NaCl和Na2CO3对不同棉花基因组的DNA甲基化影响

doi:10.3864/j.issn.0578-1752.2014.16.002

摘要/Abstract

摘要： 【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。

关键词: 棉花 , DNA甲基化 , MSAP , 盐胁迫 , 实时荧光定量PCR

Abstract: 【Objective】 The objective of the study is to research the change of genomic DNA methylation under different types of salt and to compare the variation of DNA methylation between leaves and roots, which can help us make it clear about the relation between DNA methylation and the salt tolerance in cotton.【Method】In the study, salt-tolerant variety Zhong9806 and salt-sensitive variety ZhongS9612 were used as experimental materials, which were treated with NaCl and Na2CO3 at concentration of 0.4%, respectively. DNA extracted from control materials and the treatment materials were used for double digestions, ligation reactions, pre-amplification reactions and selective amplification reactions, then MSAP technology (Methylation sensitive amplified polymorphism) was used to explore the change of DNA methylation between the control and treatment materials in cotton seedlings. The differentially methylated DNA fragments were isolated from the polyacrylamide gel, sequenced and BLAST analysis in NCBI library. Real time-PCR technology was used to test the expression quantity of polymorphic fragments in cotton seedlings.【Result】The results of salt stress indicated that the effects of different salt stresses on the growth of cotton seedlings were different. Neutral salt NaCl at 0.4% concentration had a relatively small effect on cotton seedling，and the morphological characters of different tissues changed a little, but 0.4% alkaline salt Na2CO3 had a bigger harm, resulted in the cotyledon soft, and the basal part of stem and roots of cotton seedlings black. MSAP analysis showed that after NaCl treatment, the genomic DNA methylation level of leaves of Zhong9806 and ZhongS9612 were 23.5% and 27.7%, in which full methylated ratio was 20.3% and 22.9%, respectively. The methylation level of roots of Zhong9806 and ZhongS9612 was 24.7% and 27.1%, in which full methylated ratio was 19.6% and 21.6%, respectively. After Na2CO3 treatment, the methylation level of leaves of Zhong9806 and ZhongS9612 were 28.9% and 28.1%, in which full methylated ratio was 24.3% and 24.5%, respectively. The methylation level of roots of Zhong9806 and ZhongS9612 was 25.7% and 27.6%, in which full methylated ratio was 21.5% and 24.0%, respectively. Genomic DNA methylation levels were different between leaves and roots. With the salt type changed from neutral salt to alkaline salt, the genomic DNA methylation levels went up rapidly and reached the maximum in the Na2CO3 treatment. From the result of DNA methylation status analysis, we could learn that the methylated band ratio (% polymorphic bands) in leaves of Zhong9806 was 40.00% and 50.00%, respectively, and demethylated band ratio (% polymorphic bands) was 54.12% and 46.67%, respectively. The methylated band ratio (% polymorphic bands) in roots of Zhong9806 was 35.53% and 43.59%, respectively, and demethylated band ratio (% polymorphic bands) was 56.58% and 51.28%, respectively. But the methylated band ratio (% polymorphic bands) and the demethylated band ratio (% polymorphic bands) in leaves and roots of ZhongS9612 changed a little after stress. In total, 6 fragments were obtained from the gel, which were amplified and analyzed with BLAST method. The result indicated that the 6 sequences were homologous to 6 different genes, widely distributed in coding region and non-coding region, involving different metabolic reactions. The analysis of qRT-PCR showed that these genes were differentially expressed between control and treatment.【Conclusion】The response of cotton varieties with different salt-tolerances to the stress of different salt is different. The DNA methylation level of Zhong 9806, a salt-tolerant variety, declined under salt stress, which could induce the expression of genes related to salt-tolerance, but Zhong S9612, a salt-sensitive variety, lack relevant genes to make the damage increase, reaching the most after the Na2CO3 treatment. A big difference exists in the DNA methylation level between the control and treatment，and the pattern of DNA methylation has tissue specificity. From the analysis of polymorphic fragments, homogenous genes involve many metabolic pathways, through the synergistic effect of three pathways to deal with the stress.

Key words: cotton , DNA methylation , MSAP , salt , qRT-PCR

陆许可, 王德龙, 阴祖军, 王俊娟, 樊伟丽, 王帅, 赵小洁, 张天豹, 叶武威. NaCl和Na2CO3对不同棉花基因组的DNA甲基化影响[J]. 中国农业科学, 2014, 47(16): 3132-3142.

LU Xu-Ke, WANG De-Long, YIN Zu-Jun, WANG Jun-Juan, FAN Wei-Li, WANG Shuai, ZHAO Xiao-Jie, ZHANG Tian-Bao, YE Wu-Wei. Genomic DNA Methylation Polymorphism Analysis of Cotton Under NaCl and Na2CO3 Stress[J]. Scientia Agricultura Sinica, 2014, 47(16): 3132-3142.

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