红麻不育系与保持系基因组DNA甲基化比较分析

Comparative analysis on the kenaf (Hibiscus cannabinus L.) genomic DNA methylation of its male sterility line and maintainer line

关键词:  红麻  基因组DNA甲基化  CMS  MSAP  qRT-PCR

Key words:  kenaf  DNA methylation  CMS  MSAP  qRT-PCR

摘要:

为分析红麻不育系中甲基化模式，以红麻不育系UG93A和保持系UG93B为试验材料，分别提取2个材料的苗期叶片、四分体时期和双核期花药的基因组DNA（gDNA），采用甲基化敏感扩增多态性（MSAP）技术和实时荧光定量PCR（qRT-PCR）方法分析比较其不同生长发育时期基因组DNA甲基化的变化规律，并研究甲基化变化基因的表达模式。结果表明：1）红麻生长发育过程中基因组DNA甲基化呈现一定的时空动态变化规律，不育系UG93A和保持系UG93B苗期叶片的甲基化率分别为56.79%（全甲基化率为44.25%，下同）和58.89%（43.24%）；花药发育的四分体时期的甲基化率分别为48.08%（36.24%）和44.25%（33.22%）；花药发育的双核期的甲基化率分别为45.30%（34.15%）和48.78%（37.98%）；2）不育系UG93A基因组DNA的甲基化率在苗期最高、花药败育发生前的四分体期次之、败育后的双核期最低，在整个生长发育过程中呈现先高后低的趋势；保持系UG93B基因组DNA的甲基化率在苗期最高、花药发育的四分体期最低、双核期次之，在整个生长发育过程中呈现先高后低、再缓慢上升的趋势。保持系UG93B基因组DNA的甲基化水平总体上高于不育系UG93A，但在花药败育发生前的四分体时期，不育系的甲基化水平明显高于保持系；3）ATP8、SCL13、SRF6和植物磺肽素受体2等基因在不育系UG93A与保持系UG93B中存在甲基化差异。qRT-PCR分析结果表明，甲基化发生变化的基因在不育系和保持系之间的表达差异显著，推测这些基因的甲基化变化在红麻细胞质雄性不育（CMS）中发挥重要的作用。

Abstract:

Taking the CMS line UG93A and maintainer line UG93B of kenaf as materials,gDNA(genomic DNA) were extracted from seedling leaves,tetrad stage anthers and binucleate stage anthers respectively.MSAP (methylation sensitive amplified polymorphism) technology was used to analyze and compare their methylation of gDNA.The expression patterns of methylated genes were studied by qRT-PCR.The results showed that:1) During the growth and development of kenaf,DNA methylation showed spatio-temporal variation pattern.The gDNA methylation rate (MSAP%) of UG93A and UG93B at seedling stage were 56.79% (full methylated rate was 44.25%,similar as follows) and 58.89% (43.24%);MSAP% at tetrad stage was 48.08% (36.24%) and 44.25% (33.22%);MSAP% at binucleate stage was 45.30% (34.15%) and 48.78%(37.98%), respectively.2) The ratio of UG93A was the highest at seedling stage,tetrad stage before anther abortion came the second,then binucleate stage after abortion.It displayed a trend of high to low during growth and development.The rate of UG93B was highest at seedling stage,followed by binucleate stage and tetrad stage showing a trend of high to low,and then gradually rising.Although,the rate of UG93B was higher than that of UG93A,but the UG93A was significantly higher than UG93B in the tetrad stage before anther abortion.3) Gene methylation or demethylation happened in many genes including atp8、SCL13、SRF6、phytosulfokine receptor 2.These genes in UG93A and UG93B were differentially methylated.The results from qRT-PCR analysis reviewed that the differentially expression levels of methylated fragments between the cytoplasmic male sterile line and maintainer line showed significant differences,indicating gene methylation played an important role of in kenaf CMS.