木质化介导的连作地黄块根膨大受阻的分子机制

The lignifications are one of the important immune mechanisms in response to environmental stress in plants. Our preliminary study found that the lignifications in root of consecutive monoculture Rehmannia glutinosa L. inhibited tuberous root formation. To study the role of lignifications in tuberous formation in R.glutinosa, we will conduct following analysis by comparing first and consecutive monoculture R.glutinosa based on the field and simulation experiments. (1) the difference of lignin deposition (spatial and temporal) in root will be determined by Sulfuric acid digestion and Tissue staining method, respectively. (2) the different expression of genes related to lignin biosynthesis will be detected by qRT-PCR, Northern Blot and Western Blot. (3) RACE technology was used to clone the core gene full length, whose function were confirmed by RNAi and over expressed technology. (4) the different lignin synthesis inhibitor will be applied to treat consecutive monoculture R. glutinosa to reveal the effect for the inhibitors on the inhabitation of disposition of lignin in root. The analysis on the association of lignification and root tuberization will shed light on the solving of root tuberization for the consecutive monoculture of R. glutinosa.

木质化作用是植物应对环境胁迫的重要免疫机制之一。申请者前期研究发现连作地黄根系木质化作用抑制其块根的膨大。为了深入解析木质化作用在连作地黄块根膨大受阻中的分子机制，本项目拟在大田和模拟条件下对头茬和连作地黄开展以下研究：①采用硫酸消化法和组织染色法测定头茬和连作地黄根部木质素沉积的关键期和时空分布；②运用qRT-PCR、Northern Blot技术结合Western Blot方法，研究连作地黄根系内木质素合成关键酶的表达变化，锁定阻碍连作地黄块根膨大的木质素合成通路；③利用RACE技术获取木质素合成通路关键酶的全长序列，并用基因过表达和RNAi技术验证其功能；④用不同木质素合成酶抑制剂处理连作地黄根部，观察细胞木质素合成变化及块根发育状态，筛选最佳木质素合成酶抑制剂。本项目为解决连作条件下地黄块根不能正常膨大的难题，提供重要的理论依据和技术支撑。

细胞壁中木质素的快速合成是植物面对胁迫的一种重要免疫防护机制。连作障碍作为一种复合胁迫，包含了生物和非生物等多种胁迫因子的交叉作用。地黄在响应连作胁迫过程中块根细胞木质化进程的诱发，给植物本身提供防御的同时却限制了块根的正常膨大。为了深入解析木质化作用在连作地黄块根膨大受阻中的分子机制，本研究通过对连作地黄块根发育受阻和木质素异常沉积的系统分析，精确锁定了地黄木质素生物合成途径响应连作胁迫的关键节点。同时，在地黄木质素生物合成途径节点催化酶筛选、鉴定的基础上，从转录和蛋白水平详细解析了连作地黄木质素生物合成途径催化酶基因的差异表达模式，并锁定了响应连作胁迫的关键催化酶基因。此外，利用反向遗传学手段对响应连作胁迫关键催化酶基因的生物学功能进行了验证，明确了连作条件下木质素异常沉积导致地黄块根不能正常膨大的分子机制。本研究为深入解读地黄连作障碍诱因和开展消减技术策略研究提供了关键的证据链条和有效抓手。