Study On Regulation Of BpGRP Gene By BpGRF3 In Betula Platyphylla Involved In Salt Stress

Glycine rich RNA binding proteins(GRPs)were a class of glycine rich proteins with a typical RRM domain at the N-terminal and a variable number of amino acids at the C-terminal.GRPs played an important role in flowering time,flower development,biological rhythm,chromatin modification,ABA signaling pathway and stress response.At present,the function of GRPs was mostly focused on cold stress,freezing stress and osmotic stress.However,the salt tolerance mechanism of Betula platyphylla was still unclear.Therefore,GRP gene was cloned from birch(named BpGRP).In order to reveal the function of BpGRP,the transcriptional level of BpGRP under salt stress and the phenotypic and physiological indexes of transgenic lines were analyzed.The upstream regulatory genes were screened and verified by yeast one hybrid,Chromatin Immunoprecipitation-PCR and EMSA et al.In addition,the upstream regulatory genes BpGRF3 and BpMiR396c-2 of the BpGRP gene were analyzed for bioinformatics and physiological indicators of transgenic callus.The results were as follows:1.The expression pattern of BpGRP gene showed that the expression was all down regulated in root tissues(except 12 h),and reached the lowest expression level at 6 h,which was 4.9%of the control under salt stress.However,in stems and leaves,BpGRP gene expressions were all induced.The highest expression level of BpGRP in stems reached at 36 h,which was 62.47 times higher than that of control,while in leaves,BpGRP reached the highest expression level at 24 h(38.41 times of control).In conclusion,BpGRP gene can be induced by salt stress.2.The overexpression BpGRP lines(OE1,OE2 and OE6)and the expression inhibition lines(SE1,SE5 and SE7)were obtained from birch callus mediated by Agrobacterium tumefaciens.After 7 days of salt stress treatment,WT and SE1,SE5 and SE7 showed obvious stress symptoms.Under salt stress,the growth of the expression inhibition lines was seriously affected,and the leaves were yellowing seriously,and part of them fell off.However,compared with WT lines,the growth status of OE1,OE2 and OE6 were slightly inhibited,and the yellowing of leaves were only a little at the edge of leaves.The results of physiological indexes showed that BpGRP gene could reduce the damage or death of birch cells by increasing the activity of superoxide dismutase(SOD),increasing the content of proline(Pro),reducing the content of hydrogen peroxide(H2O2),malondialdehyde(MDA)and relative electrical conductivity,so as to improve the salt tolerance of transgenic birch.3.After analyzing the promoter of BpGRP gene,ABRE element was selected to obtain the upstream regulatory gene growth regulator(BpGRF3)through yeast one hybrid experiment.It was further verified that BpGRF3 gene can bind ABRE element to regulate the expression of BpGRP gene by yeast experiment,vivo binding experiment in birch and EMSA.4.The open reading frame(ORF)of BpGRF3 was 1821 bp,encoding 606 amino acids with relative molecular weight of 65.75 KD,theoretical isoelectric point(PI)of 8.67,half-life of 4.4 hours,fat coefficient of 28.91 and instability coefficient of 52.49.The BpGRF3 protein contained two conserved domains,QLQ and WRC.Multiple sequence alignment and phylogenetic tree analysis showed that BpGRF3 protein had high homology with GRF protein from Myrica rubra(75.37%)and Juglans regia(75.08%).Physiological indexes under salt stress showed that overexpression of BpGRF3 gene could improve salt tolerance of transgenic birch by enhancing SOD activity,increasing Pro content,reducing H2O2 and MDA content,while inhibiting the expression of BpGRF3 gene was the opposite,reducing birch tolerance to salt stress.5.The results of yeast two hybrid system showed that BpGRF3 gene had no transcriptional activation activity.Two proteins interacting with BpGRF3 transcription factor were identified by yeast two hybrid system,including BpSHMS(serine hydroxymethyl transferase)and Bp14-3-3(14-3-3-like protein).The expression levels of BpSHMS and Bp14-3-3 genes under salt stress were analyzed by qRT-PCR.We found that BpSHMS could strongly respond to salt stress,and the bimolecular fluorescence complementation experiment(BiFC)once again verified that BpSHMS can interact with the BpGRF3 gene.In addition,the interaction between BpGRF3 gene and 6 BpGRFs genes in birch was studied by yeast two hybrid system.The results showed that BpGRF3 could form heterodimer with its homologous proteins BpGRF6,BpGRF7 and BpGRF8,and could also form heterodimer with BpGRF1/BpGRF2,but its specific binding ability was weak.6.Two BpmiR396c were obtained by analyzing and screening birch miRNA-seq and RNA-seq data under salt stress.The expression pattern analysis showed that the transcription levels of BpmiR396c-1 and BpmiR396c-2 were inhibited under salt stress.The corresponding seven target genes were all induced to be up-regulated under salt stress.The downstream target gene BpGRF3 reached the highest expression level at 36 h of salt stress.The above studies show that we have obtained the possible upstream regulatory factor BpmiR396c-2 of the target gene BpGRF3.In order to further confirm this conclusion,BpmiR396c-2 and the downstream target gene BpGRF3 were selected for further study.The results showed that the BpGRF3 gene expression was inhibited in the overexpressing of BpmiR396c-2 birch callus,and the SOD activity and the content of Pro in the overexpressing of BpmiR396c-2 birch callus were significantly lower than the control,while the content of H2O2 and MDA was significantly higher compared with the control,indicating that the overexpression of BpmiR396c-2 birch callus has reduced tolerance to salt stress.In summary,the expression of BpmiR396c-2 was inhibited by salt stress during the regulation of salt tolerance in birch,which reduced the ability to cut or inhibit the downstream BpGRF3 gene,releases BpGRF3 gene expression,and enhanced the effect of BpGRF3 on BpGRP,thereby improving reactive oxygen species(ROS)scavenging capability,decreasing the malondialdehyde content and lipid peroxidation cell membranes in the process of salt tolerance regulation of birch.This study provides a basis for revealing the salt tolerance regulatory network of birch and provides a theoretical reference for molecular breeding of birch.At the same time,the new salt-tolerant variety of birch will effectively utilize and develop saline soil and increase the cultivation area birch.