水稻(Oryza sativa)起源于热带,随着人们对粮食需求的增长其种植区不断向北扩展,这就要求选择耐冷能力强的水稻品种.但是目前,对水稻耐冷性的分化及其机制的研究尚少见报道.本研究以辽宁、江苏和广东3个不同气候地区水稻为实验材料,通过冷害症状及叶绿素荧光指数比较水稻耐冷性差异,采用DNA甲基化亚硫酸盐测序法(bisulfite sequencing PCR,BSP),研究CBF(C-repeat bindingfactor)冷响应转录通路的ICE1(inducer of CBF expression 1)基因区域与启动子区域甲基化水平影响不同品种水稻冷害差异的分子机制.结果表明ICE1基因区域甲基化水平无差异,但是启动子区域甲基化有显著差异(P＜0.05).辽宁丹粳17(LN004)的ICE1基因启动子区域2个胞嘧啶被甲基化,耐冷指数最高;江苏的南粳5055(JS013)的ICE1基因启动子区域7个胞嘧啶被甲基化,耐冷指数居中;广东的粤新占2号(GD008)的ICE1基因启动子区域11个胞嘧啶被甲基化,耐冷指数为最低.定量PCR检测结果显示,ICE1和其下游调控的CBF1(C-repeat DRE binding factor 1)、CBF3(C-repeat DRE binding factor 3)的基因表达量与耐冷性显著正相关(P＜0.05),但是与ICE1启动子区域甲基化水平显著负相关(P＜0.05).这表明辽宁(LN004)、江苏(JS013)和广东(GD008)水稻的耐冷性与地区气候相适应,且耐冷性受ICE1基因甲基化水平决定的CBF冷响应转录通路调节.本研究结果为进一步解释水稻耐冷的分子机制提供了理论依据.%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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