观赏羽衣甘蓝粉色叶基因克隆与功能互补验证

Color is one of the most importatnt traits in ornamental plants. A semi-dominant gene, Pi, confers pink-leaved trait in ornamental kale (Brassica oleracea var. acephala). We have localized Pi on the top of choromosome 3 of genome C in Brassica. But the DNA sequence of Pi gene is still unknown. In order to isolate Pi, we will construct a huge F2 genetic population and a recombination population depending on the previous flanking molecular markers we achived. Depending on raw map of Genome C in Brassica, we will develop DNA molecular markers in the area of Pi, then predict candidate genes by drawing a fine-scale map of Pi. At the same time, we will explore genes and transcription factors on the pathway of anthocyanin biosynthesis by semi-quantitative and real-time quantification PCR. Afterwards, we will isolate candidate genes and design specific primers depending on the DNA sequences of the candidate genes, then design specific DNA markers for pink-leaved kale in order to focus on target gene in recombinant population. In addition, we will design a functional complementation test by constructing a Pi recombinant plasmid expression vector and transgenosis to model plant Arabindopsis thaliana and ornamental kale. This program will illustrate the molecular basis of pink-leaved coloration in ornamental kale, enrich theory of anthocyanin regulation in plants, benefit marker assisted selection (MAS) breeding in ornamentle kale, provide basic information for funtional genome research in Brassica, and help the research on coloraration of landscape plants.

色彩是园林植物的重要性状，羽衣甘蓝粉色叶由不完全显性单基因控制，我们已将该基因（Pi）初步定位于芸薹属C03染色体顶端，但该基因DNA序列尚不清楚。为成功分离该基因，本研究通过构建超大F2遗传群体，利用初定位侧翼标记，构建重组群体；根据C基因组草图，在Pi初定位区段开发分子标记，逐步缩小定位区间，并预测定位区域的候选基因；同时，查找植物花青素生物合成路径基因与转录调控因子，采用半定量与实时荧光定量PCR技术，筛选羽衣甘蓝花青素生物合成候选基因；克隆上述预测到的关键候选基因，据此开发特异性分子标记，并利用重组群体进行筛选，从而锁定粉色叶目标基因；构建表达载体，分别将目标基因转化拟南芥与羽衣甘蓝，进行功能互补验证。本项目旨在揭示调控羽衣甘蓝心叶呈现粉色的分子基础，预期可以丰富植物花青素调控理论，并为羽衣甘蓝分子标记辅助育种提供技术支撑，对于芸薹属功能基因组和园林植物呈色也具有科学借鉴意义。

色彩是园林植物的重要性状。项目组通过4年时间，在前期明确羽衣甘蓝粉色叶由不完全显性单基因控制的基础上，本项目首先通过构建超大F2遗传群体，采用染色体精细定位方法，预测BoDFRs是控制羽衣甘蓝心叶呈现粉色的关键候选基因。其次我们克隆了该基因，并分析了羽衣甘蓝花青苷组分，构建了稳定的羽衣甘蓝遗传转化体系；最后我们通过转基因方法，获得了过量表达和瞬时沉默突变体，互补验证了该BoDFR1基因具有使羽衣甘蓝心叶呈现粉色的功能。本项目全部完成了预期结果，找到并佐证了BoDFR1是控制观赏羽衣甘蓝心叶呈现粉色的关键基因，回答了什么是控制观赏羽衣甘蓝心叶呈现粉色关键功能基因的科学问题，研究结果对于丰富植物呈色理论具有科学借鉴意义。本项目还开发了一个羽衣甘蓝粉色心叶的DNA分子标记，这可以为羽衣甘蓝叶色分子标记辅助选择育种提供重要依据，为本项目后期羽衣甘蓝F1种业相关成果研发奠定基础。项目成果发表了SCI收录论文4篇，中文核心期刊论文2篇，羽衣甘蓝粉色心叶DNA标记获发明专利授权，培养了已毕业博士研究生1名和硕士研究生5名。