甘蓝BoFLC2基因序列多态性调控开花期的分子机制

Cabbage is a typical plant-vernalization-responsive plant. The quality and quantity of head was seriously decreased by premature bolting, leading to huge production losses. BoFLC2 is one of major repressor in regulating flowering in Brassica oleracea. Previously, nucleotide polymorphism of BoFLC2 was identified between early flowering lines (BoFLC2E) and late flowering lines (BoFLC2L). However, the mechanism of nucleotide polymorphism of BoFLC2 regulating flowering time is unknown. In order to reveal the molecular mechanism of nucleotide polymorphism of BoFLC2 in regulating flowering time of cabbage, association analysis between nucleotide polymorphism of BoFLC2 and flowering time in segregation population of cabbage, and flowering time of transgenic plants developed by transforming BoFLC2L and BoFLC2E gene into boflc2 Rapid Cycle will be analyzed to confirm its role in regulating flowering time. What’s more, to explain how the nucleotide variation of BoFLC2 regulating flowering time, the flowering time, the flowering time related genes expression and epigenetic modification of BoFLC2 will be analyzed in transgenic plants developed by transforming nucleotide polymorphism in coding and non-coding position of BoFLC2L gene into early flowering cabbage (BoFLC2E), respectively.

结球甘蓝是典型的绿体春化植物，“未熟抽薹”严重影响叶球品质和产量，给生产上造成巨大损失。BoFLC2是调控甘蓝类蔬菜抽薹开花的关键因子之一。前期研究发现BoFLC2基因在早开花和晚开花甘蓝自交系之间存在稳定的序列多态性，但这些差异序列/位点调控甘蓝抽薹开花早晚的机理尚不清楚。本项目拟通过BoFLC2序列多态性与甘蓝开花早晚分离群体的关联分析，BoFLC2L（晚开花）和BoFLC2E（早开花）基因分别转缺失boflc2基因的甘蓝材料Rapid Cycle进一步确认BoFLC2基因调控甘蓝开花期的功能；另外，建立系列BoFLC2基因编码区和非编码区差异序列转基因甘蓝植株，通过开花期、开花相关基因的表达和BoFLC2表观修饰分析，揭示BoFLC2序列多态性调控结球甘蓝早花和晚花的分子机制。

结球甘蓝（Brassica oleracea var. capitata）是典型的绿体春化植物，只有植株生长至7片叶子或茎粗达6mm才能感受低温，经过40天以上的低温春化才能从营养生长期过渡到生殖生长期。BoFLC2是结球甘蓝响应低温反应，从营养生长阶段过渡到生殖生长阶段的一个重要转录因子。然而，关于BoFLC2基因遗传变异对结球甘蓝开花期早晚影响的报道很少。在此，我们从非常早开花和非常晚开花的结球甘蓝中克隆了BoFLC2E和BoFLC2L同源基因，二者在第一内含子处有215bp插入/缺失（indel），在第二外显子处有三个非同义SNP和3bp插入/缺失。根据BoFLC2E和BoFLC2L第一内含子215bp插入/缺失设计的分子标记（BoFLC2E/BoFLC2L：501bp/286bp）在极端早开花和极端晚开花的结球甘蓝种质、自然甘蓝群体和两个F2后代中验证，发现BoFLC2L与晚开花紧密关联。此外，BoFLC2L第一内含子中 215bp的缺失可以造成种子春化型拟南芥（Col）和无需春化的甘蓝（TO1000, boflc2）的转基因后代开花时间延迟，表明BoFLC2第一内含子的215bp插入/缺失是影响结球甘蓝开花时间的。尽管BoFLC2E和BoFLC2L第一内含子的215bp插入/缺失没有引起可变剪接，但它减缓了BoFLC2L在春化过程中的沉默并反馈到PHD-PRC2复合物的核心基因，导致它们的转录水平降低。该研究不仅为分子标记辅助鉴定耐抽薹资源和选育甘蓝良种提供了有效策略，也为探索春化响应植物开花时间机制提供了切入点。