Knockdown Expression And Molecular Analysis Of LRR-RLK Genes In Populus

Leucine-rich repeat receptor-like protein kinases (LRR-RLKs) involve in avariety of signal transduction process during the plant growth and development andenvironmental responses, which play an important role throughout the life course oforganisms. Recent studies have shown that LRR-RLKs undertake a key role inregulating morphogenesis and cell differentiation processes in plant meristem system.In this tesearch, we focus on the knockdown expression and molecular analysisof LRR-RLK genes in Populus to indentify their biological functions. At first, with thecombination of bioinformatics, comparative genomics and other research tools, wefound four key AtLRR-RLK genes (namely, PtBAM1、PtBAM2、PtPXYa、PtPXYb) inthe herbaceous model plant Arabidopsis that had close relationship with themorphogenesis of secondary vascular tissue and the differentiation process. Thenbased on the protein sequence of theese genes, searching from both Arabidopsis andP.trichocarpa database to get some candidate genes with higher levels of similarity.By way of building the phylogenetic tree, we screened four genes homologous withAtLRR-RLK genes, and named them PtBAM2, PtPXYa, PtPXYb, respectively, this wasfollowed by a series of theoretical analysis, containing gene structure, protein’sphysical and chemical properties, conserved domains, signal peptide, transmembranehelix residues and topology and subcellular positioning to identify the structures targetproteins. From the prediction results above, we made a conclusion that these targetproteins were LRR-RLKs locating on plasm membrane as single transmembraneproteins. Finally, the transcriptional map was used to determine the spatial andtemporal specificity of these functional candidate genes in poplar.Since the regulatory development mechanism of the vascular system of trees ismore complicated than Arabidopsis, in order to determine the exact function of thesegenes in the poplar, we built five RNAi expression vectors (PtBAM1_RNAiPtBAM2_RNAi, PtPXYa,&b_RNAi, PtBAM1,&2_RNAi, PtBAM12+PtPXYa-,&B_RNAi) through Gateway technology. Genetic transformation was undertakennext by Agrobacterium tumefaciens C58(pMP90) mediated method on the petiole orsegment without auxiliary buds, and we finally got the regenerative seedings withhygromycin resistant (10mg/L). For each type of RNAi seedings, with whole genome PCR we identified37,40,28,18,23independent transformants, respectively. It wasfound that, when compared with wild-type the RNAi poplar target genes expressionshowed varying degrees of down reduction through RT-PCR. However, we still needthe microscope slice technology to do some further research on the morphologicalchanges of RNAi poplar’s apical meristem, vascular tissue, and the stem vascularsystem, to make sure the exact function of these genes.