| Abiotic stress affects plant growth and yield.Plant cell membrane system is an important cellular structure that senses environmental stimulation and transmits stress signal.Proteins located in membrane system play an important role in the process of anti-stress response.At present,there are many studies on the cytoplasmic and nuclear localization proteins involved in plant stress response,but few studies on how the membrane localization proteins are involved in the regulation of plant stress response.COR413-PM1 is a cold-regulated membrane protein which has a multi-transmembrane domain,and the function of COR413-PM1 is still unclear at present.AtCOR413-PM1(Arabidopsis thaliana)and OsWCOR413(Oryza sativa)are homologous genes belonging to the cold-regulated gene(COR)family.In this study,the biological function,basic characteristics and regulatory mechanism of AtCOR413-PM1 gene of Arabidopsis thaliana were systematically analyzed.At the same time,the biological functions of OsWCOR413 gene in rice of gramineous crops were analyzed,and the differences and similarities in the functions of Arabidopsis thaliana and rice COR413-PM1 proteins were compared to provide new candidate genes for anti-stress in molecular breeding of crops.The main research results were obtained as follows:1.Biological functions of AtCOR413-PM1 gene in Arabidopsis thaliana.The main root length and fresh weight of mutant Atcor413-pm1 and wild type(WT,Col-0)were measured under different concentrations of PEG or NaCl stress treatment conditions,which proved that the mutant Atcor413-pm1 was more sensitive than WT under drought and salt stress.The sensitivity of mutant Atcor413-pm1 to ABA treatment was reduced,and the germination rate of mutant Atcor413-pm1 was higher than that of WT at different ABA concentrations,while the germination rate of AtCOR413-PM1 transgenic overexpressing lines was lower than that of WT,and the stomatal closure rate of mutant Atcor413-pm1 under ABA treatment was lower than that of WT.The results showed that AtCOR413-PM1 positively regulates the response of plants to drought and salt stress,and also positively regulates the ABA signaling pathway.2.Analysis of AtCOR413-PM1 gene characteristics in Arabidopsis thaliana.Analysis of the expression pattern of AtCOR413-PM1 showed that AtCOR413-PM1 responded to various abiotic stresses at the transcription level,including cold,ethylene,ABA,NaCl,mannitol and PEG6000.The results of subcellular localization analysis showed that AtCOR413-PM1 protein was located on the endoplasmic reticulum of the protoplast in Arabidopsis thaliana.The results of gene tissue specific analysis showed that AtCOR413-PM1 was mainly expressed in roots,root hairs,cotyledons,old leaves,stamens,stigma and petals of Arabidopsis thaliana.3.The binding of ABA to AtCOR413-PM1 protein was measured by Microscale Thermophoresis(MST),Surface Plasmon Resonance(SPR)and Biolayer Interferometry(BLI)The kinetic binding constant KD were 11.2 μM,0.66 mm and 0.21±0.048 m M,respectively.These results suggest that AtCOR413-PM1 can be directly combined with ABA hormone.AtCOR413-PM1-p BT3-STE was used as the bait to screen the interaction proteins of AtCOR413-PM1 through yeast two-hybrid method.It was found that the alignment results of BLAST were screened for AtCOR413-PM1 itself,and the number of times was the most.There were two other members of COR413 family of thylakoid membranes,COR413-TM1 and COR314-TM2,respectively,indicating that there might be a polymer formed between the AtCOR413-PM1 gene itself,and AtCOR413-PM1 may interact with COR413-TM1 and COR314-TM2,two other members of the COR413 family which targeted thylakoid membranes,and transmit signals downstream.In addition,an ABA-dependent transcription factor HB7 was also screened,AtCOR413-PM1 may also be involved in regulating ABA,drought and salt stress signaling pathways through HB7.The results of BLAST sequencing of AtCOR413-PM1 screening Arabidopsis thaliana c DNA library indicated that AtCOR413-PM1 was involved in the stress-related reaction processes of photosystem II,cold,drought,ABA,IAA,plant defense,cationic transport,etc.,which further demonstrated that AtCOR413-PM1 played an important role in plant resistance to environmental abiotic stresses.4.The resistance of mutant Atcor413-pm1 to cold stress was lower than that of WT.A proteomics method was used to analyze the differentially abundant proteins(DAPs)between Atcor413-pm1 and WT.A total of 4143 protein groups were identified and 3139 were accurately quantitated.The DAPs associated with AtCOR413-PM1 and freezing treatment were mainly involved in the metabolisms of fatty acids,sugars,and purine.Quantitative real-time PCR(q RT-PCR)confirmed the proteomic analysis results of four proteins: fatty acid biosynthesis 1(FAB1)is involved in fatty acid metabolism and might affect the plasma membrane structure;fructokinase 3(FRK3)and sucrose phosphate synthase A1(SPSA1)play roles in sugar metabolism,and may influence the ability of osmotic adjustment under freezing stress;GLN phosphoribosyl pyrophosphate amidotransferase 2(ASE2)affects the freezing tolerance through purine metabolism pathways.Proteomics studies show that the multispanning transmembrane protein AtCOR413-PM1 regulates plant tolerance to freezing stress by affecting metabolism of fatty acids,sugars,and purine in Arabidopsis thaliana.5.Basic characteristics and functions of OsWCOR413 gene in rice.OsWCOR413 was mainly expressed in leaves of rice which was different with the tissue specific expression of AtCOR413-PM1 in Arabidopsis thaliana.OsWCOR413 was induced by ABA.Rice gene deletion mutant Oswcor413 was created by CRISPR technology.Gene function analysis results show that Oswcor413 were more sensitive to PEG,NaCl and drought treatment than wild type rice(Kitaake).In addition,OsWCOR413 affected the plant height,panicle length,thousand kernel weight,the number of kernels per spike,seed setting rate,tillering number and grain weight per plant under normal growth conditions,especially the mutant Oswcor413:2-3-2.Protein structure analysis showed that OsWCOR413 and AtCOR413-PM1 had many hydrophobic domains.In conclusion,this study elucidated the biological functions,basic characteristics and regulatory approaches of the membrane localization protein AtCOR413-PM1 in Arabidopsis thaliana.It was also found that the AtCOR413-PM1 homologous gene OsWCOR413 in rice had similar biological functions,but the tissue specific expression was different.The study on the membrane localization protein AtCOR413-PM1 provides new evidence for further explaining of the response of plant membrane system and regulating the signal regulation mechanism of external stress,and provides new candidate genes for anti-stress molecular breeding of crops. |
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