Functional Study On Salt Tolerance Of PAT1 Gene From Betula Platyphylla

The GRAS(GAI,RGA,and SCR)protein family are plant-specific transcription factors and play a key role in plant growth and development and response to stress.In this study,a GRAS family gene was obtained from the salt-stressed transcriptome of Betula platyphylla and named it BpPAT1.Multi-sequence comparison of BpPAT1 with GRAS proteins of other 9 kinds of plants showed that the conserved domain of BpPAT1 is at the C-terminus;Phylogenetic tree analysis of 33 GRAS proteins in Arabidopsis showed that BpPAT1 is closely related to AtPAT1.The fluorescent localization expression vector pBI121-GFP-BpPAT1 was constructed,indicating that BpPAT1 is a nuclear localization protein.The BpPAT1 gene overexpression vector(pROK Ⅱ-BpPAT1)and the suppression expression vector(pFGC5941-BpPATl)were constructed to preliminarily explore the salt tolerance function of the BpPAT1 gene.The two and pROK Ⅱ vector were used Agrobacterium-mediated high-efficiency transient transformation system to obtain three transient expression plants.The BpPAT1 gene overexpression vector(pROK Ⅱ-BpPAT1)and the suppression expression vector(pFGC5941-BpPAT1)were constructed,and the first two vectors were combined with the pROK Ⅱ vector to obtain transient transgenic plants through an efficient transient transformation system mediated by Agrobacterium.Under salt stress,the superoxide dismutase(SOD),peroxidase(POD)activity and proline content of the overexpressed plants were the highest,and were significantly higher than the control plants;the malondialdehyde(MDA)content and electrical conductivity were the lowest,and the cell membrane structural integrity was the best:In contrast.inhibiting the superoxide dismutase(SOD),peroxidase(POD)activity and proline content of expressing plants,the highest MDA content and electrical conductivity,and the most severe cell damage.The above results indicate that BpPAT1 protects the structural integrity of cell membranes by reducing the accumulation of reactive oxygen species(ROS).thereby improving the tolerance of transient transgenic plants to salt stress.In order to further verify the results of transient transformation,the pROK Ⅱ-BpPAT1 overexpression vector was stably transferred into Betula platyphylla.and 11 transgenic plants were screened.The expression level of BpPAT1 gene is 2-31 times that of wild-type plants in pROK Ⅱ-BpPAT1 transgenic plants.Under salt stress,the SOD and POD enzyme activities and proline content of the overexpression transgenic plants were the highest.and were significantly higher than those of the control plants;the MDA content and electrical conductivity were the lowest,and the membrane structure integrity was the best.Under salt stress and normal conditions,BpPAT1 gene overexpression(OE5)and wild type were analyzed by RNA-seq.The results showed that a total of 1302 differentially expressed genes were obtained under normal conditions,838 were up-regulated,and 464 were down-regulated;under salt stress conditions,a total of 838 differentially expressed genes were obtained,with 315 up-regulated and 523 down-regulated.A total of 12 genes with 6 up-regulation and 6 down-regulation were selected for qRT-PCR to verify the results of RNA-Seq data.Among the differentially expressed genes obtained,there are more than 30 differential genes that are up-regulated under salt stress conditions,such as peroxidase,lipid transporter and other encoding proteins,as well as transcription factor genes such as MYB,AP2,DREB.etc.This indicates that these differential genes can respond to salt stress and may be regulated by the BpPAT1 gene.