NaCl和Na_2CO_3对不同棉花基因组的DNA甲基化影响  

Genomic DNA Methylation Polymorphism Analysis of Cotton Under NaCl and Na_2CO_3 Stress

作　　者：陆许可[1] 王德龙[1] 阴祖军[1] 王俊娟[1] 樊伟丽[1] 王帅[1] 赵小洁[1] 张天豹 叶武威[1] 

机构地区：[1]中国农业科学院棉花研究所/棉花生物学国家重点实验室/农业部棉花遗传改良重点开放实验室,河南安阳455000

出　　处：《中国农业科学》2014年第16期3132-3142,共11页Scientia Agricultura Sinica

基　　金：国家863计划项目(2013AA102601)

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S9【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

关 键 词：棉花 DNA甲基化 MSAP 盐胁迫 实时荧光定量PCR 

分 类 号：S562[农业科学—作物学]