Abstract：Abstract Rice (Oryza sativa) is originated from the tropics, but human is continuously exploring its suitable planting areas northward, thus, the selection of cold-tolerant rice cultivars is required. However, the studies on the mechanism of differentiation on cold tolerance among rice cultivars has not been documented. In the present study, three materials of rice germ from different climate regions in Liaoning, Jiangsu and Guangdong provinces in China were selected. Their cold-tolerance was comparatively determined by indexes of cold injury and chlorophyll florescent. Methylation sites of coding and promoter regions of ICE1 (inducer of CBF expression 1) in CBF(C-repeat binding factor) cold response transcription pathway were determined to elucidate molecular mechanism of cold differentiation in different regions using DNA methylation sequencing sulfite (bisulfite sequencing PCR, BSP). The results showed that the differences in methylation levels of ICE1 gene were only found at promoter regions(P＜0.05). Danjing-17 rice cultivar from Liaoning (LN004) had 2 cytosine methylations at ICE1 gene promoter region and the highest index of cold tolerance; Nanjing-5055 from Jiangsu (JS013) had 7 cytosine methylations, and the middle index. Yuexinzhan-2 from Guangdong (GD008) had 11 cytosine methylation and the the lowest index. qRT-PCR tests indicated that ICE1 showed significant positive correlation with downstream regulated expression of CBF1 (C-repeat DRE binding factor 1) and CBF3 (C-repeat DRE binding factor 3) on cold tolerance level (P＜0.05), but ICE1 showed significant negative relationship with the ICE1 methylation levels(P＜0.05). These indicated that cold tolerance among 3 rice cultivars may be adapted to local climate, and were regulated by CBF cold response transcription pathway, which was determined by the methylation of ICE1 gene. This study may provides theoretical basis for further explanation of the molecular mechanism of cold tolerance in rice.
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