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.

Key words： Chinese jujube; cDNA-AFLP; stoneless gene; difference expression

Acknowledgements

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

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