荔枝类受体激酶LcLRR在菌根耐受碳饥饿过程中的功能及调控机制解析

Trunk girdling is an important strategy to promote flowering in cultivated litchi (Litchi chinensis Sonn.). Girdling inhibits the carbohydrate transport to underground and decreases the root absorption, which further increases the third time physiological fruit drop and decreases the fruit quality. SYMRK is a leucine-rich repeat receptor-like kinase that is located in the cell membrane. This kinase interacts with the receptor of ‘Nod factor’ and is important in the signal transduction of nodule formation. However, its function in arbuscular mycorrhiza (AM) formation is unclear. The RNA-seq data showed 54,100 unigenes of litchi and 33,120 unigenes of AM fungi during carbohydrate starvation. The LcLRR induced by carbohydrate starvation in those unigenes possessed a 96.7% sequence similarity to SYMRK. The gene and promoter will be cloned in our project based on the litchi genome. The LcLRR will be transformed into the symrk mutant of Lotus japonicas to characterize its function. The promoter fragments will be transformed into tobacco leaves to analyse the promoter activities. The promoter and regulator of LcLRR will be analysed by yeast hybrids and electrophoretic mobility shift assay. The interacting proteins of LcLRR will be analysed by yeast two-hybrid, GST Pull-Down and bimolecular fluorescence complementation identification based on the girdling and control of the litchi mycorrhizal root. This study will investigate the function and regulation of LcLRR in response to carbohydrate starvation in litchi mycorrhizal root. This study serves as a basis for the signal transduction of AM formation and the improvement of cultivation measures for litchi.

环剥是荔枝促花措施，但环剥引起菌根碳水化合物饥饿使菌根吸收功能降低，导致第三次生理落果增加与果实品质下降。亮氨酸富集蛋白类受体激酶（LRR-RLK）SYMRK定位于细胞膜，其能与识别根瘤菌信号的受体结合，完成共生信号传递，但其在菌根形成中的作用尚不清楚。转录组数据表明共有荔枝源54,100 unigenes与真菌源33,120 unigenes参与菌根碳饥饿耐受过程。其中LcLRR被碳饥饿诱导，且其与SYMRK序列相似性达96.7%。本项目拟基于荔枝基因组克隆该基因及启动子，LcLRR转化symrk百脉根突变体，验证其功能；启动子缺失序列转化烟草，分析其活性。同时以环剥与对照荔枝菌根为材料，用酵母单杂结合凝胶阻滞分析LcLRR启动子及调控因子；用酵母双杂结合GST Pull Down与双分子荧光互补分析LcLRR互作蛋白。此研究能明晰荔枝LcLRR的功能及调控机制，具有理论与实践双重意义。

荔枝类受体激酶LcLRR基因为调控根瘤与菌根形成SymRK基因的同源基因，其表达水平在荔枝菌根耐受碳饥饿过程中被诱导。项目通过克隆荔枝类受体激酶基因LcLRR并异源互补百脉根symrk突变体验证其功能；启动子活性分析、酵母单杂、凝胶阻滞分析LcLRR调控因子；酵母双杂、Pull-down、双分子荧光互补分析LcLRR互作蛋白。以期解析LcLRR在菌根耐受碳饥饿过程中的功能及调控机制。功能分析表明LcLRR编码931个氨基酸，其中1-531为胞外Malectin like结构域，532-554为跨膜域，555-930为丝氨酸/苏氨酸胞内激酶结构域。虽然LcLRR与其它物种SymRK基因保守区段的相似度高，但其在Malectin like与丝氨酸/苏氨酸非保守区段存在较大变异。聚类分析显示LcLRR与非菌根植物拟南芥及油菜SymRK基因处于同一系统进化分支。异源互补实验表明LcLRR转化百脉根symrk突变体有菌根真菌菌丝侵入。针对调控因子筛选，采用核体系酵母文库筛选策略，成功构建以pGADT7-DEST为载体的cDNA文库，其总克隆数达到1×1000000cfu，重组率100%，插入片段平均长度1KB以上，为后续筛选、验证打下基础。互作蛋白鉴定则采用膜文库体系策略，构建以pPR3-N-DEST为载体的cDNA文库，其总克隆数同样达到1×1000000cfu，重组率100%，插入片段平均长度1KB以上。利用LcLRR-BD载体与次级文库共转化，初筛获得9个潜在互作蛋白，确定一对一验证及体外验证互作蛋白基因。项目分析了LcLRR在功能，并筛选其互作蛋白与调控因子，结果丰富了菌根共生过程中的信号转导路径；同时LcLRR功能鉴定对荔枝菌根碳饥饿机制解析有重要意义，具有减少荔枝第三次生理落果的潜在价值。