软腐病菌胁迫下的魔芋抗性基因表达差异及NBS类关键基因功能鉴定

Yunnan Province is the birthplace of one of konjac (Amorphophallus spp.). Soft rot disease of Erwinia in konjac is the most important diseases that affects the quality and yield, and it lacks the resistance of genetic resources in China, if breeding disease-resistant varieties need to look for more resources of resistance genes. Crop wild relatives, which can harbor vast reserves of quality and pest-resistance traits, are fundamental to the genetic improvement of crops. It is extremely important that these wild relatives be protected so that their desirable genes can be exploited for efficient crop breeding and sustainable development. Amorphophallus muelleri has the strong resistance to the soft rot of Amorphophallus spp. and it is an ideal material to screen resistance to soft rot genes. The project is on the basis of screening soft rot-resistant materials of Amorphophallus spp successfully in early, using the method of genomics and transcriptome analysis to carry out the following research: For study on STK resistance genes in Amorphophallus spp, degenerate primers based on conserved motif of the nucleotide binding site (NBS) region from the cloned plant disease resistance genes were used to isolate resistance gene analogues (RGAs) from genomic DNA of Amorphophallus muelleri. The expected results from the proposed study will contribute significantly to our knowledge on the molecular mechanisms underlying the interactions between Amorphophallus spp and E. carotovora subsp. carotovora to our future breeding efforts for developing Amorphophallus spp with broad spectrum and durable resistance to soft rot.

云南为中国魔芋产业发展的发源地。软腐病是魔芋生产中最严重的危害之一。目前，传统魔芋栽培种中高抗软腐病种质严重匮乏，而野生魔芋中蕴藏着大量潜在抗病耐逆基因，是魔芋遗传改良的珍贵种质资源库。项目组已开展了魔芋种质对软腐病菌抗性的前期研究，现拟通过生物信息学及RNA-seq技术，进行如下工作：一、检索EST数据库，根据已知核苷酸结合位点和富亮氨酸重复(NBS-LRR)类结构域设计特异或简并引物，扩增获得样品RGA片段；二、通过RNA-seq技术获得软腐菌胁迫下的魔芋关键抗性基因，并进行RT-PCR定量分析；采用cDNA末端扩增手段得到目标RGA序列全长；三、通过Gateway建立魔芋植株RNAi干涉及植物超表达载体的遗传转化体系，进而开展魔芋抗、感种质的遗传转化和功能验证。本研究可构建魔芋抗软腐病性状的调控网络和功能模式，也可为魔芋软腐病的持久防控开发新的抗性基因资源。

本项目批准后，整个项目期间均按计划正常进行，进展顺利。项目组持续开展了魔芋种质对软腐病菌抗性的研究，以A. konjac（高感）和A. muelleri（高抗）为植物材料，通过软腐病菌（Pcc）胁迫，基本建立了成熟的魔芋-软腐病菌互作研究系统。通过生物信息学及RNA-seq技术，本研究利用NBS抗病基因类保守域设计简并引物，经PCR扩增、克隆和测序，在Genbank上使用Blast进行同源序列搜索和比对，最终从魔芋种质中共获得12条RGAs片段，这些抗病基因同源序列均具有P-loop、Kinase-2、Kinase-3a以及GLPL等NBS抗病基因的保守序列；以魔芋抗病种质A. muelleri和感病种质A. konjac为材料，以接种0h为对照，对接种魔芋软腐病菌12h、24h和48h时间点的魔芋进行Illumina RNA测序。通过多点时序比较，分析魔芋响应软腐病病原菌侵染过程中，不同处理间的差异基因表达变化，构建了魔芋响应软腐病病原菌侵染过程的转录组表达谱，初步解析魔芋--软腐病病原菌互作的分子机制主要由SA和JA正调控因子、转录因子WRKY33、ROS等信号通路及LRR受体样丝氨酸/苏氨酸蛋白激酶等共同构成了在抗病中复杂的抗病免疫系统；通过Gateway建立魔芋植株RNAi干涉及植物超表达载体的遗传转化体系，进而开展魔芋抗、感种质的遗传转化和功能验证。本研究初步构建魔芋抗软腐病性状的调控网络和功能模式，为下一步魔芋软腐病的持久防控开发新的抗性基因资源。总体按计划完成了研究工作。