OSCA Family Analysis And Functional Validation Of PbrOSCA2.6 And PbrOSCA3.2 In Pyrus

Hyperosmolality-gated calcium-permeable channels(OSCA)are highly conserved families of ligand-gated ion channels present in plants,which are best characterized for their roles in signal communication involving a wide variety of plant-specific processes.Recently more and more researchers were involved into investigating the function of hyperosmolality-gated calcium-permeable channels(OSCA)and the majority of researches focused on the model plant and rice.Based on the pear genome database publicly announced,we used pear,Arabidopsis thaliana,humanity and other species especially pear as experiment material to do some bioinformatics analysis about glutamate receptor-like genes,meanwhile,preliminary proved the function of PbrOSCA2.6 and PbrOSCA3.2 in pear pollen.The primary results were as follows:1.Response to osmitic changes is critical for plant survival,development and reproduction.Hyperosmolality-gated calcium-permeable channels(OSCA)were identified as osmosensor in Arabidopsis,rice and human cells.To explore the potential functions of OSCAs in pear,we performed a bioinformatics and expression analysis of the PbrOSCA gene family.16 PbrOSCA members were identified from the Pyrus bretschneideri genome data base.Based on sequence composition and phylogenetic analysis,PbrOSCA family were classified into 4 clades.Protein structure analysis indicated that the 16 PbrOSCAs shared similar structures with their homologs in Arabidopsis.Multi-transmembrane pattern and ion transport pore site of PbrOSCAs were both conserved.By analyzing the expression of the PbrOSCAs in different tissues,we found that the expression patters of PbrGLRs from the same subgroup show the great resemblance and there are overlaps in organ expression profiles of different clades.Most of the PbrOSCAs were expressed in roots,8 PbrOSCAs were expressed in pollen tubes,and 8 PbrOSCA genes were expressed in roots,leaves,fruits and pollen.The expression patters provided the basis for the function study of OSCA genes in pear.2.The expression profiles of PbOSCA genes were analysed in different tissues and osmitic stress.6 PbOSCAs expression level raised during osmitic stress.These data suggest PbOSCAs could be a candicate target for generation of osmitoc stress resistiat plants.We selected PbrOSCA2.6,which is specifically expressed in pollen,and PbrOSCA3.2,which is expressed in all tissues,we constructed PbrOSCA2.6:GFP and PbrOSCA3.2:GFP vectors to affected Arabidopsis protoplasts and tobacco leaves proving the subcellular localization of PbrOSCA2.6 and PbrOSCA3.2 is in plasmamembrane which provided the basis for the study of ion channels.3.Under different osmotic stress,the growth of pear pollen tube was different.It was found that osmotic stress could inhibit the growth of pollen tube,and the greater the osmotic stress,the more the growth of pollen tube was inhibited.It has been reported that OSCAs of Arabidopsis thaliana are involved in the regulation of intracellular calcium concentration,while no report in pear.During the Arabidopsis thaliana mutant experiment,it was found that the growth of pollen tubes of OSCA-deficient plants was better than wild type.Based on this,utilizing antisense aligodeoxynucleotideand methods preliminary proved that the growth of pear pollen tubes lacking PbrOSCA2.6 and PbrOSCA3.2 was promoted.While the more functions of PbrOSCA2.6 and PbrOSCA3.2 should be further studied.