucleotides Using the WAVE? Oligo System. DOI ：http ：//www.transgenomic.
com/lib/an/612021.pdf.
［20］ Gilar M, Fountain KJ, Budman Y, et al. Ion-pair reversed-phase high-performance liquid chromatography analysis of oligonucleotides: Retention prediction [J]. Journal of Chromatography A, 2002, 958（1-2）：167-182.
［21］ Apffel A, Chakel JA, Fischer S, et al. Analysis of oligonucleotides by HPLC-electrospray ionization mass spectrometry [J]. Analytical Chemistry, 1997, 69（7）：1320-1325.
［22］ Apffel A, Chakel JA, Fischer S, et al. New procedure for the use of high-performance liquid chromatography-electrospray ionization mass spectrometry for the analysis of nucleotides and oligonucleotides [J]. Journal of Chromatography A, 1997, 777（1）：3-21.
［23］ Brakke MK, White JL, Samson RG, et al. Chlorophyll, chloroplast ribosomal RNA, and DNA are reduced by barley stripe mosaic virus systemic infection [J]. Phytopathology, 1988, 78（5）：570-574.

[1]	SHAN Ting-ting ,CHEN Xiao-mei ,GUO Shun-xing ,WANG Qian-qing ,WANG Ai-rong. Studies on Total RNA Extraction Methods from the Stems of Dendrobium Species [J]. Biotechnology Bulletin, 2018, 34(6): 54-58.
[2]	DU Wen-kai, YUAN Su-xia, HU Feng-rong. A Rapid Detection Method of Residual Genomic DNA in Total RNA Samples from Lilium regale [J]. Biotechnology Bulletin, 2018, 34(10): 81-86.
[3]	CHEN Xiao-feng, ZENG Qi-feng, DENG Xu, ZENG Guang-yao, ZHOU Ying-jun. A Method for Extracting Total RNA of Taxus chinensis at Large-scale [J]. Biotechnology Bulletin, 2017, 33(11): 60-66.
[4]	WU Chun-tai, LI Yu, FENG Wei-yu, ZENG Ri-zhong. Effects of the Latex Total RNA Precipitated with Different Methods on Amplification Efficiency [J]. Biotechnology Bulletin, 2016, 32(11): 124-129.
[5]	Chen Jing Gao Fei Zhou Yijun Zhang Zichen Li Zhanglei Cao Yuzhen Zhang Zhiwei. Extraction of High Quality Total RNA from Ammopiptanthus mongolicus with Improved Trizol Method [J]. Biotechnology Bulletin, 2013, 0(10): 87-92.