柑橘菌根信号受体蛋白基因LYK2的克隆及功能分析

Cloning and Function Analysis of Mycorrhizal Signaling Receptor Protein Lysin Motif Receptor-like Kinases 2 Gene(LYK2)in Citrus

从甜橙基因组中鉴定到12个菌根信号受体蛋白基因CsLYK,分布于chr1、chr2、chr6、chr7、chr8和chr9等6条染色体上。结构域分析表明CsLYK家族基因的3′端含有7个保守motif,且其组合也高度保守;而5′端仅有3个保守motif,且其组合变异较大。另外,CsLYK2、CsLYK3、CsLYK6、CsLYK10和CsLYK12没有内含子,其余7个成员含有1~19个内含子。系统进化分析将CsLYK基因分为3组,CsLYK2与已经报道的菌根共生信号受体基因PaNFP和SlLYK10处于同一小分支。qRT-PCR分析表明,枳实生苗中12个PtrLYK在根、茎、叶中均有表达,其中PtrLYK10在根系中高量表达,PtrLYK3、PtrLYK7、PtrLYK8、PtrLYK9和PtrLYK12在叶片中高量表达;另外,PtrLYK2在枳根系中的表达受菌根侵染的诱导,且在菌根侵染的早期表达量最高。在拟南芥原生质体中瞬时表达亚细胞定位载体35S-PtrLYK2-CFP,共聚焦荧光显微镜观察发现PtrLYK2定位于细胞膜上。通过苜蓿毛状根瞬时表达启动子表达载体pro PtrLYK2::GUS,利用Ds-Red红色荧光报告基因筛选阳性根并接种AM真菌,通过GUS组织化学染色和WGA-488菌根染色发现pro PtrLYK2主要在丛枝周围的细胞和根尖细胞表达。这些结果表明PtrLYK2可能在柑橘菌根共生早期的信号识别中发挥重要功能。

Twelve CsLYK genes were annotated from Citrus sinensis genome,which were unevenly distributed on six chromosomes,including chr1,chr2,chr6,chr7,chr8 and chr9. The conserved domain analysis showed that seven conserved motifs were detected at 3′ terminator of CsLYKs,and the constructions of these seven motifs were highly conserved. However,only three conserved motifs were detected at 5′ terminator of CsLYKs,and the constructions of these three motifs were highly variable. Gene structure analysis showed that CsLYK2,CsLYK3,CsLYK6,CsLYK10 and CsLYK12 had no introns,while other seven CsLYK family members had one to nineteen introns. Phylogenetic analysis revealed that twelve CsLYKs could be divided into three groups and CsLYK2 was clustered together with PaNFP and SlLYK10 in a small brunch. The expression analysis revealed that all PtrLYK genes were expressed in the root,stem and leaf of P. trifoliata. In details,the expression of PtrLYK10 was more abundant in the roots than leaf and stem,and the expression of PtrLYK3,PtrLYK7,PtrLYK8,PtrLYK9 and PtrLYK12 were more abundant in the leaf. Additionally,the expression of PtrLYK2 was induced in the roots of poncirus during different mycorrhizal infection periods,and the highest expression level was observed at two weeks post inoculation. The subcellular localization vector,35 S-PtrLYK2-CFP,was transiently expressed in the protoplast of Arabidopsis thaliana. The result of laser scanning Confocal microscope indicated that PtrLYK2 was expressed on the plasma membrane. The promoter expression vector,pro PtrLYK2::GUS,was transiently expressed in M. truncatula hair root,and the positive roots were screened with Ds-Red report gene and inoculated with AM fungi. After GUS histochemical staining and WGA-488 mycorrhizal staining,the expression of pro PtrLYK2 was observed in the cells around arbuscules and the root tip cells.Taken together,these results indicated that PtrLYK2 played a significant role in symbiotic signaling recognition at early stage between citrus and AM fungi.