The Transcription Factor MxMYB176 And Its Upstream Promoter Respond To Drought Stress In Malus Xiaojinensis

In this paper,we studied the function of MxMYB176 under drought stress,and analyzed the structure and function of MxMYB176 promoter to explore the molecular mechanism of the transcription factor MxMYB176 in response to drought stress.The following results were obtained:The total RNA extracted under drought stress was found to increase the expression of MxMYB176 transcription factor under drought stress by RT-PCR in Malus xiaojinensis.The growth of transgenic 1300-MYB Arabidopsis thaliana under 300 mmol/L mannitol was significantly higher than that of wild type,and the main root length was 1.84-fold of WT.Under drought stress,the MDA content in transgenic 1300-MYB Arabidopsis thaliana was significantly lower than that in transgenic?1300?and wild-type?WT?,the accumulation of proline was significantly higher,the content of H₂O₂ was significantly lower than that of transgenic?1300?and wild-type?WT?,SOD activity was significantly higher than that of transgenic?1300?and wild-type?WT?,anthocyanin and carotenoid content were also significantly higher than that of transgenic?1300?and wild-type?WT?,chla,chlb,and total chlorophyll significantly increased,but chla/chlb did not change significantly.The results showed that MxMYB176 transcription factor can significantly improve the drought resistance of Arabidopsis thaliana.MxMYB176 gene promoter contained 1275bp was cloned by using PCR amplification.Using software for bioinformatics analysis of MxMYB176 promoter sequence,the results showed that the MxMYB176 gene promoter contained various cis-acting elements such as light-sensitive elements,ABRE,CGTCA-motif,LTR,GARE-motif,Unnamed_<sub>4.The plant expression vector of three 5?-terminal deletion fragments of p9::GUS,p6::GUS and p3::GUS was successfully constructed.The transient expression analysis of tobacco showed that the GUS gene driven by different 5?-terminal deletion promoters was expressed in pF>p9>p6>p3,in which the GUS gene is hardly expressed in the p3 fragment.Different 5?-terminal deletion fragment promoters were transformed into Arabidopsis thaliana,and their stable expression was analyzed.It was found that the MxMYB176promoter activity was weakened with the increase of the deleted fragment,and the activity of the GUS protease of the full-length promoter pF was 0.0303 nM·Min^-1·?g^-1.They were 1.8,5.3,and 24.1-fold of p9,p6,and p3,respectively.The stable expression in plants showed a trend consistent with transient expression,and the activity of MxMYB176promoter in green tissues was significantly increased in plants transfected with p6 fragment promoter.In the promoter of the transgenic p3,only the expression of the GUS gene was observed in the Arabidopsis thaliana roots with weak GUS expression.It was concluded that the core region of the MxMYB176 promoter in response to the stress response was between-950 bp and-653 bp.Treatment of transgenic pF::GUS Arabidopsis thaliana under different mannitol concentration gradients,the pF promoter activity increased with the increase of mannitol concentration,and the promoter activity was the strongest under 300 mmol/L mannitol treatment,which was 1.67-fold that of the control,Arabidopsis thaliana transfected with different deletion fragment promoters was treated with 300 mmol/L of mannitol,and the GUS activities of different fragment promoters pF,p9,p6,and p3 were increased by 0.69,0.90,0.26,and 0.84-fold,respectively.The results showed that the activity of MxMYB176promoter was significantly enhanced under mannitol treatment?p<0.05?,and the transcription factor was predicted to be induced by drought stress.