Preliminary Functional Analysis Of Lrb1in Arabidopsis

Plant receptor-like kinase (RLKs) belongs to the family of transmembrane proteins, the amino-terminal islocated in extracellular, while the carboxyl-terminal owns an intracellular kinase domain, which is similar tothe animal receptor tyrosine kinase in structure, such as epidermal growth factor. Recent data shows theArabidopsis genome contains at least600homologous RLK sequences, which represents about2.5%of knownencoding protein genes in Arabidopsis. One of the subfamily, Leucine-rich Repeats RLK (LRR-RLK) haveover300members, which means that the LRR-RLKs occupy more than half of RLKs. Early studies show that,the subfamily gene play a role both in the plant growth regulation and stress resistance. Arabidopsis LRB1encodes LRB1containing625aa. Protein analysis display that, LRB1contains four regions: the extracellular Nterminal signal peptide region, leucine composite zone, single transmembrane region and cytoplasmic kinasedomain.In this study,we use wild type Col-0, mutants lrb1-1、lrb1-2, and over-expression plants as the materials.PCR and RT-PCR results shows the gene expression of LRB1in mutant lrb1-1and lrb1-2was partly downregulated but not completely. Treating wild type、mutants and over-expression plants respectively with ABA indifferent concentration of0.5μΜ,1μΜ,1.5μΜ, results show the seed germination rates was not significantlydifferent. This suggests that LRB1may not participate in the inhibition of seeds germination by ABA. It hasbeen known that ABA can promote stomatal closure. Therefore, we detected water loss rate of different plantsunder constant temperature, results show that the mutant LRB1has higher water loss rate than the wild type andsuper-expression plants. At the same time, when treating the lower epidermis of leaf with MES buffercontaining10μΜ ABA, the stomata aperture of LRB1was always greater than the other materials, whichsuggests that LRB1may participate in the impact of stomata by ABA, as when LRB1down-regulated, stomatamay not be normally closed. We predict that the LRB1gene of Arabidopsis thaliana can express in roots, stems,leaves, flowers, fruit, buds, flowers and other parts of plants. To further define that, we constracted theLRB1::pCMBIA1381-GUS recombinant vectors, by GUS staining of different organizations, LRB1was confirmed located in roots, stems, leaves, flowers, fruit, buds, flowers and other parts of Arabidopsis thaliana,and the expression quantity in the roots, stems, leaves was most abundant. Results of laser scanning for thedetection of GFP fusion protein expression suggest that, the LRB1-GFP fusion protein locates on cellmembrane. The results of staining by treating GUS transgenic plants with50μΜ ABA showed, with timeincreasing, the expression of LRB1gradually decreased, the RT-PCR results also suggests that expression ofLRB1was down-regulated after treating with ABA. At last, we constructed prokaryotic expression vector withGST tag and eukaryotic expression vector with c-myc tags, and peform preliminary protein experiments,whichmake the foundation of the further study of LRB1function.