Study On Drought Response Of Genes Related To Lignin Synthesis In Caragana Korshinskii

Caragana korshinskii is a perennial deciduous shrub widely distributed in arid and semi-arid regions of China,because of its strong drought resistance,it has become a dominant species in the returning farmland to forest on the Loess Plateau.Lignin is one of the important secondary metabolites of the plant phenylpropane synthesis pathway,which can enhance the mechanical strength of the plant and ensure the transport of water in the transfusion tissue.Previous studies in the laboratory showed that the xylem of the veins in Caragana korshinskii became more developed with the intensification of drought,RNA-Seq technology was used to sequence the leaves of Caragana korshinskii at the three sampling sites(Huangling?Yulin and Dalad Banner)with decreasing precipitation from south to north in the Loess Plateau.It was found that the phenylpropanoid metabolic pathway of Caragana korshinskii was located at the top of the differentially expressed metabolic pathways,and the expression level of the lignin synthesis-related enzyme genes was significantly up-regulated.The purpose of this study was to elucidate the drought response pattern of the key enzyme genes involved in the lignin synthesis of Caragana korshinskii,and to lay a foundation for revealing that the drought has caused the xylem to be more developed and then improved drought resistance.In this study,Caragana korshinskii leaves in the field precipitation gradients and single indoor water control treatments(control,moderate drought,and severe drought)were used as research objects to verify the response and regulation of lignin to drought from the genetic?protein and physiological levels.The CkC4 H gene in lignin synthesis pathway and transcription factor CkMYB39 gene were cloned and transformed into Arabidopsis thaliana,which aimed to verify that lignin synthesis genes and regulatory factors enhance the drought resistance of plants by strengthening xylem in Caragana korshinskii.The test results of this study are as follows:(1)Through the analysis of RNA-Seq data,it was found that the expression of seven major genes on the lignin synthesis pathway of Caragana korshinskii showed an upward trend with decreasing precipitation.Real-time fluorescence quantitative PCR was used to detect the relative expression level of each gene in the field samples and laboratory samples respectively.The results showed that the relative expression level of CkPAL?CkC4H?CkC3H?CkCOMT?CkCCR and CkCAD genes was increased with drought in field samples.The amount of Ck4 CL gene expression showed a trend of reducing first and then increasing.In lab samples,the expression levels of CkC4 H ? CkCOMT and CkCAD genes gradually increased with decreasing water gradients,the expression levels of CkPAL?Ck4CL?CkC3H and CkCCR genes showed a trend of decreasing first and then increasing.(2)The polyclonal antibodies of key enzymes CkC4 H and CkC3 H of the lignin synthesis pathway were prepared and their relative contents in the field and lab samples were detected.Western blot results showed that the expression levels of CkC4 H and CkC3 H proteins increased significantly with the deepening of the degree of drought in both field samples and lab samples.The gray value analysis of the CkC4 H and CkC3 H proteins also supports the above test results.(3)The lignin synthesis pathway mainly forms three kinds of lignin monomers.Phloroglucinol will stain both G and H lignin monomers and the M?ule reaction will color the S lignin monomers.The staining results showed that the vascular bundle area?sclerenchyma area and xylem area gradually increased with the increasing degree of drought both in the field samples and lab samples,and the statistical analysis showed that the ratio of the coloring part to the entire vascular bundle area showed a significant increase.(4)The contents of lignin in leaves of Caragana korshinskii were measured by acetyl bromide-ultraviolet spectrophotometry.The results showed that the lignin content in leaves of Caragana korshinskii in the field and lab samples showed a significant increase with the intensification of drought.(5)The 35S::CkC4H and 35S::CkMYB39 overexpression vectors were successfully constructed.The leaves of Nicotiana benthamiana were transiently transformed and the protein localization was observed by laser confocal scanning microscopy.The results showed that the CkC4 H protein was localized at the cell membrane,the CkMYB39 transcription factor was located in the nucleus.(6)The constructed 35S::CkC4H and 35S::CkMYB39 overexpression vectors infiltrates Arabidopsis thaliana using the inflorescence method.The two target genes and pCAMBIA1302 vector have been obtained the T2 plants.Based on the above findings that obtained the following conclusion: Caragana korshinskii increased lignin content under drought stress by increasing the expression of related enzyme genes and proteins in the lignin synthesis pathway.The deposition of lignin in xylem vessels and sclerenchyma is one of the important mechanisms for the development of drought tolerance in the developed xylem of Caragana korshinskii.