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【目的】探究不同盐胁迫后的棉花基因组DNA甲基化变化情况,并比较分析叶片和根部DNA甲基化变化差异,进而探究DNA甲基化与棉花耐盐性之间的关系。【方法】以陆地棉耐盐品种中9806和盐敏感品种中S9612为试验材料,分别用NaCl和Na2CO3(浓度均为0.4%)处理。提取对照和处理材料的DNA,进行双酶切、连接、预扩增和选择性扩增,然后采用MSAP技术(甲基化敏感扩增多态性)分析棉花幼苗在盐胁迫前后的DNA甲基化情况。从聚丙烯酰胺凝胶中回收纯化多态性片段并进行测序,通过NCBI进行比对分析;设计引物,用实时荧光定量PCR对多态性片段在棉花幼苗中的表达量进行验证。【结果】盐胁迫分析表明不同类型盐胁迫对棉花幼苗的影响不同;0.4%的中性盐NaCl对棉花幼苗的影响相对较小,各部分组织的形态变化不明显,而0.4%的碱性盐Na2CO3对棉花幼苗的影响较大,使棉花幼苗子叶变软,茎基部和根部发黑;MSAP分析结果表明,NaCl胁迫后中9806和中S9612叶片的甲基化比率分别为23.5%和27.7%,其中,全甲基化比率分别为20.3%和22.9%,根部的甲基化比率分别为24.7%和27.1%,其中,全甲基化比率分别为19.6%和21.6%;Na2CO3胁迫后中9806和中S9612叶片的甲基化比率分别为28.9%和28.1%,其中,全甲基化比率分别为24.3%和24.5,根部的甲基化比率分别为25.7%和27.6%,其中,全甲基化比率分别为21.5%和24.0%。叶片和根部甲基化水平存在差异,随着盐类型由中性盐向碱性盐转变的过程中,基因组DNA甲基化水平迅速增加,并在Na2CO3处达到最大值。通过分析胁迫以后的甲基化状态,在NaCl和Na2CO3胁迫后,中9806叶片的甲基化条带所占多态性条带比率分别为40.00%和50.00%,去甲基化条带所占多态性条带比率分别为54.12%和46.67%,中9806根部的甲基化条带所占多态性条带比率分别为35.53%和43.59%,去甲基化条带所占多态性条带比率分别为56.58%和51.28%。而中S9612叶片和根部中的甲基化条带比率和去甲基化条带比率的变化不明显。对多态性片段回收共获得6条序列,这6条序列涉及不同的同源基因,在基因编码区和非编码区均有分布,参与不同的代谢反应。qRT-PCR分析结果表明,6个同源基因在对照和处理之间的表达差异显著。【结论】耐盐性不同的棉花品种对盐胁迫反应不同,耐盐品种中9806在中性盐NaCl胁迫后基因组甲基化水平降低,诱导相关耐盐基因表达来抵抗胁迫而盐敏感品种中S9612则缺乏相应的耐盐基因使植株受到伤害增加,在Na2CO3处理以后受到伤害最大,对照与处理间甲基化水平差异显著,叶片和根部甲基化水平存在差异,具有组织特异性;多态性片段比对分析可知,同源性基因涉及多条代谢途径,通过多种代谢途径间的协同作用来抵抗胁迫。
【Objective】 The purpose of this study was to investigate the changes of DNA methylation in different genotypes of cotton under different salt stress and to analyze the difference of DNA methylation between leaves and roots. Then, the relationship between DNA methylation and salt tolerance of cotton was explored. 【Method】 With 9806 salt-tolerant cultivars and S9612 as salt-sensitive cultivars, Upland cotton was treated with NaCl and Na2CO3 (both at 0.4% concentration). The DNAs of control and treatment materials were extracted and double digested, ligated, preamplified and selectively amplified, and then analyzed by using MSAP (methylation sensitive amplification polymorphism) The situation. Polymorphic fragments were recovered from polyacrylamide gel and sequenced, and then analyzed by NCBI. Primers were designed and used to verify the expression level of the polymorphic fragments in cotton seedlings by real-time fluorescence quantitative PCR. 【Result】 Salt stress analysis showed that different types of salt stress had different effects on cotton seedlings. 0.4% neutral salt NaCl had less influence on cotton seedlings, and the morphological changes of various tissues were not obvious. 0.4% The effect of Na2CO3 on cotton seedling was larger, the cotyledons of cotton seedling softened and the base and root of the cotton seedling became black. MSAP analysis showed that the methylation rates of medium 9806 and medium S9612 were 23.5% and 27.7%, respectively, Among them, the methylation rates were 20.3% and 22.9%, respectively. The methylation ratios of the roots were 24.7% and 27.1%, respectively, of which the methylation ratios were 19.6% and 21.6% The methylation ratios of S9612 leaves were 28.9% and 28.1%, respectively, of which the methylation ratios were 24.3% and 24.5%, and the methylation ratios of roots were 25.7% and 27.6%, respectively. Among them, The rates were 21.5% and 24.0% respectively. There was a difference in the methylation level between leaves and roots. As the salt type changed from neutral salt to basic salt, the methylation level of genomic DNA increased rapidly and reached the maximum at Na2CO3. By analyzing the methylation status after stress, the methylation bands of 9806 leaves were 40.00% and 50.00% respectively under NaCl and Na2CO3 stress, and the demethylation bands accounted for The percentage of polymorphic bands was 54.12% and 46.67% respectively. The proportion of methylation bands in 9806 roots was 35.53% and 43.59%, respectively. The percentage of demethylated bands The banding rates were 56.58% and 51.28%, respectively. However, the changes of methylated bands and demethylated bands in leaves and roots of medium S9612 were not obvious. A total of 6 sequences were obtained for polymorphic fragment recovery. These 6 sequences involved different homologous genes, which were distributed in the coding and non-coding regions of the gene and participated in different metabolic reactions. qRT-PCR analysis showed that there were significant differences in the expression of 6 homologous genes between the control and the treatment. 【Conclusion】 Different cotton cultivars with different salt tolerance responded differently to salt stress. Among salt-tolerant cultivars, the methylation level of 9806 was decreased after NaCl stress, and the expression of salt-tolerance-related genes was induced to resist stress. The lack of corresponding salt-tolerant genes increased plant damage and was harmed most greatly after treatment with Na2CO3. There was significant difference in methylation level between control and treatment, and there was a difference in methylation level between leaves and roots, which was of tissue specificity. Polymorphism Fragment alignment analysis shows that homologous genes involved in a number of metabolic pathways, through a variety of metabolic pathways to co-play the role of co-resistance.