Analysis Of Downstream Regulation Mechanism Of ThSCL32 In Tamarix Hispida

GRAS transcription factors belong to the plant-specific protein family,in addition to participating in plant growth and development,they also participate in plant responses to a variety of abiotic stresses.In previous studies,ThGRASl(which belongs to the SCL subfamily SCARECROW-LIKE 32 protein in the GRAS family,so it was renamed ThSCL32)was cloned from Tamarix hispida.And it was confirmed that the gene can improve salt tolerance in T.hispida.In this study,the salt tolerance downstream mechanism of ThSCL32 was further analyzed.The specific results were as follows:The TpROKII-ThSCL32,pFGC5941-ThSCL32 and empty pROKII vectors were transiently transformed into T.hispida to obtain transient overexpression(OE),suppression expression(SE)and control(CON)transgentic plants.Transcriptome sequencing analysis was performed on three transgenic T.hispida.The results showed that compared with the CON group,1623 genes were up-regulated and 2275 genes were down-regulated in SE lines.The up-regulated and down-regulated genes in the OE group were 254 and 473,respectively.Among them,83 differentially expressed genes were common among the three transgenic lines.21 genes with complete sequences were screened as possible target genes of ThSCL32 after analysis of T.hispida genome.ThSCL32 was constructed on pGADT7,and 19 random sequences were obtained from the library using Y1H.These included 4 known elements(CAAT-BOX,Unnamed-4,GT1-motif,TATA-box)and 15 unknown element(W1-15)sequences.Interestingly,the 5 nucleic acids deletion of W1 from the left or right sides did not affect its normal growth.So,these possible target gene promoters were analyzed.the results showed that many deletions sequenes of the W1 were found in these promoters.In order to verify the differential genes that ThSCL32 may directly regulate in transcriptome sequencing by the series missing fragment,ChIP-PCR experiment were performed in T.hispida.The results showed that ThSCL32 may directly bind to the promoters of ThPHD and ThGH3,1.And ThSCL32 can specifically recognize the missed W1 element(ACGTTG)on the ThPHD promoter to regulate ThPHD.The salt tolerance function of ThPHD were further explored.The ThPHD overexpression vector pROKII-ThPHD,the RNAi vector pFGC5941-ThPHD were constructed and transiently transformed it with empty pROKII vector into T hispida.Transgenic plants of OE,RNAi and CON were analyzed and compared for H2O2,MDA and pro line content before and after 120 mmol NaCl stress.The results showed that under NaCl stress,OE plants had the lowest H2O2 and MDA content,the largest accumulation of free proline,and a weak response to salt stress environment.These results suggested that ThPHD overexpression under salt stress may enhance the salt tolerance of transgenic plants by reducing H2O2 and MDA accumulation and increasing proline accumulation.The results of this study lay the foundation for further understanding the salt-tolerance mechanism of T.hispida.