Establishment of cDNA-AFLP technology system and stoneless gene difference expression in Ziziphus jujuba Mill.

Abstract

An efficient and stable cDNA-amplified fragment length polymorphism (cDNA-AFLP) analysis system for Chinese jujube was established and successfully used for the studies of related stoneless gene difference expression in Ziziphus jujuba Mill. ‘Wuhejinsixiaozao’ fruit. Several main factors influencing cDNA-AFLP analysis were studied, including the preparation and purification of cDNA, restriction and ligation of cDNA, the preamplification reaction, selective amplification reaction, electrophoresis on denaturing polyacrylamide gels, and sliver staining. The results indicated that the total RNA extracted by modified SDS method was pure, complete, and suitable for reverse transcription to cDNA. Restriction digestion of cDNA was performed by using two restriction enzymes, around 150 ng DNA digested with three units of EcoRI and MseI enzymes, respectively, and incubated at 37°C for 5 h. The digested cDNA fragment was diluted 5 times and used as templates for preamplification, and the preamplification products were diluted 10 times and used as templates for selective amplification. The selective amplification fragments were subjected to PAGE electrophoresis and silver staining. By cDNA-AFLP analysis, it acquired three transcript-derived fragments (TDFs), DC1, DC5, and DC9, related with stoneless gene of Z. jujuba Mill. ‘Wuhejinsixiaozao’ fruit.

Keywords

Chinese jujube / cDNA-AFLP / stoneless gene / difference expression

References

[1] Ainsworth C (1994). Isolation of RNA from floral tissue of Rumex acetosa (sorrel). Plant Mol Biol Rep, 12(3): 198–203

[2] Bachem C W B, van der Hoeven R S, de Bruijn S M, Vreugdenhil D, Zabeau M, Visser R G F (1996). Visualization of differential gene expression using a novel method of RNA fingerprinting based on AFLP: analysis of gene expression during potato tuber development. Plant J, 9(5): 745–753

[3] Bachem C W B, Oomen R J F J, Visser R G F (1998). Transcript imaging with cDNA-AFLP: A step-by-step protocol. Plant Mol Biol Rep, 16(2): 157–173

[4] Bai R X (2008). Studies on genetic diversity and core collection construction of Ziziphus jujuba Mill. germsplasm resources using AFLP and SRAP markers, Dissertation for the Doctoral Degree. Baoding: Agricultural University of Hebei, 21–23 (in Chinese)

[5] Bassam B J, Caetano-Anollés G, Gresshoff P M (1991). Fast and sensitive silver staining of DNA in polyacrylamide gels. Anal Biochem, 196(1): 80–83

[6] Korpelainen H, Kostamo K (2010). An improved and cost-effective cDNA-AFLP method to investigate transcription-derived products when high throughput sequencing is not available. J Biotechnol, 145(1): 43–46

[7] Lewinsohn E, Steele C L, Croteau R (1994). Simple isolation of functional RNA from woody stems of gymnosperms. Plant Mol Biol Rep, 12(1): 20–25

[8] Li Z, Wang H Z, Li R F, Wei J H (2009). Lignin biosynthesis and manipulation in plants and utilization of biomass energy. Chinese Bulletin of Botany, 44(3): 262–272

[9] Liang P, Pardee A B (1992). Differential display of eukaryotic messenger RNA by means of the polymerase chain reaction. Science, 257(5072): 967–971

[10] Logemann J, Schell J, Willmitzer L (1987). Improved method for the isolation of RNA from plant tissues. Anal Biochem, 163(1): 16–20

[11] Lpez-Gmez R, Gmez-Lim M A (1992). A method for extracting intact RNA from fruit rich in polysaccharides using ripe mango mesocarp. HortScience, 27(5): 440–442

[12] Qu Z Z, Wang Y H (1993). Fruit Tree Records of China. Chinese Jujube Volume. Beijing: Chinese Forestry Publishing House, 43–47 (in Chinese)

[13] Schneiderbauer A, Sandermann H Jr, Ernst D (1991). Isolation of functional RNA from plant tissues rich in phenolic compounds. Anal Biochem, 197(1): 91–95

[14] Stölting K N, Gort G, Wüst C, Wilson A B (2009). Eukaryotic transcriptomics in silico: optimizing cDNA-AFLP efficiency. BMC Genomics, 10(1): 565

[15] Wan C Y, Wilkins T A (1994). A modified hot borate method significantly enhances the yield of high-quality RNA from cotton (Gossypium hirsutum L.). Anal Biochem, 223(1): 7–12

[16] Wang C S, Vodkin L O (1994). Extraction of RNA from tissues containing high levels of procyanidins that bind RNA. Plant Mol Biol Rep, 12(2): 132–145

[17] Wang X L (1974). Anatomy of fruit development of a preliminary observation. Chinese Bulletin of Botany, 16(2): 161–168

[18] Weiberg A, Pöhler D, Morgenstern B, Karlovsky P (2008). Improved coverage of cDNA-AFLP by sequential digestion of immobilized cDNA. BMC Genomics, 9(9): 480

[19] Welsh J, Chada K, Dalal S S, Cheng R, Ralph D, McClelland M (1992). Arbitrarily primed PCR fingerprinting of RNA. Nucleic Acids Res, 20(19): 4965–4970

Acknowledgements

This study was funded by the Provincial Natural Science Foundation of Hebei (No. C2004000363; C2007000448).

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2014 Higher Education Press and Springer-Verlag Berlin Heidelberg