Functional Studies On C-type Lectin Receptor-like Kinase Genes HbClecRLK1 And HbClecRLK2 In Hevea Brasiliensis

Receptor-like protein kinases(RLKs)play important roles in plant growth and development as well as the signal transduction pathways in response to multiple biotic and abiotic stresses.In order to investigate the function of C-type lectin receptor kinases gene in Hevea brasiliensis,two C-type lectin receptor-like kinase genes named HbClecRLK1 and HbClecRLK2 was amplified by RT-PCR based on the RNA-Seq database of rubber tree.Their functions were explored through bioinformatics analysis,expression profile analysis,subcellular localization,ROS level analysis,biological functional analysis of HbClecRLK1 and HbClecRLK2.The results were as follows:1.Bioinformatics analysis of HbClecRLK1 and HbClecRLK2HbClecRLK1 contains a 1626 bp complete open reading frame encoding a polypeptide containing 541 amino acids.The open reading frame of HbClecRLK2 is 1662 bp,encoding a polypeptide containing 553 amino acids.Bioinformatics analysis showed that HbClecRLK1 protein consists of three conserved domains: C-type lectin recognition domain,transmembrane domain and tyrosine kinase domain.HbClecRLK2 protein also has a C-type lectin recognition domain,a transmembrane domain,and a bispecific serine/threonine/tyrosine kinase domain.The homology of amino acid sequence comparison showed that the amino acid sequence of HbClecRLK1 was highly homologous to the C-type lectin receptor-like kinases of Manihot esculenta,Crantz,Ricinus communis L.and Jatropha curcas L.The similarity was 91.34%,86.63% and 86.97%,respectively.The similarity between HbClecRLK2 and aforementioned sequences was 90.42%,85.53%and 87.16%,respectively.Phylogenetic tree analysis showed that Hb Clec RLK was in the same branch with C-type lectin receptor-like kinases of other species,indicating that Hb Clec RLK belonged to C-type lectin receptor-like kinases.2.Expression profile analysis of HbClecRLK1 and HbClecRLK2We analyzed their expression patterns under different conditions and the results showed that: after Colletotrichum gloeosporioides and Oidium heveae Steinm infected the leaves of rubber tree,HbClecRLK1 and HbClecRLK2 expression increased significantly;after chitin treatment of rubber tree protoplasts,HbClecRLK1 and HbClecRLK2 expression increased significantly;after flg22 treatment of rubber tree protoplasts,HbClecRLK1 and HbClecRLK2 expression dropped significantly;after 150mmol/L salt treatment of rubber tree suspension callus,HbClecRLK1 and HbClecRLK2 expression increased significantly.It is suggested that HbClecRLK1 and HbClecRLK2 may be related to the disease resistance and salt tolerance of rubber tree.Analysis on the changes of HbClecRLK1 and HbClecRLK2 expression in rubber tree leaves treated with different hormones SA,JA,ET,GA and ABA showed that HbClecRLK1 and HbClecRLK2 were related to SA and JA mediated signal pathways,and HbClecRLK2 was also related to ET and ABA mediated signal transduction pathways.3.Subcellular localization of HbClecRLK1 and HbClecRLK2The fusion proteins of HbClecRLK1-GFP and HbClecRLK2-GFP were expressed in tobacco leaves by Agrobacterium-infiltration method.The results of laser confocal microscopy showed that the fluorescence of HbClecRLK1-GFP and HbClecRLK2-GFP fusion proteins were mainly distributed on the cell membrane,indicating that HbClecRLK1 and HbClecRLK2 proteins were mainly located on the cell membrane.The results showed that HbClecRLK1 and HbClecRLK2 proteins were mainly distributed on the cell membrane.4.Analysis of the effect of HbClecRLK1 and HbClecRLK2 on ROS in plant tissuesIn order to understand whether HbClecRLK1 and HbClecRLK2 are related to the generation and accumulation of ROS in plant leaves,tobacco leaves expressing HbClecRLK1 and HbClecRLK2 respectively were stained with DAB,the results showed that the expression of HbClecRLK1 and Hb Clc RLK2 caused obvious ROS production and the accumulation in tobacco leaves,indicating HbClecRLK1 and HbClecRLK2 were involved in the generation of ROS and accumulation in plant leaves.5.Study on biological function of HbClecRLK1In order to further study the biological function of HbClecRLK1,we generated Arabidopsis thaliana transgenic plants of HbClecRLK1 gene.Phenotypic analysis showed that the root length of transgenic plants was significantly longer control plants(Arabidopsis thaliana transgenic plants of empty vector).Under the condition of mannitol,the seed germination rate of transgenic plants was significantly lower than that of wild type and control plants.Under salt stress,the seed germination rate of transgenic plants was significantly lower than that of wild type and control plants.These results suggest that HbClecRLK1 gene is related to plant root growth and is involved in the regulation of plant resistance to drought and salt stress.6.Study on the biological function of HbClecRLK2In order to further study the biological function of HbClecRLK2,we constructed Arabidopsis thaliana transgenic plants of HbClecRLK2 gene.Phenotypic analysis showed that there is no significant difference in the root length of transgenic plants and control plants.Under the condition of mannitol,the seed germination rate of transgenic plants was significantly higher than that of wild type and control plants.Under salt stress,the seed germination rate of transgenic plants of HbClecRLK2 gene was significantly higher than that of wild type and control plants.These results suggest that HbClecRLK2 gene is related to plant root growth and is involved in the regulation of plant resistance to drought and salt stress.7.Screening and identification of HbClecRLK2 interacting proteinsIn order to further analyze the function of HbClecRLK2,we screened the interacting proteins by yeast two-hybrid system,and demonstrated that HbClecRLK2 can interact with an endonuclease chitinase in rubber tree,named Hbendo CH2.Hbendo CH2 is highly similar to the endochitinases of cassava(Manihot esculenta Crantz),Populus Euphratica,Populus Trichocarpa with the similarity of 80.43%,79.28% and 78.81%,respectively.The Bi FC results showed that the interaction between HbClecRLK2 and Hbendo CH2 occurred on the cell membrane.Hbendo CH2 is located in the cell,its related to the production and accumulation of ROS in plant leaves.These results suggest that C-type lectin receptor-like kinases genes HbClecRLK1 and HbClecRLK2 of rubber tree are widely involved in plant growth and the regulation of resistance to different biotic and abiotic stresses,which may provide reference for further research on the function of plant lectin receptor-like kinases and search for new plant disease control strategies.