Abstract：
Grapevine (Vitis vinifera L.) crops are continuously exposed to biotic and abiotic stresses, which can cause genetic and epigenetic alterations. To determine the possible effects of grapevine cryopreservation on the regulation of DNA demethylase genes, this work studied the expression of DNA demethylase genes in cryopreserved and post-cryopreserved grapevine tissues. V. vinifera DNA demethylases were characterized by in silico analysis, and gene expression quantification was conducted by RT‒qPCR. Three DNA demethylase sequences were found: VIT_13s0074g00450 (VvDMT), VIT_08s0007g03920 (VvROS1), and VIT_06s0061g01270 (VvDML3). Phylogenetic analysis revealed that the sequences from V. vinifera and A. thaliana had a common ancestry. In the promoters of responsive elements to transcription factors such as AP-2, Myb, bZIP, TBP, and GATA, the conserved domains RRM DME and Perm CXXC were detected. These responsive elements play roles in the response to abiotic stress and the regulation of cell growth. These data helped us characterize the V. vinifera DNA demethylase genes. Gene expression analysis indicated that plant vitrification solution 2 (PVS2) treatment does not alter the expression of DNA demethylase genes. The expression levels of VvDMT and VvROS1 increased in response to cryopreservation by vitrification. Furthermore, in post-cryopreservation, VvROS1 was highly induced, and VvDML3 was repressed in all the treatment groups. Gene expression differences between different treatments and tissues may play roles in controlling methylation patterns during gene regulation in tissues stressed by cryopreservation procedures and in the post-cryopreservation period during plant growth and development.
摘要：
葡萄（VitisviniferaL.）作物不断暴露于生物和非生物胁迫下，这可能导致遗传和表观遗传改变。为了确定葡萄冷冻保存对DNA去甲基酶基因调控的可能影响，这项工作研究了DNA去甲基酶基因在冷冻保存和冷冻保存后的葡萄组织中的表达。V.viniferaDNA去甲基酶通过计算机模拟分析进行表征，并通过RT-qPCR进行基因表达定量。发现了三个DNA去甲基酶序列:VIT_13s0074g00450(VvDMT),VIT_08s0007g03920(VvROS1),和VIT_06s0061g01270(VvDML3)。系统发育分析显示，紫花苜蓿和拟南芥的序列具有共同的祖先。在对转录因子如AP-2，Myb，bZIP,TBP,GATA,检测到保守域RRMDME和PermCXXC。这些响应元件在对非生物胁迫的响应和细胞生长的调节中起作用。这些数据帮助我们表征了V.viniferaDNA去甲基酶基因。基因表达分析表明，植物玻璃化溶液2（PVS2）处理不会改变DNA去甲基酶基因的表达。VvDMT和VvROS1的表达水平响应于玻璃化冷冻保存而增加。此外，在冷冻保存后，VvROS1是高度诱导的，所有治疗组的VvDML3均被抑制。不同处理和组织之间的基因表达差异可能在通过冷冻保存程序胁迫的组织中以及植物生长和发育期间的冷冻保存后时期的基因调控过程中控制甲基化模式。
