西瓜在枯萎病菌侵染过程中木质素合成及功能研究

In a recent study, most genes involved in biosynthesis shikimate-phenylpropanoid-lignin biosynthesis pathway were induced expressed from resistance watermelon root infected by Fusarium oxysporum f. sp. niveum using an Agilent custom microarray and suppression subtractive hybridization (SSH). These suggested that lignin involved in inhibiting Fusarium oxyspporum f. sp. niveum colonization in wtermelon. Results of quantitative real-time PCR in incompatible and compatible interaction also supported this case. Those results demonstrate that lignin biosynthesis pathways might play important roles in watermelon against FON infection，but the exact function and biosynthesis process of lignin is yet unclear. In this project, we intend to study function of lignin on watermelon inoculated by Fusarium oxysporum f. sp. niveum in histochemistry; The changes of gene expression of the plants are studied by quantitative real-time PCR induced Jasmonic acid and Fusarium oxysporum f. sp. niveum in order to understand biosynthesis process of lignin on watermelon inoculated by Fusarium oxysporum f. sp. niveum . Clone Cl4CL gene on the basis of preliminary studies, and validate gene function by construction and transformation of anti-sense expression vector of Cl4CL gene using CRISPR/Cas9 technology. The transgenic plants are obtained. The phenotype and physiological characteristics are researched in transgenic watermelon inoculated by Fusarium oxysporum f. sp. niveum. In addition, the regulatory factors and interaction proteins of Cl4CL gene will be identified through the yeast one-hybrid and two-hybrid. The implementation of this project will not only reveal function and biosynthesis process of lignin in watermelon in response to Fusarium oxysporum f.sp. niveum infection, but also will be helpful in molecular breeding of resistant varieties and the development of rational strategies for control of Fusarium wilt disease in watermelon.

课题组前期通过SSH和microarray 研究发现木质素合成基因在西瓜枯萎病抗性反应中被诱导表达，用 Realtime-PCR在抗、感西瓜中进行验证，确定木质素合成基因在抗性品种上调表达，而感病无变化，且4CL基因表达与西瓜枯萎病抗性进程最相关，推测木质素合成参与西瓜枯萎病抗性反应，但其具体的功能尚不明确。本项目拟在前期基础上检测枯萎病菌对西瓜维管束细胞结构、木质素含量的影响，测定在枯萎病菌和茉莉酸诱导下木质素合成相关基因表达时空性、酶类活性及代谢产物的含量，明确木质素合成在西瓜枯萎病抗性中功能；克隆木质素合成关键基因Cl4CL，通过CRISPR/Cas9技术构建载体，获得突变体植株，对木质素的功能进行验证, 最后对Cl4CL基因调控因子及互作蛋白进行鉴定。研究结果不仅可以揭示木质素合成在西瓜枯萎病抗性反应中的功能，还对西瓜枯萎病可持续防控具有重要的理论和实际意义。

西瓜是世界第五大水果，我国西瓜的栽培面积、总产量与人均消费量均居世界首位。西瓜枯萎病是由半知菌亚门镰孢属尖孢镰刀菌寄生引起的一种世界性真菌土传病害，病田一般减产 20%-30%、严重田块可达 50%-60%，甚至绝产。细胞壁的强度提升和根茎部的木质化是西瓜枯萎病结构抗病的重要内在机制。项目前期利用绿色荧光蛋白（GFP）标记西瓜枯萎病菌研究发现西瓜对枯萎病菌的抗性表现在阻止了病菌再次入侵或扩散生长。为了揭示木质素合成基因在西瓜枯萎病抗性反应中作用。项目首先确定木质素含量与西瓜幼苗对枯萎病的抗性呈正相关，明确了在枯萎病菌和茉莉酸诱导下抗病品种的木质素合成相关基因表达时空性、酶类活性及代谢产物的含量都比感病品种高，明确4CL基因是西瓜枯萎病抗性中木质素合成关键基因；克隆木质素合成关键基因Cl4CL，Cl4CL1、Cl4CL2与冬瓜Bh4CL聚为一支，同属于4CL基因ClassⅠ类参与木质素合成。项目研究发现茉莉酸含量和木质素含量与西瓜幼苗对枯萎病的抗性呈正相关（n=0.93 ，n=0.89），茉莉酸转导途径通过ERF分支参与西瓜苗期对枯萎病抗性的调控。接菌处理会使抗、感品种根茎部茉莉酸含量、合成相关基因、酶活均显著提高和木质素的含量提高，明确了木质素合成在茉莉酸调控西瓜枯萎病抗性反应的作用起一定的作用。研究结果不仅可以揭示木质素合成在西瓜枯萎病抗性反应中的功能，还对西瓜枯萎病可持续防控具有重要的理论和实际意义。