Study On The Mechanism Of Bermudagrass Transcription Factor PIF4 And Shade In Regulating Salt Stress

Bermudagrass（Cynodon dactylon L.）grows rapidly,with dense root,developed stolon,and strong resistance to stress.It is widely used in landscaping,stadium construction and soil rehabilitation areas,with high ecological,economic,and social application values.Therefore,it is crucial to study the bermudagrass growth and development process,stress response,so as to improve stress resistance using the molecular biological methods.In this study,common bermudagrass was used to analyze the photosynthetic physiological characteristics in salt,shade,and combined stress.Then,the Cd PIF4 gene was transferred to bermudagrass by Agrobacterium-mediated genetic transformation technology,and further its regulating mechanism in salt stress was verified.The results could be used to screen gene resources for salt-resistant breeding of bermudagrass.The main findings are as follows:（1）Shade,salt,and the combined stress affected the plant height,relative water content,chlorophyll content,and caused changes in photosynthetic capacity of bermudagrass.Among them,salt stress caused the most serious damage.The growth status and photosynthetic characteristics of bermudagrass under the combined stress were better than those under salt stress.Under salt stress,combined with shade treatment could reduce the damage degree of the cell membrane of bermudagrass leaves and maintain stable relative water content.Also,shade treatment improved the expression level of photosynthesis-related genes,increased the photosynthetic pigment content,allowed more light energy to be absorbed and captured,and increased the maximum photochemical efficiency of photosystem II,improved the photosynthetic performance of bermudagrass,thus alleviated the negative effects of salt stress on bermudagrass to a certain extent.（2）The expression pattern of Cd PIF4 gene in roots,stems and leaves was detected by fluorescence quantitative PCR.It was found that the expression level of Cd PIF4 gene was the highest in leaves,followed by stems,and the lowest in roots.Then the Cd PIF4 gene was cloned from bermudagrass leaves using PCR technology,whose CDS region was 1290 nucleotides,430 amino acids encoded.Bioinformatics analysis showed that Cd PIF4 encodes a hydrophilic protein containing the b HLH conservative domain.（3）The Cd PIF4 gene was constructed on the PANIC-6B vector,at least two bermudagrass transgenic lines were obtained by Agrobacterium tumefaciens-mediated genetic transformation.GUS staining and PCR showed that it has been integrated into the genome of bermudagrass,and overexpressed confirmed by q RT-PCR.（4）The overexpressed-Cd PIF4 bermudagrass were treated with salt,the phenotypic results showed that the tolerance of transgenic lines were worse than that of wild type.Furthermore,the photosynthesis related indexes under salt stress were determined.Compared with the wild type,the photosynthetic pigment content decreased significantly,the reaction center did not absorb and capture more light energy,and the maximum photochemical efficiency and photosynthetic performance indexes PI_ABS and PI_total decreased significantly in transgenic lines.Overall,the photosynthetic performance of overexpressed-Cd PIF4 bermudagrass did not improve under salt stress.Therefore,as a key transcription factor in skotomorphogenesis,PIF4had other mechanisms that were different from shade treatment in the salt stress regulatory pathway.（5）In order to further investigate the function of Cd PIF4 in the salt stress regulatory pathway,other physiological indicators were measured.The results showed that under salt stress,the growth of transgenic bermudagrass was inhibited,the electrolyte leakage and malondialdehyde content of leaves were higher,the crude protein and content antioxidant enzyme activity were lower than those of wild type.In addition,the expression levels of oxidative stress related genes SOD,POD,and CAT varied among different bermudagrass lines.Overexpression of Cd PIF4 reduced the transcription levels of SOD,POD,and CAT.Therefore,we infer that overexpressed Cd PIF4 leads to a decrease in the antioxidant capacity of bermudagrass,which in turn affects its salt tolerance.