Cloning And Functional Analysis Of Maize Receptor-like Protein Kinase Gene ZmLRLK1

The receptor-like protein kinase of plants is a kind of serine/threonine kinases,which plays an important role in plant growth,development and stress response.It is similar to the receptor protein kinase of animals,but the extracellular ligands that associated with them have not been isolated yet,so it is called receptor-like protein kinase.Based on the differences of RLKs extracellular domains,it can be divided into several sub-families.The diversity among plant RLKs,reflected in their structural and functional properties,has opened up a broad new area of investigation into cellular signalling in plants with farreaching implications for the mechanisms by which plant cells perceive and respond to extracellular signals.Maize(Zea mays L.)is the most widely grown crop in the world and represents an important source of food,feed,biofuel,and industrial products,and plays an important role in global food security and economic trade.Through consulting relevant paper before the project,Maize receptor-like kinase related genes of GI number were obtained.Based on the existing RNA-Seq data,the heat maps of the 727 Maize receptor kinase gene expression in the seed were drawed,then two high expressed in the seed or specific expressed genes as were selected as candidates.Using SMART to analyse the protein conserved domain,multiple LRR repeating units were founded,which belongs to the LRR-RLK members of the subfamily,and were named by ZmLRLK1 and ZmLRLK2 respectively.Their expression patterns were analysed by real time PCR,it was founded that ZmLRLK1 only had a higher relative expression level in the seeds,indicating that it has obvious tissue-specific expression.ZmLRLK2 has different expression levels in the seeds and leaves.Taking ZmLRLK1 gene as the research object,its function was analysed by using RNA in situ hybridization,subcellular localization,yeast two-hybrid and other molecular biology methods,which can lay the foundation for further analysing the activity of phosphorylation,screening for the extracellular ligands and the kinases that interacted with ZmLRLK1.The main results are as follows:1.The result of RNA in situ hybridization showed that ZmLRLK1 was mainly expressed in endosperms.The ZmLRLK1 gene cloned from maize endosperm cDNA contains an open reading frame which encodes 958 amino acids,SMART analysis showed that ZmLRLK1 protein has a extracellular domain,transmembrane region and a serine/threonine protein kinase catalytic domain,which belongs to the typical LRR-RLK,and the intracellular domain includes 327 amino acid residues,it also contains multiple serine/threonine sites that may phosphorylate.2.Conduct GFP fusion expression vector of ZmLRLK1,and respectively use the maize leaves protoplast to do the subcellular localization.The results showed that ZmLRLK1 was mainly located on cell membrane,possibly on the chloroplast membrane,indicating that it is performative on the membrane.The results of Yeast activation showed that ZmLRLK1 intracellular domain did not have self-activating activity,so the Yeast two-hybrid experiment could be carried out.3.Yeast two-hybrid was carried to analyze whether ZmLRLK1 can form homodimer or not.The results showed that both of them cannot interact directly,speculating that it may activates its biological activity by relating to other kinases in signal transduction pathways or after some modifications;By screening for the proteins that interact with ZmLRLK1,the cDNA library of maize endosperm was used to do the yeast two-hybrid,the results showed that the starch synthase SS?a and eukaryotic initiation factor4a(Eif4a)could interact with ZmLRLK1 in Yeast.4.ZmLRLK1 intracellular domain corresponding DNA sequences was cloned and restructured to the vector pGEX-6t-1,and the expression was induced in Transetta(DE3)strains,showing that the recombinant protein were soluble.After purification,the polyclonal antibody was prepared and ZmLRLK1 protein of seed was detected from the protein level.