WRKY调控高氧气调包装（HO-MAP）菜心木质素形成的机制

Chinese cabbage (Brassica campestris L. ssp. chinensis Makino) is one of the most cultivated vegetables for its edible stem and leaf in South China. However, the lignification of its stem during storage has become a serious problem for its quality and marketable value. In our previous study, high-oxygen modified atmosphere packaging (HO-MAP) treatment could effectively delay the accumulation of lignin, reduce the activity of key enzymes involved in lignin biosynthesis, and retard the production of H2O2, as compared to air packaging (AP). Furthermore, the relative expression levels of genes involved in lignin biosynthesis were inhibited, the WRKY transcription factors (TFs) in MAPK signal pathway were also found to be regulated. Therefore, the goal of this proposed project is to address the mechanism of WRKY TFs regulating on the lignification of Chinese cabbage during storage. We hypothesize that the WRKY TFs may regulate the lignification of Chinese cabbage directly interacting with the promoter of target genes, and/or through MAPK pathway to influence the ROS production during storage. (1) RNA sequencing is employed to screen the potential WRKY TFs, which may take part in regulating the lignification process after the Chinese cabbage were treated by HO-MAP and AP during storage. (2) Multiple molecular techniques such as Yeast 1/2 hybrid, EMSA, dual luciferase assay, ChIP-Seq, and BiFC etc., will be applied to investigate the characteristics of regulation attributes of WRKY TFs to the target genes involved in lignin biosynthesis and ROS metabolism process. In addition to the interactions between WRKY TFs and the target genes, other proteins i.e., MAPK members, which may interact with WRKY TFs to co-function on regulating the production of ROS, thus, influencing the lignification process are also going to be conducted. (3) To further verify the function of candidate WRKY TFs, over-expression and RNA interference will be manipulated. The results obtained from this proposed project are hopefully to help illustrate the mechanisms of WRKY TFs regulating on lignification of Chinese cabbage during storage; and to provide guidance for developing possible techniques to delay the shelf life.

菜心是我国华南地区栽培的特色蔬菜之一。菜心采后贮运期间，茎部容易木质化，严重影响其营养品质及商品价值。前期研究发现，高氧处理可明显延缓菜心木质素积累、H2O2的产生及木质素合成关键酶活性的提高，下调苯丙烷途径和木质素特异途径相关酶基因的表达，并发现WRKY转录因子在MAPK信号级联途径中差异表达。基于此，本项目拟对WRKY对菜心采后木质化的调控机制进行深入研究。（1）通过转录组测序及生物信息学分析筛选可能对木质素合成起调控作用的WRKY转录因子。（2）利用酵母单杂交、ChIP-Seq、EMSA等方法分析WRKY下游靶基因；并采用酵母双杂交，BiFC等筛选WRKY的互作蛋白，解析其调控网络。（3）通过超表达及RNA干扰验证WRKY参与采后菜心木质素化的调控功能。该研究有助于明确菜心木质化品质劣变的机理；并为探索菜心采后保鲜技术提供理论参考。

菜心（Brassica rapa ssp. chinensis）是我国华南地区栽培规模最广的蔬菜之一。采后木质化是果蔬衰老的重要现象，也是限制菜心采后品质劣变及贮运销售的重要因素。本项目通过研究高氧气调包装对采后菜心木质化的影响作用，并在采后菜心木质化进程中，筛选差异表达WRKY转录因子，对菜心木质化的转录调控机制进行研究。研究结果发现采后菜心衰老过程中茎组织的木质素、纤维素、半纤维素等含量上升，硬度增加。高氧气调包装调控茎部组织内部活性氧产生/清除系统，降低 H2O2 水平；抑制木质素单体聚合酶 Laccase、POD 等酶活性增加及活性氧水平上升；从而延缓木质素的积累。筛选获得了4个转录因子进行分析，WRKY70/33具有转录激活活性，而WRKY48/13可能起转录抑制作用；WRKY70可结合木质素合成基因BrPAL3、BrCAD3、BrPOD69、BrPODC3启动子，WRKY33可合BrCAD3启动子。通过构建酵母双杂核文库，筛选WRKY13的互作蛋白，互作编码蛋白包括丝氨酸苏氨酸蛋白磷酸酶，TIFY蛋白。在拟南芥过表达WRKY70，对阳性植物检测发现拟南芥茎组织木质素合成基因AtPAL1、At4CL1、AtCAD6、AtC3H、AtF5H、AtCCoAoMT、AtCOMT表达量显著上调。上述研究结果明确了WRKY参与菜心薹茎衰老过程中木质化的转录调控机制。本项目的研究结果为开展菜心采后薹茎质地劣变机制提供新的思路，可为保障蔬菜品质，延长贮运期提供重要的理论依据和实践指导。. 在本项目的资助下，已经在Postharvest Biology and Technology、食品科学技术学报等刊物上发表了7篇论文，申请中国发明专利1项。