Construction and Analysis of SSH-cDNA Library Induced by High Pressure in Rice Seeds during Germination
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摘要: 为了研究高压处理对高等植物基因表达的影响,以萌发过程中经高压处理后的水稻种子的mRNA作为检测子(Tester),以对照样品的mRNA作为驱动子(Driver),采用抑制差减杂交(SSH)结合镜像选择技术(MOS)构建了高压处理后水稻种子萌发过程中诱导表达的差减cDNA文库,并通过蓝白斑筛选获得了580个重组克隆。菌液PCR检测的结果表明这些克隆中的插入片段的大小主要分布在200~1000 bp之间,平均大小约为575 bp,其中单一扩增条带的有效重组克隆数为542个,有效的重组克隆率为93.3%。对部分重组克隆进行测序发现,受高压诱导表达的基因主要涉及到代谢、胁迫响应、转录调控等方面。为高压处理后水稻种子基因表达模式变化的研究打下了基础。Abstract: In order to study the effect of expression pattern of high hydrostatic pressure on high plant, a SSH-cDNA library was constructed with rice germinating seeds in this paper. Suppression subtractive hybridization (SSH) in combination with mirror orientation selection (MOS) was performed using the mRNA from rice germinating seeds treated by 75 MPa high hydrostatic pressure as tester and the mRNA from control (seeds untreated by high hydrostatic pressure) as the driver. 580 recombinant clones were picked through blue/white screening. PCR detection of the recombinant clones showed that the size of inserts mainly ranged from 200 bp to 1000 bp, and the average size of these inserts were about 575 bp. Among the 580 recombinant clones, 542 clones (93.3%) were identified to be effective single band ones. After sequencing some of the recombinant clones, it was found that the genes induced by high hydrostatic pressure were mainly involved in metabolism, stress response and transcriptional regulation. The research paved the way for studying the gene expression patterns of rice seeds responding to high hydrostatic pressure.
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Winter R. High Pressure Effects in Molecular Biophysics [A]//Proceedings of the International School of Physics Enrico Fermi, Course CXLVII on High Pressure Phenomena [C]. Amsterdam: IOS, 2002: 413-453. Kaarniranta K, Elo M, Sironen R, et al. Hsp70 Accumulation in Chondrocytic Cells Exposed to High Continuous Hydrostatic Pressure Coincides with mRNA Stabilization Rather than Transcriptional Activation [J]. Proc Natl Acad Sci USA, 1998, 95: 2319-2324. Dixon D R, Pruski A M, Dixon L R J, et al. The Effects of Hydrostatic Pressure Change on DNA Integrity and Embryonic Development in the Hydrothermal-Vent Mussel Bathymodiolus Azoricus: Implications for Future Deep-Sea Mutagenicity Studies [J]. Mutation Research, 2004, 552: 235-246. Iwahashi H, Odani M, Ishidou E, et al. Adaptation of Saccharomyces Cerevisiae to High Hydrostatic Pressure Causing Growth Inhibition [J]. FEBS Letters, 2005, 579: 2847-2852. Fernandes P M, Domitrovic T, Kao C M, et al. Genomic Expression Pattern in Saccharomyces Cerevisiae Cells in Response to High Hydrostatic Pressure [J]. FEBS Letters, 2004, 556: 153-160. Vogel R F, Pavlovic M, Hormann S, et al. High Pressure-Sensitive Gene Expression in Lactobacillus Sanfranciscensis [J]. Braz J Med Biol Res, 2005, 38: 1247-1252. Li G S, Bai C K, Duan J, et al. Research on Mutation in Rice (Oryza Sativa L. ) Induced by High Hydrostatic Pressure [J]. Chinese Journal of High Pressure Physics, 2006, 20(4): 421-428. (in Chinese) 李桂双, 白成科, 段俊, 等. 高静水压诱导水稻产生突变的研究 [J]. 高压物理学报, 2006, 20(4): 421-428. Rebrikov D V, Britanova O V, Gurskaya N G, et al. Mirror Orientation Selection (MOS): A Method for Eliminating False Positive Clones from Libraries Generated by Suppression Subtractive Hybridization [J]. Nucleic Acids Res, 2000, 28: E90. Sambrook J, Russell D. Molecular Clone: A Laboratory Manual(3rd ed) [M]. Translated by Huang P T. Beijing: Science Press, 2002: 93-96. (in Chinese) Sambrook J, Russell D. 分子克隆实验指南(第3版) [M]. 黄培堂, 译. 北京: 科学出版社, 2002: 93-96. Ji W, Wright M B, Cai L, et al. Efficacy of SSH PCR in Isolating Differentially Expressed Genes [J]. BMC Genomics, 2002, 20: 3-12. Diatchenko L, Lau Y F, Campbell A P, et a1. Suppression Subtractive Hybridization: A Method for Generating Differentially Regulated or Tissue-Specific cDNA Probes and Libraries [J]. Proc Natl Acad Sci USA, 1996, 93(12): 6025-6030. Li G C, Jin L P, Xie K Y, et al. The Principle of Suppression Subtractive Hybridization Technique and Its Application in Plant Gene Isolation [J]. China Biotechnology, 2004, 24(9): 26-34. (in Chinese) 李广存, 金黎平, 谢开云, 等. 抑制差减杂交(SSH)技术及其在植物基因分离上的应用 [J]. 中国生物工程杂志, 2004, 24(9): 26-34. Su P H, Yu S M, Chen C S, et a1. Spatial and Temporal Expression of a Rice Prolamin Gene RP5 Promoter in Transgenic Tobacco and Rice [J]. Plant Physiol, 2001, 158(2): 247-254. Mandal S, Mandal R K. Seed Storage Proteins and Approaches for Improvement of Their Nutritional Quality by Genetic Engineering [J]. Current Science, 2000, 79(5): 576-589. Bai C K, Li G S, Duan J, et a1. Effect of High Hydrostatic Pressure on Seeds Germination and Seedling Isoenzyme in Rice (Oryza sativa L. ) [J]. Chinese Journal of High Pressure Physics, 2003, 17(4): 283-289. (in Chinese) 白成科, 李桂双, 段俊, 等. 高静水压对水稻种子萌发及同工酶的影响 [J]. 高压物理学报, 2003, 17(4): 283-289. McCord J M, Fridovich I. Superoxide Dismutase. An Enzymic Function for Erythrocuprein(Hemocuprein) [J]. J Biol Chem, 1969, 244(3): 6049-6055. Hwang I K, Eum W S, Yoo K-Y, et al. Copper Chaperone for Cu, Zn-SOD Supplement Potentiates the Cu, Zn-SOD Function of Neuroprotective Effects against Ischemic Neuronal Damage in the Gerbil Hippocampus [J]. Free Radic Biol Med, 2005, 39(3): 392-402.
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