Carbohydrate Variation And Expression Analysis Of Related Genes In Endo-dormant Nectarine Floral Buds

RNA extraction of nectarine floral buds and the seasonal expression of carbohydrate metabolism and related genes were examined using ten-year-old field-grown'Shuguang'nectarine; the expression of related genes under low temperature treatment were detected using three-year-old potted'Shuguang'nectarine. A subtractive cDNA library was developed to study genes associated with the dormant floral buds in nectarine by using the RNA from dormant floral buds as"tester"and that from burst floral buds as"driver". The results are as following:1. The improved CTAB method gave high quality RNA from nectarine floral buds. The RNA integrity was judged by the sharpness of ribosomal RNA bands visualized on a denaturing agarose gel, the bands are clear and the ratio of 28S to 18S was 2 indicating the RNA integrity was better. The A₂₆₀/A₂₈₀ ratios ranged around 2.0 indicating little or no protein contamination, the results indicated that RNA extracted from improved CTAB method can fulfill the requirement of subsequent test such as gene expression analysis. However, RNA extracted by Trizol method cannot be detected on a denaturing agarose gel, and cannot meet our requirement.2. Soluble sugars (mainly sucrose) content of floral buds increased gradually during dormancy; however starch content showed an inverse decreasing pattern. The transcripts of genes (adenosine diphosphate glucose pyrophosphorylase (AGPase), histone H3 (HisH3), hexokinase 1 (HK1), sucrose synthase (SuSy), uridine diphosphate glucose pyrophosphorylase (UGPase)) involved in sugar metabolism were differentially regulated. The expression of HisH3 and HK1 distinctly up-regulated before endo-dormancy, SuSy displayed a contrary expression pattern; the expression of AGPase showed no significant change, the expression of UGPase was increased during dormancy state. The results demonstrated that HK1-depedent sugar signaling pathway might play an important role after entering endo-dormancy. The expression of cell division related gene HisH3 increased dramatically and then decreased after low temperature treatment, indicating that inhibition of growth during endo-dormancy was not a consequence of reduced cell division availability. UGPase had an adaptability to low temperature to some extent for it showed a parallel trend between endo-dormancy period and low temperature treatment.3. To further elucidate the molecular mechanism of nectarine floral bud dormancy, we developed the cDNA library of dormancy-related genes with suppression subtractive hybridization technique (SSH). The results indicated that the EST sequence showed a relatively high repeatability, thus we concluded that this method cannot separate dormancy-related genes effectively.