小麦网格轻链蛋白基因TaCLC1参与调控赤霉病抗性的分子机制研究

Fusarium head blight (FHB) of wheat is mainly caused by Fusarium graminearum. It not only affects the yield of wheat, but also reduces the quality of wheat, which often result in large economic losses. The resistance mechanism of wheat scab is not clear. According to the transcriptome sequencing and bioinformatics analysis of the resistance relevant genes in scab sensitive and resistance near-isogenic lines Jimai 22 and Jimai 22-Fhb7 infected with Fusarium graminearum, we found that the expression of clathrin protein genes transcription were up-regulated in Jimai 22-Fhb7. It showed that clathrin proteins were related to the resistance of fusarium head blight. We had cloned a full length cDNA sequence from wheat, named TaCLC1 (GenBank KT345966), which encode a clathrin light protein. We found the expression of TaCLC1 was up-regulated by Fusarium graminearum and DON treatments. Gene silencing induced by Barely stripe mosic virus was practiced to verify the candidate genes. Here, we will study on the molecular mechanisms of FHB regulated by TaCLC1 in wheat. The full-length TaCLC1 cDNA was subcloned into the expression vector pZP211 downstream of the Ubi promoter to form pZP211-Ubi-TaCLC1 construct. And we will construct a CRISPR-Cpf1 vector that expresses a guide RNA sequence targeting TaCLC1. All vectors will transform wheat through Agrobacterium tumefaciens. Transgenic wheat plants will be treated by Fusarium graminearum, and the incidence rate of FHB of the transgenic plants will be detected. In particular, we will investigate the key genes expression of SA synthesis and JA synthesis way of overexpressed TaCLC1 plants under Fusarium graminearum stress conditions. The specifical interaction of TaCLC1 with the target protein of TaCLC1 will be determined by a yeast two-hybrid analysis combined with GST pull-down technique. We will investigate the epigenetic characteristics of different transgenic wheat strains to analyze the molecular mechanisms involved in the regulation of resistance to scab using BS-Seq, ChIP-Seq, Hi-C and other techniques. These results will be helpful for understanding the molecular mechanisms of FHB regulated by TaCLC1 in wheat. This will offer materials for further wheat breeding for FHB resistance.

小麦赤霉病主要由禾谷镰刀菌引起，既影响小麦产量，又产生多种真菌毒素，危害人类和动物的健康。发掘抗病基因，培育抗病品种很有必要。项目组前期对感/抗赤霉病的济麦22/济麦22-Fhb7接种禾谷镰刀菌，进行转录组测序发现，网格蛋白及内吞途径相关基因在济麦22-Fhb7中上调表达，差异明显，说明网格蛋白与赤霉病的抗性相关。本研究以济麦22-Fhb7为试材，同源克隆得到网格轻链蛋白基因TaCLC1，利用BSMV-VIGS技术，对TaCLC1基因参与调控小麦赤霉病抗性进行了初步确定。在此基础上，构建过表达和基因敲除载体转化小麦，鉴定TaCLC1参与调控赤霉病抗性中的作用，从SA、JA和内吞途径，结合互作蛋白研究TaCLC1参与调控的信号通路；利用BS-Seq、ChIP-Seq、Hi-C等技术，调查不同株系表观遗传学特点来解析其参与调控赤霉病抗性的分子机制，从而为小麦抗病育种奠定理论基础和储备基因资源。

小麦赤霉病主要由禾谷镰刀菌引起，既影响小麦产量，又产生多种真菌毒素，危害人类和动物的健康。网格蛋白介导的内吞是细胞抵抗病原菌的入侵、传递胞内外信号的主要手段，对生物的生存与适应均具有重要的生物学意义。研究以济麦22-Fhb7为试材，同源克隆得到网格轻链蛋白基因TaCLC1（GenBank：KT345966），TaCLC1为单拷贝基因，位于小麦7BL染色体上。qRT-PCR结果表明，TaCLC1在根、茎、叶和穗部均有表达，在叶片中的表达量最高，同时该基因的转录受到SA、MeJA 等多种激素及禾谷镰刀菌的诱导表达。利用大麦条纹花叶病毒（Barely Stripe Mosaic Virus，BSMV）介导的基因沉默（Virus-Induced Gene Silencing, VIGS）技术，在小麦叶部进行了抗赤霉病的研究，结果显示，BSMV::TaCLC1植株中腐生斑的面积明显比对照植株和BSMV::00植株中腐生斑面积大，同时禾谷镰刀菌的相对数量也较多。同时发现沉默植株中SA、JA合成关键基因TaPAL、TaOPR的表达量不同程度地下调。将禾谷镰刀菌接种到野生型、转基因拟南芥和突变体的叶片上，统计发病情况发现，与野生型相比，转基因拟南芥具有更强的抗病性；对转基因烟草接种禾谷镰刀菌得到了类似的结果。筛选得到了TaCLC1的互作蛋白TaAP2。构建过表达TaAP2σ载体并得到了转基因拟南芥。接种禾谷镰刀菌后发现，过表达TaAP2σ拟南芥比野生型具有更强的抗性。表明TaCLC1与TaAP2σ在膜泡运输途径互作调控赤霉病的抗性。该研究结果为小麦抗病育种提供了理论基础和储备了基因资源。