逆境胁迫激发火龙果高频体细胞遗传变异的分子机制

Dragonfruit (Hylocereus undatus) had been marked as a core industry during the "twelvth five-years plan" of Guizhou Province. Our previous work proved that after exposure to abiotic stresses including drought and plant growth regulators (PGRs) etc, the higher occurrence of somatic variations were observed from field-grown and in vitro plants. Further, it also had be confirmed that there exited retrotranspons TY1- copia and Ty3-gypsy in dragonfruit, and the transcript of the former was significantly enhanced as subjected to abiotic stress, e.g. drought, PGRs, SA and ultraviolent ray (UV) etc. Up to date, no evidance had been reported that abiotic stresses may induce a higher occurrence of somatic variations in this crop. The current program will focus on the molecular mechanism of higher occurrence of somatic variations in both field-grown and in vitro plants as induced by abiotic stresses. The items include in 1) cloning the retrotransposons of dragonfruit; 2) systematically exploring the roles of various abiotic stresses in the trancript of retrotransposon; 3) investigating the effects of stresses on methylation variations, especially on that of retrotransposon; and 4) unraveling the molecular cause of frequent variation of fruit color from high temperature and droght orchards. Additionally, a new type of molecular marker will be developed based on retrotranspon sequence, consequently the somaclonic variations and genetic relationships among the core germplasms of this crop will be elucidated using this marker. Therefore, the program will unravel the molecular mechanism concerning the involvement of abotict stresses in somatic variation, and provide a substantially thoretical platform for the germplasm creation and micropropagation. Also, it may develop a new molecular marker, which will serve as a practical tool.

火龙果在贵州"十二五"规划里列为重点产业。前期研究表明，逆境能激发火龙果田间植株及组培苗产生高频的体细胞遗传变异；经检测存在，并克隆了反转录转座子，分析表明TY1类在逆境下的表达显著增强；经查新，尚无逆境激发火龙果体细胞遗传突变及分子机制的报道。本项目拟克隆、分析火龙果反转录转座子，系统、深入地探索多种逆境对其转录激活的效能；探究突变体的DNA甲基化水平，特别是反转录转座子的甲基化水平及变化特点；解析环境胁迫/组培条件影响体细胞遗传变异的分子基础，以及高温干旱地区易发生果色变异的分子机理；以反转录转座子序列为基础，开发新型分子标记，并以此检测组培苗的遗传变异及探讨重要种质的亲缘关系。项目既能揭示主要逆境因子激发火龙果高频体细胞遗传变异的分子机理，也能在分子水平求解组培条件对组培苗遗传变异的可能功效，在种质创新和组培苗培育上具有重要指导意义；火龙果新型分子标记开发，也具有重要应用价值。

火龙果在我国西南地区尤其是喀斯特地区发展迅速。本项目探讨了主要逆境因子激发火龙果高频体细胞遗传变异的分子机理。主要结果如下：分别根据Ty1-copia 类和Ty3-gypsy类反转录转座子反转录酶的保守区设计简并引物，克隆获得了火龙果39条（Ty1-copia 类）和93条（Ty3-gypsy类）反转录转座子RT片段，序列分析发现火龙果反转录转座子RT序列具有高度异质性，部分序列具有转录活性，与其他物种存在反转录转座子的横向传递；采用Southern杂交估算火龙果基因组中含有Ty1组反转录转座子22,395个拷贝，Ty3组反转录转座子9,897个拷贝；利用染色体步移法克隆了3条有转录活性的反转录转座子LTR序列，顺式作用元件分析发现其包含大量光反应元件、脱落酸、赤霉素、参与逆境诱导的MYB结合位点等应答元件；利用RT-PCR检测逆境处理下Ty1和Ty3反转录转座子RT基因的表达情况，发现均能被组培、ABA、PEG、高温低温、高盐、GA、2,4-D、SA及UV诱导激活；基于火龙果反转录转座子的长末端重复序列信息设计IRAP和SRAP引物，建立并优化了IRAP和SRAP分子标记技术，利用该分子标记技术对组培产生的变异株系进行检测，发现DNA和cDNA之间，以及野生型和突变株之间的谱带不同，说明变异来自反转录转座子转录水平的差异；建立优化了火龙果甲基化MSAP检测体系，对不同棱数变异幼苗进行检测，发现在DNA甲基化模式存在差异，对不同外源GA3处理的幼苗检测发现外胞嘧啶甲基化（CNG）位点可能参与赤霉素信号调控网络；对火龙果果实颜色变异的代谢组研究，表明干旱高温条件下，火龙果果肉甜菜红色素在果实发育26天开始迅速上升，成熟果实果肉甜菜红色素含量高于果皮，而甜菜黄质在整个果实发育期表现为果皮高于果肉。资助研究生87名，其中博士32名，硕士64名；发表相关论文14篇，其中SCI收录4篇。