桃、杏远缘嫁接杂交导致花期响应FT基因异常表达机理研究

The researches have shown that grafting can lead to genetic material exchange between stock and scion and thus emerge expected genetical viration types, which became the new breeding approach. our early studies found the blooming period was delayed 3 to 5 days when the Prunus armeniaca × sibirica was grafted on Amygdalus persica; conversely, the blooming period 3 to 5 days in advance. Delaying blooming period to avoid the late frost damage are great importance for non-timber forest industry. Thus there is an urgent need to better understand the reason of delaying blooming period and whether or not this variation is heritable. We are putative the blooming abnormal be induced by DNA level control, transcriptional-level control or protein control that is closely associated with FT gene. To explain these reasons, four parts will explored. First, the level of CpG island methylation at FT gene promoter region. Next, the expression difference of FT mRNA at transcriptional control or the post-transcriptional control by miR5200 member from small RNA family. Then, the synthesis site, transport pathway between rootstock and scion, and expression quantity at FT protein control. Finally, genetic tendency and stability will be identified by MS-HRM (Methylation-sensitive high resolution melting, MS-HRM) technology. The conclusion will be new idea and method for interaction mechanism between rootstock and scion.

嫁接会导致砧、穗间遗传物质交换，产生期望的可遗传的变异类型，是创新种质的新途径。前期研究发现，将甜仁杏嫁接到碧桃砧木上后会导致嵌合体花期推迟3-5天，反之则会导致花期提前3-5天。推迟植物花期避开晚霜危害，在经济林生产中具有重要意义。因此，我们迫切想知道导致这种花期改变的原因是什么及变异是否可遗传？我们推测嫁接的选择压力导致成花素FT基因可能在DNA水平、或转录水平、或蛋白质水平的表达受到调控而导致花期异常。因此，本项目拟开展①花期响应FT基因启动子区CpG岛甲基化程度的DNA水平研究；②FT mRNA的转录表达及small RNA家族miR5200导致的FT基因转录后沉默调控研究；③成花素的合成部位、在嵌合体的转运和表达量差异的蛋白质水平调控研究；④采用MS-HRM技术对这种花期变异的遗传倾向性进行甄别。研究结果对于破解砧、穗互作机制和“嫁接杂种优势”的利用提供新的思路和途径。

【背景】嫁接会导致砧、穗间遗传物质交换，产生期望的可遗传的变异类型，是创新种质的新途径。.【结果】项目成功构建了甜仁杏‘优一’、‘紫叶’碧桃的正反嫁接杂种，其嫁接参数为：黄淮流域在6月10日~28日采用方块芽接，砧木保留与接穗相同当量生物积累量，嫁接成功率达75%。在‘优一’ + ‘紫叶’碧桃嫁接杂种，其紫叶碧桃的花冠尺寸变小，花丝蜷曲，枝条颜色由青绿色变为红褐色，而砧木‘优一’仁用杏的果核由小变大，重要经济产量种仁由0.7g达到1.0g的极显著性增加。反向嫁接杂种‘紫叶’碧桃 + ‘优一’尚未进入结实期。通过转录组高通量测序，筛选出显著差异上调表达的mRNA共818个，下调表达457个；筛选出PAmiR520a等显著性上调表达的miRNA 79个，显著下调65个；检测到差异lncRNA为3499，预测出SSRs 17329个。通过SDS-PAGE分离和质谱测序，获得以杏作砧木显著性表达的差异为366个，以桃为砧木获得显著性表达的差异为432个。后续验证试验正在进行。.【应用】成功构建了葡萄科和木兰科科间的嫁接杂种——五味子 + 葡萄。发现嫁接杂种枝条的节间长度和果实重量位于葡萄和五味子之间，叶片背面产生白色绒毛，果实颜色由青色变成红色。转录组测序获得DEGs数222个，获得显著差异miRNAs共79个表达，其中尚未报到的52个，miRNA长度集中分布在24nt范围，比对照本砧嫁接葡萄多了3nt。联合降解组发现，221个已知miRNAs关联1，442个目标基因，32个新miRNAs关联117个目标基因，其中显著差异表达的miRNAs和目标基因共12对。至少30项代谢物存在显著差异，其中果皮色泽相关10个类黄酮代谢物存在显著差异。在嫁接杂种中，五味子主要药用成分“五味子素”显著下调，而“五味子醇甲”显著上调。.【研究结论】嫁接杂种性状变异向着砧木和接穗的中间状态转变且稳定遗传；内源生物分子跨嫁接部位转移是导致嫁接杂种性状变异的内因，嫁接杂种合成部位的信号物质最终决定了表型性状的走向。嫁接导致重要经济性状丧失的主要原因可能是反复嫁接过程中砧木的经济性状不如接穗而逐渐降低的缘故。