论文部分内容阅读
为了初步探讨黄独胚性愈伤组织冻后黑暗培养修复超低温保存伤害的分子机理,本研究构建了黄独冻后黑暗培养胚性愈伤组织和冻后光周期培养胚性愈伤组织两个cDNA文库(正,反向抑制消减杂交),并对其阳性克隆进行序列测定和分析.结果表明:所构建的正、反向消减文库有效富集了差异基因,消减效率达到要求,插入片段集中于100～500 bp之间,插入片段大小符合要求,重组率大于95％,文库质量较好.正、反向消减文库在基因表达谱上存在一定的差异,黄独冻后黑暗培养胚性愈伤组织文库在山药储藏蛋白dioscorin、线粒体蛋白、衰老相关蛋白和水解酶等基因与冻后光周期培养胚性愈伤组织文库有较大差异.正、反向消减文库的建立为进一步了解黄独胚性愈伤组织冻后黑暗培养修复超低温保存伤害的分子机制提供帮助,有利于进一步分离和筛选差异表达基因,并克隆部分与黄独胚性愈伤组织冻后黑暗培养有关的全长基因.研究其表达模式,为探索黄独胚性愈伤组织超低温保存后植株再生的机制提供了理论依据.“,”The purpose of this study was to understand the repair molecular mechanism of dark culture of Dioscorea bulbifera L.embryogenic calli after its cryopreservation damage in a certain extent.2 SSH-cDNA libraries were constructed using Dioscorea bulbifera L.embryogenic calli cultured in darkness and in photoperiod after cryopreservation.Positive clones were randomly selected for sequencing and bioinformatics analyzing from the two libraries.Results showed that differential express genes were enriched effectively in the forward and backward subtractive libraries,and subtractive hybridization efficiency reached the demand.Most of the length of inserted fragments was 100～500 bp.The size of the inserted fragment met the requirement and the recombination rate was over 95％.The quality of the SSH-cDNA libraries was better.There were differences between forward and backward libraries on gene expression profiles.In forward library,the percent of genes related with backward storage protein Dioscorin,mitochondrial protein,senescence associated protein and hydrolase was higher than backward one.The estabishment of forward and backward subtractive libraries might lay a foundation for further understanding the repair molecular mechanism of dark culture of D.bulbifera L.embryogenic calli after its cryopreservation damage,which was conducive to further isolation and screening of differentially expressed genes and cloning of full-length genes related with D.bulbifera L.embryogenic callus dark culture.Studying its expression pattern and might be also conducive to provide a theoretical basis for exploring the mechanism of plant regeneration ofD.bulbifera L.embryogenic calli after cryopreservation.