Identification And Functional Analysis Of Glycine-Rich Rna Binding Protein Gene In Chinese Cherry

Glycine-rich RNA-binding proteins(GRPs)are composed of RNA binding recognition motifs at the N-terminal and glycine rich binding domain at the C-terminal.They can respond to external stimuli,participate in plant stress response,regulate the growth and development of plants and improve the plants resistance to stress.In this study,Three GRP unigenes encoding Glycine rich RNA binding protein were identified from the transcriptome library of Chinese cherry(Prunus pseudocerasus),The bioinformatics analysis was carried out,According to the results of homology alignment,these genes were named as PpcGRP4?PpcGRP6?PpcGRP7 respectively.The expression analysis of PpcGRP4?PpcGRP6?PpcGRP7 were performed in flower buds and leaves of Chinese cherry under low temperature stress using Real-time quantitative PCR.The full length of cDNA cherry PpcGRP4?PpcGRP6?and PpcGRP7 was isolated using PCR technique,and the plant expression vectors were constructed.Then three genes were transformed into Arabidopsis thaliana by Agrobacterium tumefaciens mediated floral dip.A main results about these genes were as follows:According to the sequencing results,the annotated glycine-rich RNA-binding protein gene sequence was obtained,and the open reading frame of the gene was analyzed using ORF Finder(Open Read Frame Finder),PpcGRP4?PpcGRP6?PpcGRP7 encoded 141?140 and 179 amino acid,respectively.The amino acid quantity and proteins secondary structure analysis showed that the isoelectric point of PpcGRP4?PpcGRP6 and PpcGRP7 were all more than 7 and all of them might be alkaline proteins.The instability index of PpcGRP4?PpcGRP6 and PpcGRP7 were 39.3?55.07 and 62.92,respectively.The molecular weight and the atomic composition of amino acid were different.The fat index and total average hydrophobicity showed that the proteins might be hydrophobic.The secondary structure analysis showed that the three proteins had a helix,(3 rotation angle,extended chain,random curl and other structures.However,PpcGRP4 and PpcGRP6 accounted for a large proportion of a helix.According to the predicted results of the transmembrane region of TMHMM,the proteins of PpcGRP4?PpcGRP6 and PpcGRP7 didn't contain transmembrane,and the proteins are basically located outside the membrane.The subcellular localization analysis indicated that PpcGRP4 is mainly located in the nucleus,PpcGRP6 was mainly located in the mitochondria.and PpcGRP7 was mainly distributed in cytoplasm and had different proportions in nucleus,mitochondria and cytoplasm.Homology analysis of genes related to other plants,found that they have high phylogenetic relationship with GRP in Prunus mume and Prunus persica.2.The expression of PpcGRP4?PpcGRP6?PpcGRP7 gene in flower buds and leaves of Chinese cherry under low temperature stress was analyzed using Real-time PCR technique,respectively.The results showed that the expression of PpcRP4?PpcGRP6 and PpcGRP7 genes in flower buds and leaves increased first and then decreased,and could be up-regulated by 34 fold.It concluded that PpcGRP4?PpcGRP6 and PpcGRP7 genes have different ability of responsiveness to low temperature.PpcGRP7 had the highest expression level,followed by PpcGRP6 and finally PpcGRP4.3.The full-length cDNA of PpcGRP4.PpcGRP6 and PpcGRP7 were separated by PCR technique,and the plant expression vectors were constructed and transformed into Arabidopsis thaliana by floal dip method,the T1 generation of genetically modified transgenic lines was obtained.Wild types and transgenic Arabidopsis seeds were used to conduct seed germination tests.The results showed that the germination rates of PpcGRP6 and PpcGRP7 were 38.9%and 25.1,respectively.The germination rate of wild type seeds was 9.3,which significantly overexpression PpcGRP6 and PpcGRP7 promoted the germination of seeds.However,the germination rate of Arabidopsis thaliana seeds with overexpression of PpcGRP4 was 14.8%,which was not significantly different from that of wild type Arabidopsis thaliana seeds.4.In order to further study the function of PpcGRP4?PpcGRP6 and PpcGRP7 genes in Chinese cherry,electrolyte permeability and MDA in transgenic Arabidopsis thaliana at different time after low temperature treatment were analyzed.The results showed that,under low temperature the rate of electrolyte exosmosis rate and MDA of the T1 generation transgenic plants increased first and then decreased.According to the change rate of electrolyte exosmosis rate in Arabidopsis thaliana leaves and the accumulation of MDA in Arabidopsis leaves at 8 h,it is inferred that the expression of the glycine rich RNA binding protein gene PpcGRP4,PpcGRP6 and PpcGRP7 gene in transgenic Arabidopsis thaliana can enhance the ability of cell membrane oxidation.And then improve the ability of cold resistance.The structure,expression and function of Glycine rich RNA binding protein gene in some Chinese Cherries were confirmed by the above results,which provided a theoretical basis for the study of plant stress resistance.