Study On The Stress Resistance Of Overexpressing Brassica Rapa BrMAPK4 In Arabidopsis

Plant growth is effected by various stresses(such as drought,high/low temperature,salt,UV and heavy metals,etc),which severely restricts the growth quality,and yield of plants,especially some economic crops(such as Oryza sativa L,Brassica rapa L,Solanum tuberosum,Cucumis sativus L,Solanum lycopersicum,etc).Therefore,plants have established a variety of defense mechanisms to adapt to various adversity stresses,including signal recognition,transduction,transmission and expression of related gene.Mitogen-activated protein kinase(MAPK)cascade signal plays an important role in response to stress.MAPK cascade signal is activated mainly through step-by-step phosphorylation to amplify the extracellular signal cascade and regulate the transcription level of downstream genes.In addition,MAPK cascade signals also interact with other signal transduction pathways or signal molecules(H2O2、Ca2+、NO、H2S、etc)in response to plant stress.In this study,based on the reported MAPK information of Brassica rapa L,using Brassica rapa L of "Longyou 6" with strong cold resistance as the material,firstly carried out bioinformatics analysis of BrMAPK4 protein,and then carried out expression analysis of BrMAPK4 gene induced by different stress,and successfully cloned BrMAPK4 gene.The overexpression vector of BrMAPK4 gene was constructed by Gateway technology,and Arabidopsis plants with overexpression of BrMAPK4 gene were obtained with Agrobacterium tumebii as medium.The plants were treated with salt stress and low temperature stress,and the biological function of the overexpression of BrMAPK4 gene plants were further analyzed.To explore the regulatory mechanism of the plants overexpressing BrMAPK4 in response to adversity stress.The results are as follows:1.Bioinformatics analysis of BrMAPK4 proteinMultiple sequence alignment and phylogenetic analysis of BrMAPK4 protein showed that BrMAPK4 protein was more conserved than the MAPK protein of other species,with 11 conserved domains,and the double phosphorylation motif of TEY between the VII and VIII domains.Phylogenetic analysis showed that BrMAPK4 has a high similarity with AtMAPK4,and AtMAPK4 has been shown to respond to a variety of stresses.2.Analysis of BrMAPK4 expression under different stress treatmentsIn order to explore the expression of BrMAPK4 in Brassica rapa,qRT-PCR quantitative detection revealed that BrMAPK4 gene was expressed in root,stem and leaf tissues of Brassica rapa,and the expression levels were different in different tissues,with the highest expression level in leaves and the lowest in stems.Therefore,its leaves were selected for the subsequent experimental study.The stress induced expression analysis leaves showed that the expression level of leaves increased significantly under 4℃ and 200 mM NaCl treatment,both of which showed a trend of first increasing and then decreasing.After 20%PEG and UV treatment,the expression of BrMAPK4 gene increased slowly,but showed a trend of continuous increase.In conclusion,the expression pattern of BrMAPK4 gene in Brassica rapa were different under different stresses.3.Construction of BrMAPK4 gene overexpression vector and genetic transformation in ArabidopsisIn this study,the BrMAPK4 gene was cloned from Brassica rapa,and an overexpression vector was constructed through Gateway clone technology to connect the vector with the cDNA sequence of BrMAPK4,and the Col-0 Arabidopsis was transformed by Agrobacterium tumefaciens impregnation method,we successfully obtained four BrMAPK4 transgenic Arabidopsis plants.The relative expression level of BrMAPK4 in the T2 generation transgenic lines were detected by qRT-PCR,and its expression at the protein level were detected by Fluorescence microscopy.Finally,two transgenic lines with high expression levels were selected as the subsequent experimental materials to further study the function of BrMAPK4 gene under different stresses.4.Functional study of transgenic Arabidopsis under salt and low temperature stressesUnder salt stress,compared with wild-type Arabidopsis,overexpression of BrMAPK4 significantly enhanced the root elongation and growth,indicating that overexpression of BrMAPK4 significantly enhanced the resistance to salt stress.The overexpression of BrMAPK4 gene in Arabidopsis was treated with salt and low temperature stress,and the malonaldehyde(MDA)content and relative electrical conductivity(%)of the transgenic plants were lower than that of the wild-type plants,indicating that the overexpression of BrMAPK4 gene in Arabidopsis could reduce the degree of membrane damage.Further detection of reactive oxygen species(ROS)and antioxidant enzymes(SOD,CAT and APX)activities showed that compared with wild-type plants the contents of H2O2 and O2·-in transgenic plants were lower,while the activities of antioxidant enzymes(SOD,CAT and APX)were higher than those in wild-type plants.In conclusion,overexpression of BrMAPK4 in Arabidopsis can effectively eliminate ROS and enhance antioxidant enzyme activity.In addition,the detection of proline and soluble sugar showed that wild-type plants were lower than transgenic lines,while the determination of chlorophyll content showed that wild-type were also lower than transgenic lines,indicating that transgenic Arabidopsis could regulate osmotic balance,improving photosynthetic pigment content and enhancing plant tolerance.