Study On Salt Tolerance Mechanism Of BpGRF3 Gene In Betula Platyphylla

Growth-regulating Factors（GRFs）are a class of unique transcription factors involved in plant growth,development and abiotic stress response.In previous studies,we identified and cloned a BpGRF3 gene from birch,and demonstrated that overexpression of BpGRF3 in birch callus could increase SOD（Superoxide Dismutase）activity and proline content,decrease MDA and H₂O₂ content in birch callus.In this study,the salt tolerance function and mechanism of BpGRF3 gene were further studied.And the specific results are as follows:（1）The plant overexpression vector p CAMBIA1302-BpGRF3 was transformed into birch,and 10 BpGRF3 overexpression lines of birch were identified by q RT-PCR.Three lines with the highest expression levels（OE1,OE7 and OE11）were selected for further phenotype observation and physiological index determination.Under normal conditions,the growth state of OE lines were basically the same as that of WT lines.After 14 days of Na Cl stress,the growth of WT was severely inhibited,such as the leaves withered and fell off,while the growth inhibition of OE lines was slight and the leaf growth state was better.The results of physiological indexes showed that overexpression of BpGRF3 could increase the activities of SOD and POD（Peroxidase）and reduce the contents of MDA and H₂O₂.These results indicated that BpGRF3 overexpressed lines could enhance the salt tolerance of transgenic plants by scavenging reactive oxygen species,reducing the accumulation of H₂O₂ and alleviating cell damage..（2）The gene expression profiles of overexpressed BpGRF3 lines and WT were analyzed using RNA-Seq.The results showed that after Na Cl stress,there were 2130 up-regulated genes and 2677 down-regulated genes in OE lines compared with WT.The results of GO analysis showed that up-regulated genes enrichment reached 155 pathways and down-regulated genes enrichment reached 254 pathways.KEGG pathway results showed up-regulated genes enrichment to 36 pathways and down-regulated genes enrichment to 44 pathways.Previous studies showed that BpGRF3 gene was combined with ABRE element.Furthermore,promoter element was analysis on the differential genes in the pathways closely related to salt stress（such as glycolysis/gluconeogenesis,photosynthesis,etc.）.Ch IP-PCR was used to further explore whether BpGRF3 could regulate the expression of downstream target genes by combining with ABRE.The results showed that Bp AKR（aldo-keto reductase,AKR）and Bp Glu TR（Glutamyl-t RNA reductase,Glu TR）promoter fragment containing ABRE element can be enriched in large quantities,indicating that BpGRF3 can directly regulate the expression of Bp AKR and Bp Glu TR.（3）Previous experiments confirmed that BpGRF3 has no transcriptional activation activity,indicated BpGRF3 may play roles in salt stress by interacted with the other proteins.So in this study,the proteins interacted with BpGRF3 were searched.4 proteins that may interact with BPGRF3 were found,including Bp APX（Ascorbate peroxidase,APX）,Bp CHS（Chalcone Synthase,CHS）,Bp FBA（Fructose-1,6-bisphosphate aldolase,FBA）,and Bp LHCP（Chlorophyll a/b-Binding Protein,LHCP）.And the interaction of BpGRF3 with Bp APX,Bp CHS,Bp FBA and Bp LHCP proteins were further confirmed in vivo/in vitro by yeast two-hybridization,Bi FC and Pull down experiments.（4）In order to explore whether BpGRF3 interaction protein responds to salt stress,q RT-PCR was used to analyze its expression pattern under salt stress.The results showed that the expression levels of Bp APX,Bp CHS,Bp FBA and Bp LHCP in the roots,stems and leaves of birch had varying degrees of change.,suggested that Bp APX,Bp CHS,Bp FBA and Bp LHCP genes also may respond to salt stress.In addition,after Na Cl stress,compared with the WT,the As A content,total flavonoids content,chlorophyll content and FBA activity of BpGRF3 overexpression lines were significantly higher than those of the WT.These results indicate that BpGRF3 may interact with Bp APX,Bp CHS,Bp FBA and Bp LHCP to regulate key genes in corresponding metabolic pathways and increase the content of corresponding metabolites,and further improve the antioxidant capacity and photosynthesis of plants,and thus improve the salt tolerance of transgenic lines.These results provide a new idea for further revealing the salt tolerance mechanism of BpGRF3 gene.