【摘要】 【目的】分析绿色棉纤维发育过程中DNA甲基化状态差异。【方法】利用2组限制性内切酶Eco RⅠ/HpaⅡ和Eco RⅠ/MspⅠ对绿絮棉1号纤维基因组甲基化位点进行识别和切割,并采用甲基化敏感扩增多态性引物对切割产物进行扩增,分析基因组内5’-CCGG-3’位点的甲基化状态;同时对扩增的差异片段进行测序,分析其生物学功能。【结果】66对甲基化敏感扩增多态性引物共扩增出4112个条带;平均每个样品共扩增出822.4个带型,平均每对引物扩增出12.46个片段。随着绿色棉纤维的发育,甲基化条带总数、甲基化条带比率、全甲基化条带百分比均逐渐升高;其中,开花后10、15、20和25 d时甲基化条带总数分别比开花后5 d时增加11.45%,13.86%,20.10%和33.13%。与开花后5 d相比,开花后10、15、20和25 d时纤维DNA发生甲基化变化位点的比例分别为3.15%,3.43%,3.65%和2.52%;而去甲基化位点的比例分别为12.87%,14.72%,13.31%和48.81%。通过测序和Blast分析表明,17个片段与已知的功能基因同源性较高,包括棉花线粒体基因组、丝氨酸蛋白酶基因和酯酶基因,且这些基因均在开花后25 d发生去甲基化。【结论】绿色棉纤维发育过程中DNA发生了甲基化与去甲基化现象。更多还原

【Abstract】 [Objective] The aim of this study was to analyze the DNA methylation during fiber development in green cotton.[Method] The products resulting from Eco RI/Hpa II and Eco RI/Msp I double-digestions were amplified by methylation-sensitive amplified polymorphism primers to analyze the methylation status of the 5’-CCGG-3’ locus in the ’Lüxumian No. 1’ genome.Meanwhile, the amplified fragments were sequenced to characterize their biological functions. [Result] A total of 4 112 fragments were amplified by 66 primer pairs, with an average of 822.4 fragments each. Additionally, each primer amplified an average of 12.46 fragments. The number of methylated fragments at 10, 15, 20, and 25 days post anthesis(DPA) was 11.45%,13.86%, 20.10%, and 33.13% higher than the number of methylated fragments at 5 DPA, respectively. At 10, 15, 20, and 25 DPA, 3.15%, 3.43%, 3.65%, and 2.52% of the green cotton fiber genomic loci were methylated, respectively. In contrast, at the same time points, 12.87%, 14.72%, 13.31%, and 48.81% of the loci were demethylated, respectively. The sequencing results and BLAST searches revealed that 17 gene fragments were homologous to known functional genes, including those from the cotton mitochondrial genome as well as the genes encoding a serine protease and an esterase. These genes were methylated at 25 DPA.[Conclusion] Genes are methylated and demethylated during green cotton fiber development.更多还原