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| To gain a better understanding of gene expression in early flowering trifoliate orange mutant (precocious trifoliate orange, Poncirus trifoliata), suppressive subtractive hybridization (SSH) and bioinformatics were applied to identify differentially expressed genes in precocious trifoliate orange. However, isolation of high quality RNA from buds, internodal shoots, and mature leaf tissues of trifoliate orange is difficult because of its high levels of polyphenols, polysaccharides and other secondary metabolites. An modified extraction procedure gave satisfactory results, proteinase K and high concentrations of β- mercaptoethanol were used in the extraction buffer to improve RNA quality and yeild. In addition, proteinase K involved in the procedure avoided badly injury caused by diethyl pyrocarbonate (DEPC), too. RNA isolated using above method was of sufficient quality for construction of suppression subtraction hybridization (SSH) library, which allowed the isolation of several early flowering ESTs. |
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| Keywords:Expressed sequence tags (ESTs);Flowering time;Precocious trifoliate orange;RNA isolation |
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