Genome-wide Identification Of The Banana GLR Family, Expression And Functional Validation Of GLR1.1 At Low Temperature

Banana（Musa spp.）is a large perennial herb that grows in tropical and subtropical regions,and is susceptible to low temperature stresses that can lead to waterlogging of the leaves and even death of the whole plant.In Fujian Province,the northern edge of the banana growing region,winter cold waves and sudden temperature drops seriously affect the normal growth and development of bananas,resulting in a sharp decline in banana quality and yield.The plant glutamate receptor-like（GLR）is an important class of Ca²⁺channel protein that is actively involved in plant growth and development as well as stress response to low temperature.In this study,we investigated the effects of different GLR ligands on the physiological properties of banana under low temperature stress,and conducted a genome-wide identification and expression pattern analysis of banana GLR gene family members under low temperature based on the banana genome database,cloning and expression analysis of key members under low temperature,and constructed subcellular localization fusion vectors,promoter expression vectors and overexpression vectors for functional validation of key members.This study will lay the foundation for further research and application of the cold resistance mechanism of banana GLR family genes under low temperature.The main findings of the study are as follows:1 Effects of different treatments on physiological characteristics of bananas under low temperature stressThe effects of exogenous GLR agonists（Glu,Gly,Ser）and antagonists（DNQX）on chlorophyll fluorescence parameters,photosynthetic pigments,relative conductivity and malondialdehyde content of banana leaves under low temperature stress were investigated by foliar spraying method using banana seedlings as test materials.The results showed that low temperature stress decreased F_v/F_m,Y（II）,q P,ETR and chlorophyll a,chlorophyll b and carotenoid contents of banana leaves,and increased F₀,relative conductivity and MDA contents of banana leaves.The F_v/F_m,Y（II）,q P,ETR and photosynthetic pigment contents of CK and DNQX groups under low temperature stress decreased significantly,the massive exudation of electrolytes,and The effects of low temperature stress on chlorophyll fluorescence parameters,photosynthetic pigments,relative conductivity and MDA content of banana leaves were more significantly reduced by exogenous Glu treatment than Gly and Ser under low temperature.This indicates that the exogenous Glu treatment reduced the degree of damage to the photosynthetic pigment synthesis organ of banana leaves by low temperature and alleviated the stress on banana leaves,resulting in less damage to the cell membrane.2 Genome-wide identification and expression analysis of banana GLR gene family under low temperatureIn this study,the whole genome of the banana GLR gene family was identified and analyzed based on banana genomic data,and its expression pattern under different low temperature stresses was analyzed based on the low temperature transcriptome data of wild banana.The results showed that there were 19 MaGLR family members in Musa acuminata,16MbGLR family members in Musa balbisiana,and 14 MiGLR family members in Musa itinerans;most of them were stable proteins with signal peptides,all with 3-6 transmembrane structures and subcellular localization predicted to be localized at the plasma membrane.The banana GLR promoter presence region is enriched with hormone,stress,and growth-and development-related response elements.Based on the low-temperature transcriptome of wild banana,MaGLR1.1,MaGLR1.3,MaGLR3.1,and MaGLR3.5 were found to be significantly differentially expressed,with MaGLR1.1 and MaGLR3.5 expression significantly increased.It is hypothesized that banana GLR is a highly conserved ion channel family,which may play an important role in banana growth and development and participate in hormone metabolic pathways and resistance to stresses such as low temperature.3 Cloning and expression analysis of banana MaGLR1.1 and other genes at low temperatureThe results of cloning and expression analysis of MaGLR1.1 and MaGLR3.5 showed that MaGLR1.1 encodes 961 aa and MaGLR3.5encodes 925 aa.Both MaGLR1.1 and MaGLR3.5 are hydrophobic proteins with signal peptides and stable transmembrane structures.The transcriptomic data and RT-q PCR validation of MaGLR1.1 and MaGLR3.5showed that both MaGLR1.1 and MaGLR3.5 responded positively to low temperature at 4℃.A large number of hormone-responsive elements were found in the promoter region of MaGLR1.1 and MaGLR3.5,and the overall up-regulated expression of MaGLR1.1 and MaGLR3.5 under exogenous ABA and Me JA treatment was verified by RT-q PCR,which was presumed to be involved in ABA and Me JA hormone signaling to improve plant cold resistance.The relative expression of MaGLR1.1 and MaGLR3.5 was increased by spraying exogenous Glu under low temperature stress,indicating that Glu was an effective activating ligand for banana GLR,and its activation effect was better than Gly and Ser,thus improving the low temperature tolerance of banana,while DNQX inhibited the expression of MaGLR1.1 and MaGLR3.5 under low temperature.4 Functional validation of the MaGLR1.1 gene in bananaA fusion expression vector was constructed using Agrobacterium-mediated infiltration of the lower epidermis of tobacco and MaGLR1.1 was localised to the cell membrane by laser confocal microscopy.The MaGLR1.1 promoter expression vector was constructed,and the tobacco leaves were found to be stained blue and darker than empty by GUS staining,indicating that MaGLR1.1 has strong promoter activity.Low temperature and Me JA treatment promoted the expression of MaGLR1.1gene promoter,while GA₃ treatment inhibited the expression of MaGLR1.1gene promoter,presumably LTR and Me JA response elements positively regulated the gene expression,while GA₃ negatively regulated the expression.To further verify the function of MaGLR1.1,MaGLR1.1 was transferred into wild-type Arabidopsis thaliana using the flower dipping method,and it was found that the transgenic strains had nearly 3 d earlier days to shoot than the wild type,and the growth and development rate was faster in the first 6 weeks.Glu treatment significantly increased the expression of the transgenic lines,while DNQX suppressed this phenomenon.